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Archives of Biochemistry and Biophysics 505 (2011) 131–143

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Archives of Biochemistry and Biophysics

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Review

Enzymes of the mevalonate pathway of isoprenoid biosynthesis

Henry M. Miziorko ⇑Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri – Kansas City, 5007 Rockhill Road, Kansas City, MO 64110, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 9 September 2010and in revised form 28 September 2010Available online 7 October 2010

Keywords:Mevalonate pathwayIsoprenoid biosynthesisHMG-CoASterol biosynthesis

0003-9861/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.abb.2010.09.028

⇑ Fax: +1 816 235 5595.E-mail address: [email protected]

1 Abbreviations used: MVA, mevalonic acid/mevalondiphosphate; HMG-CoA, 3-hydroxy-3-methylglutaryl-Cphate; FPP, farnesyl diphosphate; FSPP, farnesyl thiodikinase; PMK, phosphomevalonate kinase; MDD, mevaylase; IPK, isopentenyl phosphate kinase; GHMP, galamevalonate kinase, phosphomevalonate kinase; NMPAMPPNP, 50-adenylyl-beta, gamma-imidodiphosphate.

The mevalonate pathway accounts for conversion of acetyl-CoA to isopentenyl 5-diphosphate, the versa-tile precursor of polyisoprenoid metabolites and natural products. The pathway functions in mosteukaryotes, archaea, and some eubacteria. Only recently has much of the functional and structural basisfor this metabolism been reported. The biosynthetic acetoacetyl-CoA thiolase and HMG-CoA synthasereactions rely on key amino acids that are different but are situated in active sites that are similarthroughout the family of initial condensation enzymes. Both bacterial and animal HMG-CoA reductaseshave been extensively studied and the contrasts between these proteins and their interactions with statininhibitors defined. The conversion of mevalonic acid to isopentenyl 5-diphosphate involves three ATP-dependent phosphorylation reactions. While bacterial enzymes responsible for these three reactionsshare a common protein fold, animal enzymes differ in this respect as the recently reported structureof human phosphomevalonate kinase demonstrates. There are significant contrasts between observationson metabolite inhibition of mevalonate phosphorylation in bacteria and animals. The structural basis forthese contrasts has also recently been reported. Alternatives to the phosphomevalonate kinase and mev-alonate diphosphate decarboxylase reactions may exist in archaea. Thus, new details regarding isopente-nyl diphosphate synthesis from acetyl-CoA continue to emerge.

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Introduction enzymes, with the possible exception of HMG-CoA reductase, the

The mevalonate (MVA)1 pathway for biosynthesis of isoprenoidsfrom acetate (Scheme 1) represents the initial steps in a series ofenzymatic reactions [1] that have, for decades, been established toaccount for production of polyisoprenoids (e.g. dolichol) and sterols(e.g. lanosterol, ergosterol, cholesterol) in fungi, plant cytoplasm,animals, most other eukaryotes, archaea and some eubacteria.

In recent years, much attention has been generated by the elu-cidation of an alternate pathway (the deoxyxylulose phosphate,methylerithritol phosphate, or nonmevalonate pathway [2]) thatis operative in many bacteria, plant chloroplasts, and some eukary-otic parasites. This pathway represents an alternative series ofreactions for production of isopentenyl diphosphate.

While much progress continues to be made in work on thedeoxyxylulose phosphate pathway, there had been relatively mod-est effort invested in the application of recombinant DNA method-ology and structural investigation to the mevalonate pathway

ll rights reserved.

ate; MVAPP, mevalonate 5-oA; IPP, isopentenyl diphos-

phosphate; MVK, mevalonatelonate diphosphate decarbox-ctokinase, homoserine kinase,

nucleoside monophosphate,

target of the family of statin inhibitors of cholesterol biosynthesis.These enzymes had been isolated and classical enzymological andbiochemical characterization had been performed prior to the evo-lution of experimental tools that have expedited the understandingthe molecular basis of enzyme function. These enzymes are signif-icant in animals since several have been identified as potentiallyuseful targets for modulation of polyisoprenoid and sterol biosyn-thesis. Also, defects of some of these enzymes have been impli-cated in human inherited disease. These include the cytosolicmevalonate kinase as well as mitochondrial isoforms of biosyn-thetic acetoacetyl-CoA thiolase and HMG-CoA synthase. The en-zymes occur in several gram-positive bacteria, including somethat are pathogenic to humans. Genetic work [3] has demonstratedthat disruption of genes encoding various enzymes in the mevalo-nate pathway blocks proliferation of these pathogens. These obser-vations suggest the merit of working toward a more detailedunderstanding of the mevalonate pathway enzymes.

Much of the work on identification and functional assignmentsof catalytically important active site residues, high resolutionstructure determinations, as well as the investigation of the basisfor the inhibition or regulation of some of these enzymes has onlyappeared in recent years. This review will primarily focus on someof these more recent developments. While the characterization ofseveral enzymes is still in progress, the contributions of these re-cent studies in addressing the molecular basis for isoprenoid bio-synthesis by these enzymes are interpreted in the context of the

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Scheme 1. Mevalonate (MVA) pathway for isopentenyl diphosphate biosynthesis.

ES- + CH3-C-S-CoA||O

CoAS-

ES-C-CH3

||O

132 H.M. Miziorko / Archives of Biochemistry and Biophysics 505 (2011) 131–143

earlier reports to provide an updated understanding of the mevalo-nate pathway. Reviews of selected enzymes and reactions in thispathway have also appeared [4–6].

ES-C-CH3

||O

+ CH3-C-S-CoA||OB:

ES- + CH3-C-CH2-C-SCoA|| ||

O O

acetyl-SCoA acetyl-S-enzyme

acetoacetyl-SCoA

Scheme 2. Chemical steps in the biosynthetic acetoacetyl-CoA thiolase reaction.

Acetoacetyl-CoA thiolase

The thiolase catalyzed Claisen condensation and cleavage reac-tions [7,8], whereby an acyl-CoA molecule is extended or short-ened (respectively) by a two carbon acetyl module are widelyobserved in metabolism with the enzyme being found in a widevariety of prokaryotes and eukaryotes. Thiolytic cleavage, cata-lyzed by 3-ketoacyl-CoA thiolases (EC 2.3.1.16) is perhaps mostfamiliar as a key step in Knoop’s pathway of beta oxidation of fattyacids. In the context of the mevalonate pathway, the biosynthetic(condensation) reaction to form acetoacetyl-CoA from two acet-yl-CoA molecules will be emphasized. This biosynthetic reactionis catalyzed by acetoacetyl-CoA thiolase (or acetyl-CoA acetyltransferase), classified by the International Union of Biochemistryin the EC 2.3.1.9 category:

2 Acetyl-CoA ¢ acetoacetyl-CoAþ CoASH

The chemistry of this reaction, depicted in the following scheme(Scheme 2), involves a tranferase step, i.e. formation from substrateacetyl-CoA of a covalent acetyl-S-enzyme and release of CoASH. Thesubsequent condensation step involves deprotonation of C2 of thesecond acetyl-CoA substrate and attack of the resulting carbanionon C1 of the acetyl-S-enzyme reaction intermediate to producethe product acetoacetyl-CoA and release free enzyme.

An interesting homologous reaction has been recently reported[9] to involve condensation of acetyl-CoA with malonyl-CoA to formacetoacetyl-CoA, catalyzed by the NphT7 protein from Streptomyces.Such a reaction appears chemically similar to the formation ofacetoacetyl-acyl carrier protein in the initial condensation step of

bacterial fatty acid biosynthesis. The NphT7 protein is encoded byan open reading frame mapping within a gene cluster proposed toaccount for other mevalonate pathway enzymes. It will be interest-ing to learn whether other examples of this variation in more typicalacetoacetyl-CoA biosynthesis exist.

Functional observations

Identification of the specific cysteine involved in acetyl-S-en-zyme intermediate formation was accomplished for a degradativeketoacyl-CoA thiolase [10]. Soon afterwards, a cytosolic liver aceto-acetyl-CoA thiolase was highly purified and characterized [11].Much of the more recent mechanistic and mutagenesis work onbiosynthetic thiolase, reported by the Walsh, Sinskey, and Masa-mune labs, has employed a recombinant form of the Zoogloearamigera protein, which supports bacterial polyhydroxybutyrateproduction. For this protein, Cys-89 is involved in formation ofthe acetyl-S-enzyme reaction intermediate [12]. The active sitebase (B:) that activates the second acetyl-CoA substrate moleculefor condensation with the reaction intermediate has been identi-fied as Cys-378 [13].

Fig. 1. Active site residue triad in Z. ramigera acetoacetyl-CoA thiolase, based on thestructural coordinates 1DM3. The structure [14] indicates the acetyl-enzymeintermediate formed at Cys-89 as well as the acetyl-CoA that condenses with thereaction intermediate. His-348 interacts with the C1 carbonyl of bound acetyl-CoAto provide a charge sink that stabilizes the C2 carbanion formed after protonextraction by the general base Cis-378. The carbanion is in close proximity to C1 ofthe acetyl-enzyme intermediate and supports efficient condensation to formacetoacetyl-CoA and regenerate free enzyme.

H.M. Miziorko / Archives of Biochemistry and Biophysics 505 (2011) 131–143 133

Structural observations

Several reports of structural work from the Wierenga lab haveinvolved the Z. ramigera protein. In particular, results were ob-tained using crystals that were flash frozen after soaking with acet-yl-CoA to produce a crystal containing the acetylated enzymereaction intermediate and a bound acetyl-CoA molecule [14]. Thisapproach led to structures that confirmed Cys-89 as the site ofreaction intermediate formation and also Cys-378 as the base thatdeprotonates the second acetyl-CoA substrate prior to condensa-tion (Fig. 1). Cys-378 is situated within 3.3 Å of C2 of substrateacetyl-CoA, supporting a functional assignment as general basecatalyst. The C2 of acetyl-CoA is closely juxtaposed (3.0 Å) to C1of acetyl-enzyme, as required for an efficient condensation reac-tion. A positively charged conserved His-348 could interact withthe thioester carbonyl of this acetyl-CoA (3.3 Å between His-348and the C1 carbonyl oxygen) to stabilize the carbanion that is pro-duced after proton abstraction. Such stabilization by a basic aminoacid residue of the thioester carbonyl of acyl-CoA metabolites atwhich negative charge develops during the reaction is a recurringtheme in a variety of enzyme catalyzed Claisen condensation/cleavage reactions [15–19].

H3C S-CoA

O

H3C S-Enz

OCoA-S-

HO S-CoA

OO CH3HO

S

H2O

ES- +

ESH +

acetyl-SCoA acetyl-S-enzyme

HMG-SCoA

B:

Scheme 3. Chemical steps in b

The Cys–His–Cys ‘‘triad” of thiolase active site residues evokessimilar motifs found in the family of ‘‘initial condensation en-zymes” and may be compared with the Cys–His–Asn triad de-scribed for the initial condensing enzyme of bacterial type twofatty acid biosynthesis [20]. Another variation of such a triad is ob-served for the enzyme that catalyzes the next mevalonate pathwayreaction, HMG-CoA synthase.

HMG-CoA synthase

The biosynthesis of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) by condensation of acetyl-CoA with acetoacetyl-CoA wasdemonstrated using a preparation of yeast protein [21]. HMG-CoA synthase (EC 2.3.3.10; formerly EC 4.1.3.5) catalyzes a reactionthat is physiologically irreversible but has recently been demon-strated [18] to also support slow catalysis of the cleavage ofHMG-CoA:

acetyl-CoAþ acetoacetyl-CoAþH2O!3-hydroxy-3-methylglutaryl-CoAþ CoASH

Work with yeast enzyme [22] supported formation of a covalentreaction intermediate. The purified avian liver enzyme was usedto identify cysteine in formation of acetyl-S-enzyme and enzyme-S-HMG-CoA covalent reaction intermediates [23,24] that are de-picted in Scheme 3, which outlines the chemistry of the reaction.

A mitochondrial isoform [25] supports the ketogenic pathwayfor acetoacetate biosynthesis while a cytosolic isoform [26]participates in the mevalonate pathway for isoprenoid biosynthe-sis. Several bacteria utilize the mevalonate pathway and theEnterococcus faecalis mvaS protein catalyzes the HMG-CoA syn-thase reaction [27].


The condensation reaction proceeds with inversion of stereo-chemistry to produce the S-isomer of HMG-CoA [28]. Mechanismbased inhibitor labeling [29] and protein sequencing [30] ap-proaches have been used in our lab to identify Cys-129 (cytosolicenzyme numbering) as the residue involved in formation of thereaction intermediates. Recombinant forms of the avian, human,and bacterial enzymes have become available [31,32,17,33]. Usingthese tools, the essential nature of Cys-129 was demonstrated and,in contrast to acetoacetyl-CoA thiolase, replacement with serine

H2C S-Enz

O

O

S-CoA

O

Enz-S S-CoA

OO CH3HO

H2C S-Enz

O

acetoacetyl-SCoA

enzyme-S-HMG-SCoA

BH

iosynthesis of HMG-CoA.


did not support measurable reactivity [31]. Inhibition of cytosolichuman enzyme by the beta lactone containing natural product,hymeglusin (structure shown below), also involves this active sitecysteine in formation of a covalent enzyme-inhibitor adduct [32].

This compound has been demonstrated to be a potent inhibitor ofhepatic steroidogenesis [34]. Mutagenesis and mechanistic enzy-mology approaches were used in our lab to implicate a histidine(His-264) in substrate acetoacetyl-CoA binding [35] as well as a glu-tamate residue (Glu-95) in general acid/base catalysis [36]. The roleof Glu-95 was confirmed as the general base in studies whichdemonstrated that substitutions of this glutamate by alanine or glu-tamine impair enolization of the acetyl-CoA analog, acetyldithio-CoA [37].


The first three dimensional structural data for HMG-CoA syn-thase were generated using recombinant bacterial (mvaS) enzymes[17,18,33]. The structure of plant enzyme with bound beta lactoneinhibitor has been subsequently reported [38]. Only very recentlyhave structures of animal cytosolic and mitochondrial isoformsbeen published [39]. There is reasonable agreement between struc-tures of prokaryotic and eukaryotic enzymes in terms of confirma-tion/assignment of catalytic residues and functional roles.

Structural results [17] on the Staphylococcus aureus proteinnoncovalent complex with acetoacetyl-CoA indicated a thiolase-like fold and confirmed the close juxtaposition of active site cys-teine, histidine, and glutamate residues that, as mentioned above,had previously been identified. Work on the E. faecalis enzyme

Fig. 2. Active site residue triad in S. aureus HMG-CoA synthase (mvaS), based on the sintermediate formed at Cys-111/129 (bacterial/animal protein numbering). The enzymeand C3 oxygens, providing a charge sink to facilitate attack by a carbanion on C3. This cageneral base Glu-79/95.

[33] detected a substrate derived covalent adduct between an ace-toacetyl moiety and the active site cysteine; this novel species hadnever been reported for animal enzyme. In a subsequent paper[40], the structure of protein containing substitution of glycinefor the conserved alanine that precedes the active site cysteinewas reported by Stauffacher and colleagues; a structural rationalefor the marked increase in catalytic activity documented for thismutant was provided. Collaborative work between the Harrisonand Miziorko labs produced unexpected observations when solu-tions containing product HMG-CoA and S. aureus enzyme werecrystallized and structures determined [18]. Structures not onlyof enzyme-product complex but also of a complex of acetyl-enzyme reaction intermediate with substrate acetoacetyl-CoAwere obtained. Solution experiments under crystallization con-ditions demonstrated that enzyme dependent cleavage of14C-HMG-CoA occurs slowly (0.3 per day) and that HMG-CoA isactivated to form the E-S-HMG-CoA reaction intermediate. Thisactivation proceeds through a tetrahedral carbon. Reversibility ofthis activation step accounts for incorporation into reisolatedHMG-CoA of two 18O atoms at C5 when the reaction is performedin H2O18 [18]. The structure of the acetyl-S-enzyme complex withacetoacetyl-CoA was particularly noteworthy. These results clearlyconfirmed (Fig. 2) not only the important assignment of the activesite cysteine but also the function of glutamate, in close proximity(3.0 Å) to C2 of acetyl-S-enzyme, as the general base catalyst. Addi-tionally, close interactions (both 3.1 Å) of the active site histidinewith the C1 and C3 oxygens of bound acetoacetyl-CoA were ob-served. C3 of acetoacetyl-CoA is situated 3.3 Å from C2 of acetyl-enzyme so that, after deprotonation by the general base glutamate,efficient condensation to form enzyme-S-HMG-CoA will occur. Sol-vent hydrolysis releases product and reforms free enz-SH.

The structural data have been interpreted in the context of acatalytic Cys–His–Glu triad, which contrasts with the triads pro-posed for thiolase (Cys–His–Cys; [14]) and other condensing foldenzymes. Comparison of the thiolase triad with the HMG-CoA syn-thase triad (Figs. 1 and 2) reveals a contrast in positions of the gen-eral base residues, in accordance with the different chemical steps

tructural coordinates 1XPL. The structure [18] depicts the acetyl-enzyme reaction’s second substrate, acetoacetyl-CoA is bound with His-233/264 interacting with C1rbanion is produced upon abstraction of a proton from C2 of acetyl-enzyme by the

Fig. 3. Active site residues in the soluble catalytic domain of human HMG-CoAreductase, based on the structural coordinates 1DQA. The structure [49] includesliganded hydroxymethylglutarate and indicates the positions of three residues(Asp-767, Lys-691, and Glu-559) implicated in enzyme function. Both lysine andglutamate would be in close proximity to the thioester carbonyl of HMG-CoA that issubjected to a two reductive steps, resulting in formation of the C5 alcohol ofmevalonate. There are different proposals [48,49] regarding the precise roles ofthese residues in substrate carbonyl polarization and/or the proton transfers thataccompany NADPH reduction.


in these reactions and the particular roles for the particular resi-dues that support acetyl-CoA deprotonation in thiolase versusacetyl-S-enzyme deprotonation in HMG-CoA synthase.

HMG-CoA reductase

In elucidation of the pathway that accounts for incorporation ofacetate into isoprenoids and sterols, it was important to accountfor the observation that mevalonic acid is a good isoprenoid/sterolprecursor. The investigation [41] of a yeast HMG-CoA reductase ac-counted for mevalonate production:

ðSÞ-HMG-CoAþ 2 NADPHþ 2 Hþ !ðRÞ-mevalonateþ 2 NADPþ þ CoASH

The enzyme (EC 1.1.1.34) is found in eukaryotes, archaebacteria,and some eubacteria. The conversion of the thioesterified HMG-CoA carboxyl to an alcohol represents a two step reduction,accounting for the stoichiometry of NADPH in the reaction. Thereaction thus proceeds through the successive reduction steps tofirst produce bound mevaldyl-CoA, collapse of the thiohemiacetalto release CoASH and form mevaldehyde; the second reduction stepthen forms product mevalonate (Scheme 4).

The eukaryotic proteins (class I HMG-CoA reductases) are asso-ciated with the endoplasmic reticulum and interact through mem-brane spanning helices in the N-terminal domain [42]. It follows,therefore, that the catalytic domain follows this membraneanchoring sequence. These class I enzymes are potently inhibitedby the class of statin drugs that effectively modulate sterol synthe-sis and, as a result, have been heavily investigated. There is nohomologous sequence for membrane association at the N-terminusin the bacterial HMG-CoA reductases (class II) and a few of these(some in recombinant form) have been isolated as soluble proteins.The Pseudomonas mevalonii enzyme [43] has a degradative func-tion, allowing this microbe to grow on mevalonate as a carbonsource. In contrast, the Staphyloccus aureus enzyme [44] has a bio-synthetic function and is encoded by a gene within a mevalonatepathway gene cluster.


The soluble P. mevalonii enzyme has been a useful model formutagenesis and functional investigations; a recombinant solublehamster catalytic domain has also been employed. These studiesin the Rodwell lab aimed at identification of functionally importantactive site residues [45,46] and used not only mutagenesis but alsoemployed the reaction intermediate mevaldehyde to study partialreactions catalyzed by wild type and mutant forms of this enzyme.These results implicated active site histidine, aspartate, and gluta-mate residues prior to the availability of three dimensional struc-tural information.


The structures of various dead-end complexes of P. mevaloniienzyme were elucidated in collaborative work from the Rodwelland Stauffacher labs [47,48] and an active site lysine was alsoidentified. These results were followed by the Deisenhofer lab’s

HO S-CoA

OO CH3HO

S

S-HMG-SCoA

NADPH NADP+

HO S-CoA

O-HO CH3HO

mevaldyl-SCoA

Co

Scheme 4. Chemical steps in the

publication of structures of the soluble catalytic domain of thehuman enzyme, also liganded to substrates or products [49].Despite the low overall sequence homology (<20%) and overall pro-tein structure architecture between class I and class II enzymes,there is considerable similarity between these enzymes in thepositioning of active site residues important to catalytic function.Residues from two different subunits contribute to an active site.The histidine proposed to function in protonation of productCoenzyme A is appropriately positioned for this role. The aspartateimplicated by mutagenesis is located within the active site andinvolved in a hydrogen bond network with the lysine and the glu-tamate that have been identified by functional studies (Fig. 3).While both lysine and glutamate are in close proximity to theHMG-CoA thioester carbonyl that is reduced to form mevalonate,there are different proposals [47,48] concerning their precise rolesin substrate carbonyl polarization and/or the proton transfers thataccompany substrate reduction by NADPH.

Efficacy of inhibition by various compounds in the family of sta-tin inhibitors is markedly dependent (e.g. nanomolar versus milli-molar inhibitor affinity) on whether class I or class II enzyme isused. The inhibitors are characterized by hydroxymethylglutaryl(HMG)-like moieties linked to an extensive hydrophobic scaffold(e.g. a decalin ring in the case of mevastatin and simvastatin). Com-plexes of human enzyme catalytic domain with a variety of statinshave been crystallized and X-ray structures have been published[50]. The structural results indicated binding of the HMG moietyin the active site pocket where the catalytic glutamate and lysine

ASH

HO H

OO CH3HO

mevaldehyde

NADPH NADP+

HOH

OHO CH3HO

R-mevalonate

H

HMG-CoA reductase reaction.


residues are located. In contrast, the NADPH substrate site is notoccupied upon inhibitor binding. The structural results indicatingthat access to the substrate HMG-CoA is blocked by inhibitor bind-ing are in accord with the observation of competitive inhibitionwith respect to HMG-CoA [51]. A variety of additional polar inter-actions with the HMG moiety have also been documented. Thehydrophobic portion of the inhibitors is bound in a shallow hydro-phobic groove of the human protein. A large number of van derWaals contacts between nonpolar amino acids in this groove andthe diverse hydrophobic substituents that are a common featureof the various statins are proposed to represent the dominant con-tribution to high affinity binding. A structure of lovastatin bound tothe class II P. mevalonii enzyme has also been reported [52]. As inthe case of the class I enzyme, the structure indicates interactionswith residues (e.g. lys, glu) identified in catalysis as well other po-lar residues in this pocket, with some hydrogen bonds mediated bywater molecules. The hydrophobic decalin ring component of theinhibitor blocks closure of the C-terminal flap domain of the pro-tein, which includes the histidine residue that has been implicatedin catalysis. Thus, inhibitor binding both blocks the active site andmakes correct orientation of active site amino acids impossible. Alarge difference between class I and class II enzyme interactionswith the statin inhibitor is suggested to involve a pocket formed,in part, by the alpha helix of the P. mevalonii enzyme that containsa conserved ‘‘THNK motif”. The structural results [51] could expe-dite potential design of HMG-CoA reductase inhibitors that can dis-criminate between Class I and Class II enzymes. Additionally, anapproach proposed to increase inhibitor affinity [50] involvesderivatization of the parent inhibitory compound to incorporatechemical substituents effective in interaction with the NADPH site.

Mevalonate kinase

Mevalonate kinase (MK; MVK; ATP:mevalonate 5-phospho-transferase; EC 2.7.1.36) catalyzes transfer of ATP’s c-phosphorylto the C5 hydroxyl oxygen of mevalonic acid, resulting in forma-tion of mevalonate 5-phosphate and ADP. The reaction was charac-terized in yeast [53]; the protein is found in eukaryotes, archaea,and certain eubacteria. The enzyme was highly purified from por-cine liver and was demonstrated to catalyze a sequential reactionwith mevalonate substrate binding first and MgADP product re-leased last [54]:

The enzyme is subject to potent feedback inhibition by geranyldiphoshophate and farnesyl diphosphate, downstream intermedi-

Fig. 4. The MgATP binding site in rat mevalonate kinase [67], based on thestructural coordinates 1KVK. ATP is bound in an anti conformation. Boundmagnesium is coordinated to Glu-193 and Ser-146 as well as the beta and gammaphosphoryls of ATP. The catalytic residue Asp-204 is positioned to support transferof the ATP gamma phosphoryl to an acceptor substrate. Conserved Lys-13 interactswith both Asp-204 and the gamma phosphoryl of substrate ATP. Dashed linesindicate coordination to magnesium; dotted lines indicate hydrogen bonds.

ates in the polyisoprenoid biosynthetic pathway [55]. High affinity(10�8 M) feedback inhibition has also been observed using a recom-binant human protein and contrasted with lower potency inhibition(10�5 M) of the Staphylococcus aureus enzyme [56]. Recently, therehas also been a report that mevalonate 5-diphosphate is a potent(submicromolar) inhibitor of the Streptococcus pneumoniae enzyme[57]; this metabolite has not been observed to be effective in inhi-bition of the Staphylococcus enzyme [56]. Inherited human mevalo-nate kinase (MVK) mutations are correlated with two diseases,accounting for mevalonic aciduria (MIM6103770) and Hyper-IgDsyndrome (MIM260920).


Cloning of the cDNA encoding the rat [58] and human proteins[59] was a prelude to large scale production of recombinant forms

of these enzymes [60,61]. Subsequent mutagenesis work estab-lished Ala-334 as the basis for an inherited enzyme deficiency[62]. Other mutagenesis experiments on rat and human MVK wereconducted in our lab prior to the availability of any high resolution3-D structures for the protein. The results implicated conservedLys-13, Ser-146, Glu-193, and Asp-204 as functionally importantactive site residues [60,61,63]. Ser-146 is the second of two tandemserines that map within a glycine rich ATP consensus sequence.Subsequent work indicated the importance of His-20 in rat MVK[64] and Arg-196 in Methanococcus jannaschii MVK [65].


The X-ray structure of unliganded M. jannaschii MVK [66] indi-cated the anticipated fold for an enzyme in the family of galactoki-nase/homoserine kinase/mevalonate kinase/phosphomevalonate(GHMP) kinase proteins. A monomer was observed in the crystalinstead of the dimeric form typically reported for MVKs in solution.Based on the active site residues that had been previously identi-fied and the features of the substrate binding sites observed forother GHMP kinase proteins, a model was constructed (a syn con-formation of bound ATP was presumed) and offered to explainMVK reaction chemistry. The structure of dimeric rat MVK li-ganded to Mg-ATP was also elucidated [67]. ATP is bound to ratMVK in an anti conformation. Glu-193 and Ser-146 were confirmedto coordinate to bound cation ligand. Lys-13 interacts with the c-phosphoryl group of ATP and makes a salt bridge with Asp-204.Thus, Asp-204 is reasonably positioned to support the predictedcatalysis of phosphoryl transfer and modeling of mevalonate intothe vacant pocket for this substrate produces a ternary complexorientation that is compatible with such a function for Asp-204.The following view (Fig. 4) of Mg-ATP bound to rat MVK illustratesthe orientation of active site residues.

Fig. 6. A solvent accessible surface representation of the feedback inhibitor(farnesylSP/farnesylSPP) binding site of mammalian mevalonate kinase, based onthe structural coordinates 2R42. The animal MVK embeds farnesyl atoms C6–C15 ina pocket to which the side chains of I196, T104, L53, N54, and I56 contribute [69].This observation explains the high affinity (10�8 M; [56]) observed for feedbackinhibition, which is competitive with respect to ATP. The image is adapted from Fuet al., Biochemistry 47 (2008) 3715–3724 [69], with permission of the AmericanChemical Society.


Structural information has also become available for enzymewith bound metabolites that negatively regulate MVK activity.The structure of a Streptococcus pneumoniae MVK binary complexwith mevalonate 5-diphosphate has been elucidated [68]. Thebound metabolite was originally characterized as an allostericinhibitor of this bacterial enzyme [57]. It is situated in the catalyticcleft of the crystallized protein in such a way that it interacts withaspartate, serine, and lysine residues that are homologous to thosedemonstrated to interact with bound MgATP in rat MVK [67]. Addi-tionally, mevalonate diphosphate interacts with a threonine previ-ously shown to influence the Km for mevalonate [63]. Thus, thestructural results [68] are interpreted to suggest that mevalonatediphosphate interacts with MVK as a partial bisubstrate analog,with its pyrophosphoryl moiety mimicking a linkage betweenmevalonate and the ATP phosphoryl donor.

Structural characterization of a feedback inhibitor bound to ani-mal MVK relied on the observation [56] that farnesyl thiodiphos-phate (FSPP), a thio analog of farnesyl diphosphate (FPP), is aseffective a competitive inhibitor with respect to ATP as the natu-rally occurring metabolite. The original observation that crystalli-zation of human MVK in the presence of FPP led only to thestructure of an enzyme-pyrophosphate complex prompted theuse of rat MVK with FSPP, which is expected to be more resistantto hydrolysis.

Crystallization of enzyme-FSPP solutions in the presence ofMg2+ produced a structure [69] (Fig. 5) of rat MVK bound to far-nesyl thiophosphate (FSP), indicating either some disorder of theb phosphoryl group or some hydrolysis of inhibitor during crystalgrowth. Nonetheless, enzyme occupancy was adequate for detec-tion of electron density for the entire FSP molecule. The inhibitorbinds in the ATP site with its thiophosphoryl situated in the posi-tion observed for the b phosphoryl group of ATP. Asp-204 and

Fig. 5. Feedback inhibitor binding to rat mevalonate kinase, based on the structuralcoordinates 2R42. Residue numbering is based on human mevalonate kinase.Inhibitor ligand electron density is fit using farnesyl thiodiphosphate (FSPP),although due to disorder or hydrolysis the beta phosphoryl is not well defined. Theinhibitor binds in the ATP site [69] with its alpha phosphoryl group situated wherethe beta phosphoryl group of ATP would be located. Asp-204 and Ser-146 functionas ligands to cation. Lys-13 would be expected to interact with the beta phosphorylof inhibitor. The last 10 carbons of the farnesyl moiety interact with a number ofnonpolar residues (e.g. Leu-53, Val-56, Val-133, Ile-196).

Ser-146 function as cation ligands and K13 is expected to interactwith the beta phosphoryl of FSPP (or FPP). The FSP polyisoprenoidchain overlaps the site at which the adenosine moiety of ATP binds.FSP’s last 10 carbons (C6–C15; two isoprenyl units) interact withnonpolar residues (e.g. Leu-53, Val-56, I-196). Previously, a 1000-fold inflation in inhibitor constants for FPP and FSPP had been ob-served upon comparison of human and Staphylococcus MVKs [56].Comparison of the binding surface measured for FSP/FSPP bindingto animal MVK and modeled for Streptococcus MVK reveals the ba-sis for this differential affinity. Animal MVK contains a pocket thatembeds inhibitor atoms C6–C15 (Fig. 6). In contrast, the C15 chainof the feedback inhibitor is largely solvent exposed in the model forthe bacterial MVK.

Phosphomevalonate kinase

Phosphomevalonate kinase (PMK; EC 2.7.4.2) catalyzes thereversible reaction of mevalonate 5-phosphate and ATP to formmevalonate-5-diphosphate and ADP:

Mevalonate 5-phosphateþMgATP ¢

mevalonate 5-diphosphateþMgADP

Activity of this enzyme was demonstrated in pig liver [70] and thepig liver enzyme has subsequently been isolated and more exten-sively characterized [71]. Phosphomevalonate kinase is found ineukyaryotes and some eubacteria. The amino acid sequences foranimal and low homology invertebrate PMK proteins are not orthol-ogous to those for PMK in plants, fungi, and bacteria [72]. Thus, theproteins that catalyze the enzymatic reaction differ widely, depend-ing on their source. Animal and invertebrate PMK proteins exhibitthe fold typical of the nucleoside monophosphate (NMP) kinasefamily while the other PMK proteins are members of the GHMP ki-nase family. The tissue-isolated pig enzyme is reported to catalyzean ordered sequential bi–bi reaction with mevalonate 5-phosphateassigned as the first substrate bound and ADP as the last product re-leased [73]. A recombinant form of Streptococcus pneumoniae hasbeen characterized [74] and reported to catalyze a random sequen-tial bi-bi reaction. A recombinant form of Enterococcus faecalis PMKhas also been isolated and characterized [75]. The sequence of hu-man PMK has been deduced [76]. The protein encoding DNA wasused to produce a GST-PMK fusion construct and this recombinantfusion protein was used to test the site of feedback inhibition in themevalonate pathway [77]. The recombinant human protein has also

Fig. 7. The active site cavity of human phosphomevalonate kinase and the locationof conserved amino acid side chains implicated in enzyme function. The figure isbased on the structural coordinates 3CH4, as reported by Chang et al. [85]. Whilethe unliganded enzyme exhibits an open active site, the bound sulfate is interpretedas a phosphoryl group marker for the binding site of phosphorylated substrates.Sulfate is located in proximity to the N-terminal P-loop and interacts with the sidechain of Arg-141, which has been shown [78] to influence ATP binding. Mutagenesisof Arg-18, Lys-22, Arg-84, Arg-110, and Arg-111 has resulted in observation ofsignificant catalytic or substrate binding effects for these conserved residues.


been expressed and isolated in an N-terminal His-tagged form. Thisenzyme has been used for mutagenesis studies on active site func-tional residues [78] as well as for solution structure experiments.


The N-terminus of the protein harbors a variation on the P-loopmotif associated with ATP binding. Work in our lab with the re-combinant human protein indicated that Lys-22, as well as Arg-18, have a substantial influence on catalysis in accordance withtheir location in a P-loop [78]. Evaluation of the importance ofother conserved basic residues [79] implicated Arg-110 as makinga large contribution to catalysis, Arg-111 and Arg-84 as influencingmevalonate 5-phosphate binding, and Arg-141 as affecting ATPbinding. Using the recombinant Streptococcus pneumoniae enzyme,mutations in a series of proposed solvent accessible residues havebeen characterized [80] and interpreted in the context of a struc-tural model of protein with MgATP and phosphomevalonate li-gands. Mutation of Asp-105 mainly results in a kcat effect, whilemutations at Lys-9 and Ser-291 have considerable affects on theKm for phosphomevalonate. An enormous effect (17,500 fold) re-flected as inflation of the Km for phosphomevalonate is reportedupon mutation of ala-293, which is harbored in a glycine rich sub-strate recognition loop.


Uniformly 15N-labeled recombinant human PMK protein hasalso been used for NMR measurements on substrate induced struc-tural changes [81]. NMR estimates of equilibrium binding con-stants were in reasonable accord with affinity estimates fromkinetic characterization studies. Dynamics results were interpretedin the context of regions that may influence domain closure, whichis well precedented in NMP kinase fold proteins. In contrast withthe observation of comparable ATP or ADP dependent conforma-tional changes, the conservatively substituted analog AMPPNP1 in-duces few chemical shift perturbations, complicating evaluation ofthe effect of active site occupancy by the gamma phosphoryl moi-ety of an adenine nucleotide. Based on observed chemical shift per-turbations, binding of either mevalonate 5-phosphate or ATPinduces conformational changes in human PMK. These biophysicalobservations contrast with expectations for an ordered sequentialmechanism, based of steady state kinetic studies on pig PMK[73]. The possibility of random substrate/product binding to hu-man PMK does, however, agree with the observation of productinhibition of human PMK by mevalonate 5-diphosphate that iscompetitive with respect to substrate mevalonate 5-phosphate[79]. The preparation of a series of 15N labeled human PMK pro-teins containing single arginine substitutions (e.g. R48M, R111M,R130M, R141M) has facilitated NMR assignments of arginine sidechains. Dynamics studies on wild type PMK and these PMK argi-nine mutants [82] indicate that substrate binding to PMK corre-lates with a transition from a flexible to a more rigid protein. Theobservation extends to the arginine side chains, including severalthat are somewhat remote from the active site. The pervasive rigid-ification suggested by the results of these dynamics studies seemsreasonable in the context of the substantial structural changes doc-umented for NMP kinase proteins as the substrate binding regionsmove into the proximity of the P-loop domain upon occupancy ofphosphoryl donor and/or acceptor binding sites.

Three dimensional structure information has become availablefrom X-ray diffraction experiments on bacterial and human PMK.The structure of unliganded Streptococcus pneumoniae PMK, whichexhibits a GHMP kinase fold, was initially reported [83] and subse-quently the structure of a ternary MPK-mevalonate phosphate-MgAMPPNP complex was published [84]. Comparison of apo and

ternary complex structures indicates octahedral coordination ofMg that involves Asp-197, Ser-213, mevalonate 5-phosphate, andthree solvent waters. An active site water pentamer is noted asinteracting extensively with reactive groups of the bound substrateand ATP analog. Lys-9 is observed to interact with the gammaphosphoryl group of bound AMPPNP, an observation similar to thatreported for a lysine in a complex of ATP with another GHMP ki-nase protein, rat mevalonate kinase. Ser-147 is noted to hydrogenbond to the carboxyl group of bound phosphomevalonate.

The structure of an unliganded form of human PMK has recentlybecome available [85]. The results confirm the assignment of theprotein as an NMP kinase family member. Consequently, the unli-ganded structure is expected to reflect an ‘‘open” form of the en-zyme, i.e. the substrate binding regions or ‘‘lids” are not as closein proximity to the P-loop containing core as would be expectedin samples containing ligands in the phosphoryl donor and/oracceptor sites. There is, however, a sulfate ion bound in close prox-imity to the P-loop (Fig. 7); this is interpreted as a phosphorylgroup analog and a marker for the active site. The N-terminal P-loop assignment that was the subject of earlier functional studies[78] was confirmed in the human PMK structure.

The bound sulfate ion interacts with the amide nitrogen of P-loop Gly-21 and also the side chain of Arg-141, which has beenshown [78] to influence binding of substrate ATP. The conservedresidues which exhibit, upon mutagenesis, the largest functionaleffects on PMK catalytic activity or substrate Km can be mappedin the open cavity that exists between the P-loop containing coreand the ‘‘lids” expected to harbor binding sites for the phosphoryldonor and acceptor substrates (Fig. 7). More refined functionalassignments would be facilitated by the availability of structuralinformation on liganded forms of human PMK.

Mevalonate diphosphate decarboxylase

Mevalonate diphosphate decarboxylase (EC 4.1.1.33; variousabbreviations appear in the literature: MDD, MVD, MPD, DPM-DC) catalyzes the ATP dependent decarboxylation of mevalonate


5-diphosphate (MVAPP) to form isopentenyl 5-diphosphate [86],as indicated in the equation below:

O O

O HO CH3

P O

O

O

P O

O

O

O P O

O

O

P O

O

O

----

---

Mevalonate-5-diphosphate Isopentenyl Diphosphate

MgATP MgADP + Pi + CO2

Mevalonate DiphosphateDecarboxylase

This reaction is essential to the mevalonate pathway of polyisopr-enoid and sterol synthesis [87]. Activity has been measured in ani-mals [88], plants [89], and yeast [86]. Genetic complementationhas implied activity in the Staphylococcus aureus and Trypanosomabrucei [90] proteins. Highly purified active enzyme has been pre-pared from avian [88], porcine [91], and rat [92] tissue and in re-combinant form from bacteria expressing the yeast [93], human[94], [77,95], Trypanosoma brucei, and Staphylococcus aureus [90]proteins. Characterization of the tissue isolated protein impliedthe presence of an arginine that influences activity of the avian en-zyme [96] and documented the selectivity for divalent cation [97].The avian enzyme was also used to demonstrate that the transientphosphoryl transfer to the C3 oxygen proceeds with inversion ofstereochemistry [98] and, thus, no covalent E–P intermediateforms.

Enzyme inhibitors

Early observations that metabolite analogs could block polyi-soprenoid/sterol biosynthesis suggested that the MDD reaction isinvolved and prompted synthesis and evaluation of a variety ofMDD inhibitors. The use of 6-fluoromevalonic acid in tissue ex-tracts [99] implicated MDD as the target for a downstream metab-olite (6-fluoro-MVAPP) that blocked sterol production. In a surveyof several fluorinated mevalonic acid derivatives, Reardon andAbeles [100] showed with purified MDD that 6-fluoro-MVAPPwas the potent inhibitor that accounted for the block in sterol bio-synthesis. These studies were extended [101] to indicate that a3-phospho-6-fluoro-MVAPP analog of the normal reaction inter-mediate could be trapped as an MDD-bound species. In addition,a nitrogen substituted transition state analog (N-methyl-N-car-boxymethyl-2-pyrophosphoethanolamine) was synthesized andcharacterized [101]; results suggested that a positively chargednitrogen atom mimicked a reaction intermediate with carbocationcharacter. This carbocation proposal was supported by synthesis ofN-diphosphoglycolyl proline and the compound’s potent inhibitionof MDD was attributed to its positively charged nitrogen atom[102]. On the basis of these various studies, a relatively detailedmechanism for the MDD reaction could be proposed well in ad-vance of the availability of recombinant enzymes that could beused to investigate how MDD active site amino acids might sup-port the predicted chemistry.

Voynova et al. [95] used recombinant human MDD to confirmthe high affinity inhibition (competitive with respect to MVAPP)of enzyme by both 6-fluoro-MVAPP and diphosphoglycolylproline.Qiu and Li [103] proposed that 2-fluoromevalonate diphosphatecould irreversibly inactivate MDD in a time dependent fashion buta subsequent report [104] indicated that 2-fluoro- and 2-difluoro-MVAPP are reversible competitive inhibitors with respect toMVAPP. Recently, a series of mevalonate analogs were preparedand tested as substrates for Streptococcus pneumoniae MVK, PMK,and MDD [105]. Results suggested that this bacterial MDD isvery tolerant of replacement of the 6-methyl group with small

substituents. Resistance of MDD to compounds designed tocovalently modify and inactivate the enzyme has prompted the

hypothesis that decarboxylation and phosphate elimination of a3-phospho-MVAPP intermediate are concerted rather than disso-ciative processes. The hypothesis [105] included the prediction thatlittle carbocation character develops in the reaction intermediate.


Mutagenesis of yeast MDD and characterization of the mutantproteins by kinetic and biophysical methods [93,106] initially fo-cused on the Lys-18/Asp-305 pair of conserved residues that arehomologous to a similar pair implicated in rat MK function.Replacement of the Asp-305 side chain carboxyl (D305N; D305A)was observed to result in large (103–105 fold) decreases in catalyticactivity. Extension of this approach to alanine substitution of a ser-ies of conserved serine residues suggested that Ser-121 had a largeinfluence on catalysis (>104 fold effect decrease upon mutation toalanine). The selective influence of this second of two tandem con-served serines in an ATP-consensus motif had previously been ob-served for rat MK [63]. The results also implicated Ser-153 in apossible interaction with ATP and Ser-155 in MVAPP binding. TheSer-155 mutant exhibited inflated Km and Ki values for bothMVAPP and diphosphoglycolylproline. In work on human MDD,Voynova et al. [95] demonstrated that conservative mutation ofArg-161 (R161Q) decreased catalytic activity by 1000-fold. Suchan observation is in agreement with the earlier implication of anactive site arginine by protein modification methods [96]. Muta-tion of Asn-17 (N17A) results in inflated Km and Ki estimates forMVAPP, diphosphoglycolyl proline, and 6-fluoro-MVAPP, suggest-ing that this residue participates in interactions that affect bindingof the phosphoryl acceptor substrate. Mutation of recombinant ratMDD [104] resulted in observation of no detectable activity uponmutation of Lys-23 (K23A) or Arg-162 (R162A).


A molecular docking and simulation-base approach [107] hasrecently been reported; the study used structural information fromthe initial X-ray structure for MDD (yeast enzyme; [108]). While nostructure of liganded MDD has yet been elucidated, this computa-tional study modeled a ternary complex of enzyme with ATP andphosphoryl acceptor. The conclusions suggest that a series of con-served residues implicated by functional studies are situated in theactive site. In particular, a water molecule is predicted to mediateinteraction of a catalytic aspartate with the C3 hydroxyl of MVAPP.The critical serine in the glycine rich consensus ATP motif is pre-dicted to interact with MVAPP.

Structures of Trypanosoma brucei and Staphylococcus aureusMDD proteins have been empirically determined [90] by Hunter’slab. The proteins are unliganded except for a sulfate anion. In thehigh resolution structure of the T. brucei protein, a Lys-18/Asp-293 salt bridge is detected; this is analogous to the observationfor rat mevalonate kinase. ATP and MVAPP were positioned intothe protein structure to produce a ternary complex model, which


predicts an Arg-77 interaction with the beta phosphate of MVAPP.Also, interaction of Tyr-19 and Arg-149 with the C1 carboxyl ofMVAPP is proposed.

The structure of human MDD has been elucidated [95]. Proteinin the crystal is also unliganded except for a sulfate/phosphate an-ion (from the crystallization buffer); the anion is bound to Lys-26and Arg-78. The Z-dock algorithm (http://zdock.bu.edu) was usedto model a binary complex of human MDD with mevalonatediphosphate. The beta phosphate of modeled substrate is within1 Å of the Lys-26, Arg-78 bound anion (a putative phosphoryl mi-mic); this observation suggests that the binary complex model isreasonable. Overlay of the binary ATP-MK structure allows thepositioning of ATP into a MDD-ATP-MVAPP ternary model withoutany steric conflicts, supporting the positioning of the docked li-gands. This model (Fig. 8) predicts juxtapositioning of the Arg-161 guanidinium group within 3.5 Å of the C1 carboxyl of MVAPP;Asn-17 has been observed to form a hydrogen bond with Arg-161.

Fig. 8. A model of the ternary complex of human mevalonate diphosphatedecarboxylase with ATP and mevalonate 5-diphosphate. A binary complex modelwas generated using the Z-dock algorithm (http://zdock.bu.edu) and coordinates ofmevalonate diphosphate (from 2OI2) and the human enzyme (3D4 J). The positionof ATP is based on an overlay of a structure of the mevalonate kinase-ATP complex(1KVK) on the human MDD structure [95]. The predicted position of S127 agreeswith the previous suggestion of its interaction with the phosphoryl chain of ATP[106]. The juxtapositioning of ATP’s gamma phosphoryl group with respect toMVAPP’s C3 oxygen is in accord with production of a 3-phosphoMVAPP reactionintermediate and supports the docking position of MVAPP in the binary complexmodel. The model predicts juxtapositioning Arg-161 and the C1 carboxyl of MVAPP.Asn-17 has been observed to hydrogen bond to Arg-161. The side chain of Asp-305is close to the substrate’s C3 hydroxyl. Such interactions are in accord withfunctional roles for these conserved residues, as suggested by characterization ofmutant MDD enzymes. The image has been adapted, with permission (Elsevier)from Voynova et al. Arch. Biochem. Biophys. 480 (2008) 58–67.

O O-

OH

OPO3PO3=

ADPOPO3

ADP

O O-

OPO3PO3=

OPO3=

Pi

D305

R161 N17

S127

MVAPP

Scheme 5. Mevalonate diphosphate decarboxylase reaction ch

These interactions are in agreement with functional/mutagenesisresults for these residues. The carboxyl side chain of Asp-305 is sit-uated within 4 Å of the substrate’s C3 hydroxyl; this prediction isin accord with a catalytic function for Asp-305. Ser-127’s position-ing near ATP’s phosphoryl groups is also appropriate for the pre-dicted active site role of this residue; the homologous serine inrat mevalonate kinase interacts with ATP and divalent cation.

Other unliganded MDD structures (Streptococcus pyogenes(2GS8) and Legionalla pneumophila (3LTO) proteins) have been gen-erated and deposited in a public database but not published in ref-ereed literature. Information from liganded MDD structures wouldclearly expedite more detailed functional assignments.

Scheme 5 integrates available information and hypotheses con-cerning the reaction chemistry and the participation of conservedMDD residues. Asp-305 is shown juxtaposed to MVAPP’s C3 hydro-xyl; this is consistent with its observed large contribution to cata-lytic efficiency as well as positional homology with the proposedgeneral base catalyst in mevalonate kinase. Similar observationson the functional contribution to MDD catalysis and homologywith a serine implicated in the mevalonate kinase reaction suggestthat Ser-127 interacts with ATP to orient the phosphoryl chain forproductive transfer of ATP’s gamma phosphoryl group to MVAPP.Arg-161 has been established to hydrogen bond to Asn-17. Thelarge contribution of Arg-161 to catalysis is compatible with its de-picted interaction with the MVAPP carboxyl group. Recent struc-tural studies on PEP carboxykinase [109] provide precedent forthe proposed function of Arg-161. The scheme depicts four chem-ical species that are proposed to participate in the reaction: (1)substrate MVAPP; (2) a transient 3-phosphomevalonate 5-diphos-phate intermediate (in which the C3 alcohol is transformed to animproved leaving group needed for formation of the next interme-diate; (3) a transient carbocation intermediate, which provides anelectron sink to drive decarboxylation; (4) the reaction productisopentenyl diphosphate. The inhibition studies of Dhe-Paganonet al. [101] and Vlattas et al. [102] on N-derivatized substrate ana-logs have been interpreted to support participation of the posi-tively charged carbocation intermediate in the MDD reaction.

Variations on the mevalonate pathway

Isoprenoids represent a major component of archaeal lipids. Notonly do they contribute to the usual isoprenoid derived metabo-lites like dolichol, but ether linked long chain (e.g. C20) polyisopre-noids are abundant in membranes of archaeal cells. As genomesequences for these organisms became available, it seemed likelythat, like most eukaryotes, they utilized the mevalonate pathwayfor isoprenoid biosynthesis. However, in most archaeal genomes,

O O-

OPO3PO3=

OPO3PO3=+

CO2

R161 N17 Isopentenyl-PP

emistry and possible contributions of active site residues.

http://www.zdock.bu.edu

http://www.zdock.bu.edu

HO O

O HO CH3

P O

O

O

--

Mevalonate 5-phosphate

MgATP MgADP + Pi

HO O

O HO CH3

P O

O

O

P O

O

O

---

Mevalonate 5-diphosphate

O P O

O

O

P O

O

O

---

Isopentenyl diphosphate

MgATP MgADP + Pi + CO2

Mevalonate DiphosphateDecarboxylase

PhosphomevalonateKinase

CO2

Isopentenyl 5-phosphate

O P O

O

O

--Mevalonate 5-phosphate

Decarboxylase

MgATP MgADP + Pi

Isopentenyl 5-phosphate kinase

Scheme 6. Proposed alternative reactions in biosynthesis of isopentenyl diphosphate.


it was unclear whether genes encoding phosphomevalonate kinase(PMK) and mevalonate diphosphate decarboxylase (MDD) proteinscould be assigned [72]. This may have been anticipated given theobservation of nonorthologous PMK proteins upon comparison ofthe enzyme from higher eukaryotes with PMKs from fungi, plants,or bacteria. For MDD, where homology between the animal andbacterial enzymes is more obvious, failure to detect the proteinin most archaea is more surprising.

The observation of a protein encoded by Methanocaldococcusjannaschii that catalyzed an ATP-dependent phosphorylation ofisopentenyl 5-phosphate to produce isopentenyl 5-diphosphate(IPP) [110] seemed to provide an explanation for the lack of detec-tion of a PMK in this organism. The heterologously expressed pro-tein was purified and partially characterized. The ATP-dependentphosphorylation could be detected using isopentenyl 5-phosphateas acceptor substrate but not with mevalonate or mevalonate 5-phosphate acceptors. On this basis, the protein was assigned asan isopentenyl phosphate kinase (IPK). While the absence of amore traditional pathway in M. jannaschii for synthesis of isopen-tenyl 5-diphosphate was not excluded, these observations didprompt the hypothesis of an alternative route to IPP biosynthesis.

The alternative route requires decarboxylation of mevalonate 5-phosphate to produce the substrate for IPK, which would catalyzeformation of IPP (Scheme 6).

Recently, there have been several reports that confirm the func-tion of an isopentenyl 5-phosphate kinase. Recombinant forms ofIPK from Methanothermobacter thermautotrophicus and from Ther-moplasma acidophilum have been produced and characterized[111]. These IPK enzymes catalyze reversible interconversions ofsubstrates and products and exhibit maximum activity at 70 �C.A sequential mechanism was indicated on the basis of kinetic re-sults. Sequence alignments and pH/rate profiles have been inter-preted to suggest that a conserved histidine may be functionalwithin the active site. These proteins were demonstrated to utilizevarious C4 and C5 monophosphates as substrates, with good activ-ity supported by dimethylallyl phosphate, n-butyl phosphate, and3-butenyl phosphate. While isopentenyl 5-phosphate was the opti-mal substrate of several compounds that were tested, these en-zymes’ ability to utilize various phosphorylated compounds doesraise a question about whether their major physiological role in-volves support of the proposed mevalonate metabolism. Futurework may allow a more definitive answer to this question.

These demonstrations of isopentenyl kinase enzymes have beenfurther supported by the elucidation of X-ray structures forsubstrate/product complexes of these enzymes from several

organisms. Work from Poulter’s lab [112] led to structures of theADP-liganded Methanothermobacter thermautotrophicus IPK as wellas the Thermoplasma acidophilum IPK liganded to either isopente-nyl phosphate and ATP or IPP and ADP. The structures establishthese IPKs as members of the large amino acid kinase (AAK) familyof proteins. The liganded enzyme structures confirm the presenceof histidine and lysine active site residues. The active site lysineexhibits multiple interactions with the phosphoryl groups of donorand acceptor substrates to support an in-line phosphoryl transfer.Structures of Methanocaldococcus jannaschii IPK have also beenelucidated [113]. While no bound adenine nucleotide has beenobserved, the structures contain bound isopentenyl phosphate,isopentenyl diphosphate (IPP), or IPPbS. A bound sulfate anion isproposed to indicate the position of an adenine nucleotide phos-phoryl. A conserved His-60 has been mutated to produce H60Aand H60 N proteins that are not observed to support reaction catal-ysis. The H60Q mutant exhibits a �40-fold decrease in catalyticefficiency as well as modest inflation of Km values for ATP andisopentenyl phosphate substrates. Mutations of a hydrophobicsubstrate pocket have resulted in ‘‘chain length” mutants withability to phosphorylate the C15 substrate, farnesyl phosphate.

No detailed characterization of a protein with mevalonate phos-phate decarboxylase activity (the initial enzyme required for theproposed variation in the mevalonate pathway; Scheme 6) hasbeen reported. However, the collected observations on archaealIPK proteins and the possibility of IPK homologs in organisms forwhich all of the traditional mevalonate pathway proteins are alsopredicted raise intriguing questions about the potential complexityof mevalonate metabolism. These issues suggest that future inves-tigation of this area of biosynthesis will lead to interestingdevelopments.

Acknowledgments

Parts of the work performed in my laboratory were supportedby NIH DK21491 or DK53766. The author acknowledges the impor-tant contributions of structural collaborators Prof. Jung-Ja P. Kimand Prof. David H.T. Harrison and their lab members. Major con-tributors to my laboratory’s work on mevalonate pathway en-zymes include Ila Misra, Timothy Herdendorf, Prof. NataliaVoynova, Kelly Chun, Christine Behnke, and Chang-Zeng Wang.The author greatly appreciates the generosity of the many col-leagues who provided the DNA samples or plasmids used for prep-aration of recombinant forms of some of the mevalonate pathwayenzymes investigated in my lab.


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