unraveling the multispecificity and catalytic promiscuity of taxadiene monooxygenase

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ARTICLE IN PRESSG ModelOLCAB-3042; No. of Pages 11

Journal of Molecular Catalysis B: Enzymatic xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Molecular Catalysis B: Enzymatic

j ourna l ho me pa ge: www.elsev ier .com/ locate /molcatb

nraveling the multispecificity and catalytic promiscuityf taxadiene monooxygenase

ikramaditya G. Yadava,b,c,∗

Department of Chemical Engineering, Massachusetts Institute of Technology, 25 Ames St., Cambridge, MA 02139, USADepartment of Chemistry & Chemical Biology, Harvard University, 12 Oxford St., Cambridge, MA 02138, USADepartment of Chemical & Biological Engineering, The University of British Columbia, 2360 East Mall, Vancouver, BC, V6T 1Z3, Canada

r t i c l e i n f o

rticle history:eceived 26 February 2014eceived in revised form 8 September 2014ccepted 7 October 2014vailable online xxx

eywords:ytochrome P450 monooxygenaseaxane oxidationultispecificity

atalytic promiscuityomputational biochemistry

a b s t r a c t

The enzyme taxadiene synthase of the paclitaxel biosynthetic pathway cyclizes geranylgeranyl pyrophos-phate (GGPP) to produce taxadiene, which is then believed to be stereo- and regiospecifically oxidizedby taxadiene 5�-hydroxylase to form taxadien-5�-ol. However, when the metabolism of Escherichia coliwas re-engineered in the present study to express both aforementioned plant enzymes, it was observedthat taxadiene 5�-hydroxylase acts on 4 substrates and forms 12 products, of which taxadien-5�-ol isonly a minor constituent. Additionally, 2 of the 4 substrates of taxadiene 5�-hydroxylase are products oftaxadiene synthase, and it is very likely that this behavior by the two enzymes is independent of the host.Enzyme multispecificity and promiscuity are entirely new propositions in metabolic engineering, andcanonical enzyme engineering strategies for modulating the activity of enzymes are largely inapplicablefor addressing this phenomenon. To this end, a novel computational methodology was specifically devel-oped to investigate the catalytic mechanism of taxadiene 5�-hydroxylase – hereinafter re-christened astaxadiene monooxygenase – and potentially guide rational re-engineering of its active site. The method-ology could be extended to other P450 systems, is faster, and comparably insightful as more elaborate
structure-guided approaches such as X-crystallography. In addition, the methodology can also be used toinfer the influence of mutations in the active site of the enzyme on its catalytic promiscuity. The insightsprovided by such computational analyses of multispecificity and promiscuity could greatly enhancethe scope and impact of enzyme engineering for over-production of plant secondary metabolites inmicroorganisms.
. Introduction

The yield of the blockbuster anti-cancer drug paclitaxel fromrees of the Taxus genus is estimated to be only a few hundredthsf a percent of dry biomass [1]. As a consequence, it is widelyelieved that the paclitaxel biosynthetic pathway comprises a sig-ificant number of transformations, several of which still remainoorly understood or entirely uncharacterized. Despite the limited

nformation available about the pathway, microbial metabolic engi-eering for synthesis of paclitaxel is still a topic of great interest to

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he pharmaceutical industry [2]. Not only are there clear advan-ages to producing the molecule in microorganisms over existing

anufacturing routes [3], but plant biosynthetic pathways such as

∗ Correspondence to: Department of Chemical & Biological Engineering, The Uni-ersity of British Columbia, 2360 East Mall, Vancouver, BC, V6T 1Z3, Canada.el.: +1 604 827 2706.

E-mail address: [email protected]

ttp://dx.doi.org/10.1016/j.molcatb.2014.10.004381-1177/© 2014 Elsevier B.V. All rights reserved.

© 2014 Elsevier B.V. All rights reserved.

the paclitaxel pathway that comprise a long, albeit linear sequenceof reactions are quite amenable to the application of canonicalmetabolic engineering strategies such as adjusting either the con-centrations or the activities of the rate-limiting enzymes to increasethe flux through the pathway [4].

In keeping with the linear vision of the pathway [5–7], muchof the early work on assembly of the paclitaxel biosyntheticpathway in microorganisms such as Escherichia coli has centeredon the expression of two of its most well-understood enzymes– geranylgeranyl pyrophosphate (GGPP) synthase and taxadienesynthase. The former catalyzes the synthesis of the linear, C20molecule GGPP [8]. Taxadiene synthase then cyclizes GGPP toproduce taxa-4(5),11(12)-diene (or simply, taxadiene), the firstintermediate in the paclitaxel pathway that bears the character-istic taxane scaffold [9,10]. Since cyclization of GGPP by taxadiene

2014), http://dx.doi.org/10.1016/j.molcatb.2014.10.004

synthase proceeds via the formation of a radical-stabilized tran-sition state species, taxa-4(20),11(12)-diene (or isotaxadiene) isalso synthesized as the minor product. Accordingly, when GGPPsynthase and taxadiene synthase were introduced during a recent

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nvestigation into a strain of E. coli that had also been re-ngineered to over-express the rate-limiting enzymes of theative non-mevalonate pathway, namely 1-deoxy-d-xylulose-5-hosphate synthase, 4-diphosphocytidyl-2-C-methyl-d-erythritolynthase, 2-C-methyl-d-erythritol-2,4-cyclodiphosphate synthasend isopentenyl pyrophosphate isomerase, nearly 1 g of taxa-iene was synthesized over 5 days in 1 L fed-batch cultures of thetrain [11]. This result was successfully replicated in the presenttudy, and onward assembly using taxadiene 5�-hydroxylase –he enzyme that supposedly succeeds taxadiene synthase in theathway – was continued in the aforementioned strain. Taxa-iene 5�-hydroxylase is a cytochrome P450 monooxygenase thatas been reported to stereo- and regiospecifically hydroxylateaxadiene to taxadien-5�-ol [12,13]. This transformation too pro-eeds via the formation of a radical species, and taxadien-20-ols formed as the minor product. When taxadiene 5�-hydroxylase

as introduced into the strain in this study, however, it wasbserved that the cultures synthesize as many as 16 eicosanoidolecules. Moreover, taxadien-5�-ol, previously reported to be

he major product of taxadiene 5�-hydroxylase, is, in fact, a minorroduct of the pathway and commands less than a tenth of theux that makes its way into the paclitaxel biosynthetic path-ay.

A detailed assessment of the unexpected product profile sug-ested that catalytic promiscuity and multispecificity of taxadiene�-hydroxylase, and catalytic promiscuity of taxadiene synthaseere responsible for synthesizing the chemical diversity that was

eing observed. The propensity of enzymes to catalyze the synthe-is of multiple products through the formation of vastly differentransition state species is termed as catalytic promiscuity, and theirbility to act on multiple substrates is commonly referred to asultispecificity [14]. Incidentally, the formation of minor products

wing to radical stabilization within the transition state species,uch as the synthesis of taxa-4(20),11(12)-diene (or isotaxadiene)y taxadiene synthase, does not constitute catalytic promiscuityased on these definitions. Furthermore, it was also detected thataxadiene synthase exhibits a stress-dependent proclivity for cat-lytic promiscuity.

It is now suspected that most plant cytochrome P450s can actn multiple substrates and forms a spectrum of products [15–18].his observation is potentially significant for metabolic engineerseeking to manufacture valuable secondary metabolites in simpleicroorganisms. In the case of paclitaxel biosynthesis, the dissipa-

ive tendency of a single enzyme de-commits as much as 90% ofhe flux away from the desired product. Enhancing the substratend product fidelities of dissipative enzymes such as taxadiene�-hydroxylase could not be a more urgent problem in metabolicngineering. Unfortunately, the absence of an efficient screeninglatform renders the conventional methodology for modulating thectivity of an enzyme – which involves selecting similar examplesn nature for modification using mutagenesis or directed evolution

inapplicable to the current scenario. More elaborate structure-uided approaches are required to effectively tune the productelectivity of promiscuous and multispecific enzymes such as taxa-iene 5�-hydroxylase.

To this end, a computational methodology was specificallyeveloped during the present study to investigate the catalyticechanism of taxadiene 5�-hydroxylase and explain the fate of

axadiene once it binds within the active site of the enzyme. Thelgorithm via which the methodology is deployed also allows pre-iction of the influence of mutations in the active site of taxadiene�-hydroxylase on its catalytic promiscuity. The methodology


s faster and comparably insightful as more elaborate structure-uided approaches such as X-crystallography. Significantly, sincehe sequences of the P450 monooxygenases of the paclitaxel path-ay are ∼85% similar to one another (the sequence identity is as

PRESSs B: Enzymatic xxx (2014) xxx–xxx

high as 65%), the insights that are borne by the approach detailedherein can be readily applied to modulate the properties of theremaining P450s in the pathway. The methodology can also beemployed to infer the influence of mutations in the active site oftaxadiene 5�-hydroxylase on its catalytic promiscuity. It is hopedthat as such tools are refined further they will compensate for lim-itations in screening capabilities and reduce strain developmenttimes.

2. Materials and methods

2.1. Strains and plasmids

1-Deoxy-d-xylulose-5-phosphate synthase (Dxs), 4-dipho-sphocytidyl-2-C-methyl-d-erythritol synthase (IspD), 2-C-methyl-d-erythritol-2,4-cyclodiphosphate synthase (IspF) and isopentenylpyrophosphate isomerase (Idi) have been previously identified asthe rate-limiting enzymes in the non-mevalonate pathway [19]. Inthis study, an operon comprising dxs, ispD, ispF and idi expressedunder the control of the Trc promoter, and a second operoncomprising the genes encoding geranylgeranyl pyrophosphate syn-thase (Ggps) and taxadiene synthase (Txs) under the control ofthe T7 promoter were separately integrated into the chromosomeof an E. coli K12 MG1655 �recA �endA strain using the FRT sys-tem. Both operons were inserted at the strain’s arabinose operonwith the aid of a kanamycin antibiotic marker. The strain was thenelectro-transformed to express the fusion protein comprising taxa-diene monooxygenase (GenBank AY289209) and its redox partner(GenBank AY571340) on a 5-copy, pACYCDUET-1 plasmid.

2.2. Construction of the fusion protein

Cytochrome P450 monooxygenases can only function in con-cert with their flavoprotein redox partners, the cytochrome P450reductases. The latter shuttle electrons and protons derived fromNADPH to the former’s active sites, whereat molecular oxygen isprotonated and heterolytically cleaved to form water and a reac-tive iron-oxygen species that then inserts the second oxygen atominto the substrate [20]. Cognizant of this requirement, a truncatedversion of taxadiene 5�-hydroxylase was fused to its reductasepartner. The first 24 and 74 amino acids at the amino termini oftaxadiene monooxygenase and the P450 reductase, respectively,were excised in order to eliminate their transmembrane bindingregions.

The truncated enzymes were then fused together with a 5-amino acid GSTGS linker [21]. An 8-amino acid MALLLAVF leadersequence derived from a modified bovine microsomal steroid 17�-hydroxylase (GenBank NM 174304) was also appended to thefusion complex to improve stability and facilitate expression [22].The fusion protein was then introduced into the strain of E. colithat had been previously engineered to over-produce taxadiene(Fig. 1).

2.3. Enzyme mutagenesis

The monooxygenase was mutagenized using the Quick ChangeII Site-Directed Mutagenesis kit. A software package hosted on themanufacturer’s website was utilized to design the mutagenesisprimers, and the manufacturer’s protocol was faithfully followedto construct the mutants.


2.4. Culturing conditions

The activity of taxadiene monooxygenase was investigated in2 mL batch cultures of the engineered E. coli strain. The production


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Fig. 1. (A) The enzymes dxs, ispD, ispF and idi are rate-limiting enzymes in the non-mevalonate pathway. (B) The E. coli strain used in the study was engineered to chro-mosomally express additional copies of the rate-limiting enzymes, along with geranylgeranyl pyrophosphate synthase (ggps) and taxadiene synthase (txs). In addition, afusion P450 complex that putatively catalyzes synthesis of taxadien-5�-ol was expressed on a plasmid. (C) The fusion protein was carefully constructed to achieve optimale

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edium comprised 5 g/L yeast extract, 20 �g/mL chloramphenicol,.66 g/L �-aminolevulinic acid, 13.3 g/L KH2PO4, 4 g/L (NH4)2HPO4,.7 g/L citric acid, 0.0084 g/L EDTA, 0.0025 g/L CoCl2, 0.015 g/LnCl2, 0.0015 g/L CuCl2, 0.003 g/L H3BO3, 0.0025 g/L Na2MoO4,

.008 g/L (CH3COO)2Zn, 0.06 g/L Fe(III) citrate, 0.0045 g/L thiamine,

.3 g/L MgSO4, 10 g/L glycerol and 0.024 g/L IPTG. The productionedium was inoculated at a starting OD of 0.1 using pre-

nocula propagated in 20 �g/mL chloramphenicol-supplementedB medium. The starting pH was set at 7.0, and 20 vol.% dode-ane was added to the production medium to scavenge the nascenticosanoids. The culture tubes were agitated at 200 rpm at 22 ◦C inn oxygen-controlled environment. Production was ceased after 5ays by adding 2 mL of a 80–20 vol.% solution of hexane and diethylther to each culture tube. The liquid mixtures were subsequentlyortexed for 45 min, followed by centrifugation at 3000 rcf at roomemperature.

.5. GC–MS analysis

The organic layer in each culture tube was isolated and analyzedn a Varian Saturn 3800 GC 2000 MS system housing an Agilent HP-

ms column (30 m length, 0.25 mm ID, 0.25 �m film thickness).ltrapure helium at a flow rate of 1 mL/min was utilized as thearrier gas. Each analysis lasted 30 min. The oven temperature was


aintained at 50 ◦C for the first minute of the analysis, ramped upt a rate of 10 ◦C/min until it reached 220 ◦C, and then maintainedt 220 ◦C for 12 min. The injector and transfer line temperaturesere maintained at 200 ◦C and 250 ◦C, respectively.

3. Results and discussion

3.1. Taxadiene 5˛-hydroxylase is exceptionally promiscuous andmultispecific, and ought to be re-annotated as taxadienemonooxygenase

It was observed that the cultures synthesize as many as 16eicosanoid molecules, including taxadiene and isotaxadiene, andthat taxadiene is converted to not just taxadien-5�-ol but, instead,forms several oxygenated species (Fig. 2). Isotaxadiene too isacted upon by taxadiene 5�-hydroxylase to produce a comparablediversity of oxygenated products. Taxadiene (species B in Fig. 2),isotaxadiene (species D) and their oxidation products account for14 of the eicosanoids, and, of the 12 oxygenated products, 9 aremonooxidized and 3 are dioxidized.

The overall titer of oxidized eicosanoids was determined usinga taxadiene standard to be 200.1 mg/L following production for 5days at the 2 mL scale in a specially formulated growth medium. Aswas mentioned earlier, taxadien-5�-ol (species K) is not even themajor product of the pathway. The cyclic ethers, 5(12)-oxa-3(11)-cyclotaxane (OCT, species I) and 5(13)-oxa-3(11)-cyclotaxane(iso-OCT, species G), are the two most abundant products of theheterologous pathway (Fig. 3). The titers of taxadien-5�-ol, OCTand iso-OCT are 18 mg/L, 53 mg/L and 40 mg/L, respectively.

The structures of taxadien-5�-ol, OCT and iso-OCT were con-firmed using 1H NMR spectroscopy (see Appendix). OCT and


iso-OCT are posited to be derivatives of taxadiene, and disparitiesbetween the structures of taxadien-5�-ol, OCT and iso-OCT sug-gests that these molecules originate from vastly different transitionstate species. As for the remaining species, their low abundance and


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4 V.G. Yadav / Journal of Molecular Catalysis B: Enzymatic xxx (2014) xxx–xxx

Fig. 2. The cultures produce 16 eicosanoids. (A) Typical gas chromatograms of the organic extracts from fermentation broths populated by strains expressing only thetaxadiene synthesis module (black) and the taxadiene-P450 modules (red) reveal the presence of as many as 16 eicosanoids (labeled A-P), all of which elute from the columnafter approximately 20 min of the analysis has been completed. The katharometric signal strengths of the species (y-axis) have been expressed in kilocounts and the retentiontimes have been reported in minutes (x-axis). (B) Fragments with the highest m/z ratio were normalized to an intensity of 100% in the mass spectral plots of the 16 eicosanoids(spectral plots in black – non-hydroxylated C20s, red – monooxidized C20s, and blue – dioxidized C20s). (For interpretation of the references to color in this figure legend, thereader is referred to the web version of this article.).




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Fig. 3. Oxidation products of taxadien

nstability precluded characterization of their structures. Neverthe-ess, the comparable number of oxidized derivatives of isotaxadieneuggests that it too forms multiple transition state species in thective site of taxadiene 5�-hydroxylase.

Incidentally, this study is not the first to challenge Croteau andis colleagues’ conclusions regarding taxadiene 5�-hydroxylase.hen this P450 monooxygenase was recombinantly expressed by

ontein et al. along with taxadiene synthase in cell cultures of theoodland tobacco, Nicotiana sylvestris [23], they observed that onlyCT is produced. In light of the observations made in this study and

hose by Rontein et al., taxadiene 5�-hydroxylase will hereinaftere referred to as taxadiene monooxygenase.

.2. The catalytic promiscuity of taxadiene synthase is


tress-dependent

The remaining 2 molecules that are produced by the engineeredtrain (species E and F) are unsubstituted and presumably are

ig. 4. The catalytic promiscuity of taxadiene synthase is stress-dependent. (A–C) Gas cxpressing only the taxadiene synthesis module (A), the taxadiene synthase-taxadiene monooxygenase and a non-functional P450 monooxygenase (C). Peaks corresponding to

reen, blue and purple, respectively. The pie charts adjacent to each chromatogram revroportionately with the overall titer of the taxanes. (For interpretation of the references t

duced by taxadiene monooxygenase.

products of taxadiene synthase. This observation implies that taxa-diene synthase is catalytically promiscuous as well. Moreover, itwas observed that the catalytic promiscuity of taxadiene synthasewas commensurate to the metabolic stress experienced by theE. coli host. In the initial study on engineered E. coli that expressesonly GGPP synthase and taxadiene synthase, the selectivity towardtaxadiene was 96.1% (mg/L basis). However, when taxadiene 5�-hydroxylase was introduced into the strain, the selectivity towardtaxadiene dropped to 74.9%, and the introduction of an additional,albeit non-functional, monooxygenase further reduced the selec-tivity to 68.1% (Fig. 4). It is unclear whether this phenomenonis only observed when taxadiene synthase is expressed heterolo-gously in E. coli [24]. Nevertheless, it has implications for channelingthe flux within the taxane pathway, and, it appears that the cat-


alytic promiscuity of taxadiene synthase can potentially be tunedby efficiently managing the metabolic stress experienced by thehost through strategies such as enriching the growth medium andregulating transcription and translation, to name a few.

hromatograms of organic extracts from fermentation broths populated by strainsonooxygenase module (B), and a module comprising taxadiene synthase, taxadiene

taxadiene derivatives, isotaxadiene derivatives, species E and F are colored red,eal the product distribution on a mg/L basis, and the sizes of the pie charts scaleo color in this figure legend, the reader is referred to the web version of this article.)



6 V.G. Yadav / Journal of Molecular Catalysis B: Enzymatic xxx (2014) xxx–xxx

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ig. 5. Concentrations of all 16 eicosanoids (in mg/L) were measured on each datrengths using a calibration curve for taxadiene. Error bars represent standard dso-OCT (G), H, OCT (I), taxadien-5�-ol (K), M and N were detected 24 h into the pro

.3. Taxadiene is oxidized by taxadiene monooxygenase to formve products

The strain was then cultured for 5 days at the 2 mL scale and dailyroduction of the 16 eicosanoids was measured (Fig. 5). Taxadieneas not detected in the fermentation broth on any of the 5 days

f production. Amongst the other molecules, only taxadien-5�-ol,CT, iso-OCT, and species H, M and N are observed to accumulatefter the first day. The remaining species are detectable only afterroduction has completed two days. These observations suggesthat taxadien-5�-ol, OCT, iso-OCT, and species H, M and N originaterom taxadiene, whereas species A, C, J, L, O and P are derived fromsotaxadiene.

Additionally, since taxadiene and isotaxadiene are products ofhe same catalytic cycle, one would have also expected to detectsotaxadiene after the first day. This discrepancy may be attributedo insensitivity of the GC–MS that was used in the experiments.

.4. Taxadiene monooxygenase possibly acts on as many as fourubstrates

As molecular oxygen is reductively cleaved by the heme pros-hetic group to form water and an oxidized product, in situioxygenation during the course of a single P450 catalytic cycle

s neither mechanistically nor thermodynamically feasible. Thismplies that dioxygenated species can only be synthesized via re-iffusion of one or more nascent monooxygenated products intohe active site of a P450 enzyme, and, in addition to taxadiene andsotaxadiene, it appears that taxadiene monooxygenase acts on ateast one additional substrate.


Unfortunately, the low abundance and instability of the vastajority of the eicosanoid species synthesized by the cultures not

nly precluded characterization of their structures, but experi-ental validation of substrate–product relationships that would

e 5-day runs. The concentrations were calculated from the katharometric signalns (n = 3). Of the 16 species, taxadiene (B) is undetectable on all 5 days and only

on run.

normally proceed by incubation of the individual species with eachof the pathway enzymes too was infeasible in the current sce-nario. Nevertheless, the daily rate of change in the concentrations ofeach species was assessed (Fig. 6) in order to hypothesize the reac-tion landscape of taxadiene monooxygenase and gain an insightful,qualitative snapshot of the propensities of the species to participatein additional P450 catalytic cycles. Although speculative, the analy-sis still raises some intriguing questions, especially when assessedin conjunction with the data presented in Fig. 4.

Barring C, isotaxadiene and H, the concentrations of all speciesexhibit similar trends in their daily rates of change. Moreover,these trends are qualitatively similar to those exhibited by thenascent species that are consumed in downstream reactions, whichsuggests that C and H are the likeliest sources of the 3 dioxy-genated molecules. Of the remaining species, the concentrationsof taxadien-5�-ol, OCT, iso-OCT, species J, L, P and O in the brothappear to peak on the second day of production, whereas the con-centrations of species A, E, F, M and N peak on the third day. Theformation of species E and F is particularly intriguing. It is specu-lated that these molecules are formed in response to the concertedeffect of inhibition by the oxygenated taxanes and a high fluxthrough the prenyl pyrophosphate biosynthetic pathway. The lat-ter is possibly a physiological response to the heightened metabolicstress experienced by the bacterial host during expression of theP450 reaction complex.

3.5. Computational assessment of the reaction mechanism

Oxidation of the substrate typically proceeds via the oxy-gen rebound mechanism [25]. Herein, proton abstraction from


the substrate by the oxygen atom of oxyferyl heme generates ahydroxy-iron complex and a transition state species bearing a car-bon radical. The former then dissociates via a rebound mechanismto form the carbon oxygen bond, and regiospecificity of oxidation




Fig. 6. Daily variations in the forward derivative of the concentrations of the species with respect to time (in mM/day) have been plotted for 15 eicosanoid species – A, C-P.T en-5�C ar to

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rends suggest that the compounds fall into three groups. Iso-OCT, OCT (I), J, taxadi, isotaxadiene (D) and H form a third group. Species from two groups do not appereater inhibitory effect than those of the latter. The variation in the rate of the cha

s determined by radical stabilization (Fig. 7). It is hypothesized thatatalytic promiscuity arises from competing, regiospecific protonbstractions by the oxyferyl species, and a computational modelf the reaction mechanism was duly developed to validate thisypothesis.

To this end, a homology model of the apoenzyme of thengineered taxadiene monooxygenase was first constructed inODELLER [26] using the A-chains of the P450 enzymes, 2CIB,

EQM, 3GW9, 3DBG, 3LD6 and 2VE3 as templates. The apoenzymeequence was truncated by 50 amino acids from the N-terminalnd to ensure that it did not exceed the length of the templates.equence similarities between the truncated apoenzyme sequencend the aforementioned templates are 48.3%, 37.7%, 46.7%, 44.4%,5.3% and 56.1%, respectively. The use of multiple templates foromology modeling greatly improves the quality of the models27]. The more templates one uses, the more variations in rotameronfigurations and folds that one can account for, thereby enhanc-ng model quality. The reductase domain was also excluded fromhis analysis. The active site was then located using the pocketearching algorithm, POCASSA [28]. The result outputted by theocket search was validated by comparing it with crystal structuresf homologs available in the DALI database [29]. The homologyodel was then augmented with the heme prosthetic group to con-

truct the holoenzyme. This aim was achieved by first connectinghe heme iron to the cysteinyl sulfur in the active site using a thio-ate linkage and then optimizing the geometry of the heme moietysing the BFGS energy minimization algorithm [30]. The numberf iterations of the BFGS algorithm was left uncapped in order tonsure convergence to the optimal geometry. This process yields aoloenzyme model with a thiolate bond length of 2.559 A, whichompares quite favorably with the average thiolate bond length of.47 A for the 6 templates used to construct the homology modelf the apoenzyme.


Taxadiene was then computationally docked into the active sitef the holoenzyme model using AUTODOCK [31]. Since docking onlyrovides a static representation of the substrate in the active sitet a particular instant in the reaction, the docking algorithm was

ig. 7. The oxygen rebound mechanism involves the formation of a hydroxy-iron specieond-forming electron migrations are depicted in red and blue, respectively. (For interpreb version of this article.)

-ol (K), L, P and O comprise one group. The second group includes A, E, F, M and N.react further, but that members of the first group are more volatile and/or exert a

C and H suggests that they are likeliest sources of the dioxygenated molecules.

successively implemented using models of each of the four statesthat the heme prosthetic group assumes during the catalytic cycle.This approach enables tracking of the pose of taxadiene in the activesite as it evolves during the P450 cycle much in the same way staticsnapshots are used to describe dynamic phenomena. Additionally,since AUTODOCK outputs as many as 9 potential poses from a sin-gle input, it was executed iteratively using the most stable outputof one run as the input for the program’s next implementation. Thisexercise is similar to an unconstrained optimization, and the algo-rithm was terminated when 3 successive sets of the poses exhibitedthe same binding affinities. The true evolution of the pose of taxa-diene within the active site was then inferred by assessing thediffusional trajectory of oxygen into the active site. The diffusionof oxygen into the activated heme-containing active site greatlylimits the translational and rotational degrees of freedom that thenow-destabilized taxadiene has in order to assume a more stableposition, which, in turn, serves to effectively discriminate the posesoutputted by AUTODOCK. The likeliest path that oxygen takes todiffuse into the active site was calculated using CAVER [32]. Lastly,the aforementioned modeling algorithm was validated by compar-ing its predictions to the crystal structure of CYP105A1 bound toits ligand. CYP105A1, like taxadiene monooxygenase, is multispe-cific and catalytically promiscuous, and hydroxylates vitamin D3at either the 1�- or 20-position [33]. CYP105A1 then acts on bothmonohydroxylated products to form 1�,20-dihydroxy vitamin D3.The model’s outputs are identical to the X-ray crystallographic data.

The model reveals that taxadiene assumes one of the two pos-itions within the active site just prior to its participation in the oxy-gen rebound mechanism. In reality, it is speculated that taxadienecould be positioned in the active site in a state that is in dynamicequilibrium between these two poses. In both poses, however, theoxyferyl heme iron abstracts protons, in their order of averagefavorability, attached to carbons at the 3-, 13�- and 20-positions


(Fig. 8). Abstraction of any one of the three protons attached to thecarbon at the 20-position produces allyl-stabilized radical speciesat the 20- and 5-positions (Fig. 9), which then yield taxadien-20-oland taxadien-5�-ol, respectively. Taxadien-5�-ol is more abundant

s, which then dissociates to insert oxygen into the substrate. Bond-breaking andetation of the references to color in this figure legend, the reader is referred to the


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8 V.G. Yadav / Journal of Molecular Catalysi

Fig. 8. The energy-averaged structure of the two likeliest orientations of taxadieneib

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alignments with the other Taxus P450 monooxygenases [34].

Faioo

n the active site (poses A and B) suggests that as many as 3 proton abstractions mighte occurring. In the order of preference, these are at the 3-, 13�-, and 5�-positions.

han taxadien-20-ol as the 5-position secondary carbon radical ishe more stable of the two allyl-stabilized radical intermediates.

Taxadien-5�-ol is also produced from isotaxadiene via abstrac-ion of the sole proton that is connected to the carbon at the�-position. In this instance, however, there is no allylic stabi-

ization in the radical species. In comparison, abstraction of theole proton that is attached at the 3-position produces a tertiaryarbon radical. However, as insertion of oxygen at this positionppears to be sterically hindered, the lone electron shifts to theertiary carbon at the 12-position. This transfer does not mitigatehe steric hindrance to the oxygen rebound mechanism either. Its speculated that the close proximity of the alkenyl moiety to theydroxy-iron species and the tertiary carbon radical facilitates aoncerted sequence of electron exchanges that results in the addi-ion of the oxygen atom to the carbons at the 5- and 12-positions toorm OCT. Similarly, proton abstraction from the carbon at the 13�-


osition produces taxadiene-13�-ol and iso-OCT. Our explanationor ether formation is different from the mechanisms proposed byontein et al. [23]. However, oxidation of taxadiene in the reaction

ig. 9. The model of the association between taxadiene and taxadiene monooxygenasend (c) 13�-positions. Radical species formed via proton abstractions from the 20- ands sterically inhibited. Formation of OCT (8) following proton abstraction from the 3-posxygen to the 5- and 12-positions. The formation of iso-OCT (13) follows a similar mechxygen rebound mechanism.


mechanism detailed by Rontein et al. does not proceed via the oxy-gen rebound mechanism and neither can it explain formation oftaxadien-5�-ol and OCT via a common reaction mechanism in thesame active site.

3.6. Computational assessment of mutants as validation of thealgorithm

The computational methodology was then applied to evaluatethe influence of mutations made to the active site of the monooxy-genase. Mutations to amino acid residues located at the peripheryof the active site cavity were arbitrarily made, and the influenceof these mutations on product distribution was closely investi-gated. 26, 15 and 12 mutants bearing single, two and three aminoacid substitutions, respectively, were constructed and tested. Itwas observed that while a number of enzyme mutants exhibitedimprovements in either selectivity or turnover over the parentalenzyme, only 6 of the 53 mutants had improved in both aspects(Fig. 10).

Of these, 5 mutants bore single amino acid substitutions,whereas the sixth was a triple mutant. Selectivity is defined asthe ratio of the concentration of taxadien-5�-ol to the concentra-tions of all oxidized derivatives of taxadiene. Turnover, on the otherhand, is calculated by summing the molarities of all oxygenatedeicosanoids produced by the enzyme. Based on these definitions, asignificant variation between the fold changes in selectivity towardproducts of the same catalytic cycle would be indicative of directmanipulations to the catalytic cycle viz. improvements in reactionselectivities.

In the case of taxadiene monooxygenase, however, it wasnoticed that the magnitudes of changes in the concentrations of iso-OCT (G), OCT (I) and taxadien-5�-ol (K) were quite similar, whichsuggests that all mutants that were constructed effectively desta-bilized binding of competing substrates but did not drastically alterthe reaction mechanism. Similar effects were observed when theactive site of taxadiene monooxygenase was manipulated by ran-domly mutating amino acid residues based on multiple sequence


To confirm this hypothesis, the binding of taxadiene in the activesites of K131R, S302A and V374L was computationally evaluated.The computational models of the mutants were constructed using

predicts competition between the abstraction of protons from the (a) 20-, (b) 3- 13�-positions are allyl-stabilized. Insertion of oxygen to the 3- and 12-positionsition is hypothesized to proceed via the constrained Markovnikov-like addition ofanism. On the other hand, the formation of taxadien-5�-ol (5) is explained by the




Fig. 10. Percent changes in the selectivities and turnover of the 53 mutants are compared to those of the parental enzyme. Selectivity is defined as the ratio of the concentrationo the cot and tha he rea

tasoospot

Fl

f taxadien-5�-ol to all hydroxylated eicosanoids. Activity is defined as the sum of

o nM. Solid, hollow and hatched boxes represent the mutants bearing single, two

re colored red. (For interpretation of the references to color in this figure legend, t

wo complementary approaches. In the first approach, the aminocid residue targeted for mutation was directly replaced in theequence of the parental enzyme, and the exact same sequencef steps that was followed earlier during construction of the modelf the taxadiene monooxygenase holoenzyme was repeated. In the


econd approach, the target amino acid was replaced in the com-utational model itself with the most sterically favorable rotamerf the alternative residue, followed by geometry optimization ofhe new residue using the BFGS energy minimization algorithm.

ig. 11. The model successfully predicts variations in product selectivities of the 3 mutaikeliest poses predicted by the model (represented as dotted clouds) exhibit visible diffe

ncentrations of all hydroxylated eicosanoids. Concentrations have been convertedree amino acid substitutions, respectively. The mutants K131R, S302A and V374L

der is referred to the web version of this article.)

PyMOL’s in-built mutagenesis wizard was utilized for amino acidreplacement in the computational models [35]. The root meansquare deviation (RMSD) between the models constructed via thetwo approaches was negligible. However, the second approach wasmuch faster and easier to execute.


The 3 mutants exhibited deducible differences in their reactivitywith taxadiene (Fig. 11 and Table 1). The energy-averaged orienta-tions of taxadiene in the active sites of the parental P450 enzymeand the 3 mutants are visibly different. The proton abstraction

nts that were investigated. Electron densities of the average structures of the tworences between the 4 enzymes.


ARTICLE ING ModelMOLCAB-3042; No. of Pages 11

10 V.G. Yadav / Journal of Molecular Catalysi

Table 1Comparison of the product distribution and activity of selected mutants with theparental enzyme.

V374L K131R S302A

% Change in the distribution oftaxadien-5�-ol amongsttaxadiene derivativescompared to parentalenzyme

24.4 −23.4 −53.5

% Change in the totalproduction of oxygenated

−11.6 13.5 1.1

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nceeb5Mcrd

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egbtowaatagtelm

mefimd

14 (2012) 233–241.

eicosanoids compared toparental enzyme

engths predicted for V374L and S302A correlate quite well withhe differences observed in the relative proportion of the monooxy-enated taxadiene derivatives produced by each enzyme. For374L, whereas the abstraction length for the proton connected

o the carbon at the 20-position is unchanged, abstraction of therotons connected to the carbons at the 3- and 13�-positions isore hindered compared to the parental enzyme (the situation is

eversed for S302A).For K131R, although the abstraction lengths for the protons con-

ected to the 3- and 13�-position carbons and to the 20-positionarbon are greater and shorter, respectively, than in the case ofnergy-averaged pose of taxadiene in the active site of the parentalnzyme, production of taxadien-5�-ol is lower compared to thease case. Instead, production of species M is higher than taxadien-�-ol. This trend is also observed in S302A. It is likely that species

might be taxadien-20-ol (although this conclusion remains to beonfirmed by NMR), and the increase in its production may be theesult of the transition state species adopting an orientation thatisfavors formation of taxadien-5�-ol.

These inferences underscore the limitations of traditional orstructure-blind’ mutagenic approaches in identifying mutants thatruly diminish the catalytic promiscuity of enzymes. Neverthe-ess, phylogeny-guided and even random mutagenesis are valuableools for enzyme engineering, not least because these methodsften rapidly identify mutants with higher turnovers and dimin-shed affinities for competing substrates.

. Conclusions and significance

The present study underscores the mutual dependence betweennzyme engineering and metabolic engineering for the heterolo-ous production of secondary plant metabolites. Given that mostiological experimentation still proceeds with a ‘black box’ men-ality, and that each assumption that one makes in the formulationf the model diminishes the closeness of fit that the model hasith the phenomenon that it describes, empirical approaches such

s directed evolution and phylogeny-guided mutagenesis, whenpplicable, are superior to model-guided approaches. However, inhe case of manipulating taxadiene monooxygenase, the absence of

screen that can efficiently couple the selectivity of the enzyme toene expression or platforms such as phage-display or the yeastwo-component bait-prey system precluded the use of directedvolution. This challenge is envisaged to become a recurring prob-em in metabolic engineering for the production of plant secondary

etabolites.Additionally, the selectivity improvements exhibited by enzyme

utants bearing random active site mutations (constructed usingither purely random or phylogeny-guided mutagenesis) resulted


rom destabilizing binding of competing substrates rather thanmproving the selectivity of the catalytic mechanism. In the case of

ultispecific and catalytically promiscuous enzymes such as taxa-iene monooxygenase, the benefit of being able to rapidly probe


enzyme reactivity using random or phylogeny-guided mutagene-sis is soon lost over the inability of the methodology to identifydesirable amino acid substitutions without having to construct anexhaustive collection of mutant enzymes.

In comparison, the computational methodology that has beenoutlined herein conclusively identifies the mechanistic basis under-lying catalytic promiscuity. It is also generalizable and much morerapid than canonical structure-guided approaches. The method-ology can also be applied to identify the influence of structuralfeatures on stability, activity, substrate specificity and catalyticpromiscuity. In addition, one can possibly extend the methodologyto prescribe mutational targets within the active site to controllablymodulate substrate specificity and product selectivity. For this tooccur, though, NMR characterization of all interacting substratesand their oxidation products is required. The algorithms utilizedin the methodology can then multiplicatively compare the bind-ing affinities and orientations of all characterized substrates withinthe active site to guide the construction of mutants that favor onlyspecific orientations of desired substrates.

It must be noted that formulation of the computational method-ology assumed that only a single taxadiene substrate docks intothe active site of the monooxygenase. While this assumption iswell-grounded in literature, questions still remain as this enzymeis somewhat of a novelty compared to other P450 monooxyge-nases studied in literature. The diffusion of two molar equivalentsof taxadiene into the active site of the enzyme, if it were to occur,would not be constrained by space. We have estimated the vol-ume of the active site of taxadiene monooxygenase to be roughly474 A3 using the pocket searching algorithm, POCASSA [28]. Evalu-ation of this hypothesis necessitates isolation and purification ofthe enzyme, followed by an in vitro assessment of the spectralshift that is induced by substrate binding. Comparing the spectralshift to P450 standards known to bind to single or multiple sub-strate molecules simultaneously would provide the much-neededconstraint that is required to include or eliminate the possibilityof multiple substrates binding simultaneously within the activesite. As the computational methodology can account for the simul-taneous binding of several substrates within the active site, thisconstraint could improve the predictions of the model.

Acknowledgements

The author thanks Gregory Stephanopoulos, Kristala JonesPrather, Narendra Maheshri and Bruce Tidor for their valuable com-ments and insights. The author also thanks Christina White forassistance with acquisition of the 1H NMR spectra of taxadiene-5�-ol, OCT and iso-OCT. This work was supported by research fundsfrom the MIT Legatum Center.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.molcatb.2014.10.004.

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unraveling the multispecificity and catalytic promiscuity of taxadiene monooxygenase

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