Vol. 171, No. 12JOURNAL OF BACTERIOLOGY, Dec. 1989, p. 6800-68070021-9193/89/126800-08$02.00/0Copyright X 1989, American Society for Microbiology

Homology between Hydroxybutyryl and Hydroxyacyl Coenzyme ADehydrogenase Enzymes from Clostridium acetobutylicum

Fermentation and Vertebrate Fatty Acid 1-Oxidation PathwaysJONATHAN S. YOUNGLESON, DAVID T. JONES,t AND DAVID R. WOODS*

Department of Microbiology, University of Cape Town, Rondebosch 7700, South Africa

Received 5 June 1989/Accepted 13 September 1989

The enzymes NAD-dependent 13-hydroxybutyryl coenzyme A dehydrogenase (BHBD) and 3-hydroxyacetylcoenzyme A (3-hydroxyacyl-CoA) dehydrogenase are part of the central fermentation pathways for butyrateand butanol production in the gram-positive anaerobic bacterium Clostridium acetobutylicum and for the 1(oxidation of fatty acids in eucaryotes, respectively. The C. acetobutylicum hbd gene encoding a bacterial BHBDwas cloned, expressed, and sequenced in Escherichia coli. The deduced primary amino acid sequence of the C.acetobutylicum BHBD showed 45.9% similarity with the equivalent mitochondrial fatty acid "-oxidationenzyme and 38.4% similarity with the 3-hydroxyacyl-CoA dehydrogenase part of the bifunctional enoyl-CoAhydratase:3-hydroxyacyl-CoA dehydrogenase from rat peroxisomes. The pig mitochondrial 3-hydroxyacyl-CoA dehydrogenase showed 31.7% similarity with the 3-hydroxyacyl-CoA dehydrogenase part of thebifunctional enzyme from rat peroxisomes. The phylogenetic relationship between these enzymes supports acommon evolutionary origin for the fatty acid 13-oxidation pathways of vertebrate mitochondria andperoxisomes and the bacterial fermentation pathway.

Clostridium acetobutylicum P262 is a gram-positive anaer-obic bacterium that produces acetone, butanol, and ethanolfrom a variety of carbohydrate substrates (28, 43). In batchculture, the acetone-butanol fermentation is characterizedby an initial acid-producing growth phase, followed by asolvent-producing phase. The shift to solvent production isassociated with the induction of solventogenic enzymes anda decrease in activity of acidogenic enzymes (3, 22).The central fermentation pathway in C. acetobutylicum

resulting in the synthesis of butyrate and butanol involveseight enzymes (Fig. 1B). The four enzymes responsible forthe formation of butyryl coenzyme A (butyryl-CoA) fromacetyl-CoA are thiolase, ,3-hydroxybutyryl-CoA dehydroge-nase (BHBD), crotonase, and butyryl-CoA dehydrogenase.Similar enzymes, thiolase, 3-hydroxyacyl-CoA dehydroge-nase (HAD), enoyl-CoA hydratase, and acyl-CoA dehydro-genase (Fig. 1A), are involved in the 1 oxidation of fattyacids in eucaryotes. In mitochondria, the enoyl-CoA hy-dratase and HAD are separate enzymes (49), but animalperoxisomes and plant glyoxysomes usually contain a bi-functional enzyme that has enoyl-CoA hydratase and HADactivities (Fig. 1A) (33). In the microbodies of the yeastCandida tropicalis there is a trifunctional enzyme that has3-hydroxyacyl-CoA epimerase activity in addition to theernoyl-CoA hydratase and HAD activities (36). A multifunc-tional enzyme complex that contains thiolase, HAD, croto-nase, epimerase, and isomerase activities (42) and is en-cloded by the fadAB genes is also found in Escherichia coli(48). The amino acid sequence of the pig mitochondrial HAD(MHAD) has been determined (7). The cDNA encoding ratMHAD has been isolated (2), and the genes encoding thebifunctional enzyme from rat peroxisomes (26, 41) and thetriunctional enzyme from Candida tropicalis peroxisomes(39) have been cloned and sequenced.

* Corresponding author.t Present address: Department of Microbiology, University of

Otago, Dunedin, New Zealand.

In view of the importance of the acid and solvent path-ways in the industrial production of solvents by C. acetobu-tylicum, we have investigated the cloning and structure ofacid and solvent pathway genes. Previously, we reported thecloning and sequencing of the terminal enzyme in the buta-nol pathway (NADPH-dependent butanol dehydrogenase)(54; J. S. Youngleson, W. A. Jones, D. T. Jones, and D. R.Woods, Gene, in press). This paper describes the cloning,characterization, and amino acid similarity of the BHBDfrom the butyrate synthesis pathway of C. acetobutylicum.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions. Thebacterial strains and plasmids used have been describedelsewhere (Youngleson et al., in press). E. coli strains weregrown aerobically at 37°C on Luria medium (34).

Preparation of DNA. Plasmid DNA from E. coli strainswas prepared by the method of Clewell (11).DNA sequence analysis. Plasmid pCADH100 (54), which

contained the C. acetobutylicum adh-i and hbd genes, wasused as the primary source of DNA. Standard moleculargenetic techniques (34) were used to subclone DNA frag-ments into the plasmid vectors pUC18 and pUC19 (53). Allplasmid constructions were verified by restriction analysisand electrophoresis in 0.8 to 1.0% agarose Tris-acetate gels.Competent E. coli cells were prepared and transformed bythe calcium chloride method (13). DNA was prepared forsequencing by a combination of subcloning from availablerestriction sites and the construction of ordered deletions,using the nucleases BAL 31 (35) or exonuclease III (25).DNA sequencing of both strands was done by the dideoxy-chain termination method (47), with overlapping templates(Fig. 1). E. coli LK111 was used as the lacZ recipient strain.The DNA was radiolabeled with [35S]dATP (>1,000 Ci/mmol) and primed as specified by the manufacturers, using aSequenase kit (U.S. Biochemical Corp.). The nucleotidesequences were analyzed with an IBM XT computer, usingthe DNA tools and Genepro (version 3.1) programs. De-

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BHBD HOMOLOGIES 6801

A Fatty acid activation

Fatty acyl-CoA thioester

Al Fa

trans-Enoyl-CoA

-d........- A2 MH20O-Oxidation * AS f%-14vdmYvaCvI-rCnA

spiral............-----...-* A3 pND

NAM

3-Ketoacyl-CoA

A4 COA

Fatty acyl-CoA Acetyl-CoAthioester(2-C SHORTElt)

B ButyrateATP -ADP B7Butyryl PCoA 8 BScoA

Butyryl-CoA 7 Butyraldehyde 7 ButanolNAD NADH NAe NADPH NADPB'NADH

Crotonyl-CoA

120j B2

3-Hydroxybutyryl-CoANADIA B3NADH

Acetoacetyl-CoA

CoAl B4

Acetyl-CoA

|I GlycolysisFIG. 1. Comparison of the metabolic pathways utilized for the P oxidation of fatty acids in eucaryotes (A) and butyrate and butanol

synthesis in C. acetobutylicum (B). Enzymes involved in the reactions: Al, acyl-CoA dehydrogenase; A2, enoyl-CoA hydratase; *A3,MHAD; A4, thiolase (acetyl-CoA acetyltransferase); *A5, enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase (bifunctional enzyme);Bi, butyryl-CoA dehydrogenase; B2, crotonase; B3*, BHBD; B4, thiolase (acetyl-CoA acetyltransferase); B6, butyraldehyde dehydrogeflase;B7, butanol dehydrogenase (ADH-1).

duced amino acid sequences were analyzed and comparedby using the Microgenie (Beckman version 299) proteinalignment subroutine and the GenBank DNA data base(release 52) and the PIR protein data base of Genepro(release 13) via an IBM XT microcomputer. Adjustmentswere made to optimize the alignment of the enzymes.

Preparation of cell extracts. Cell extracts of E. coli wereprepared from overnight cultures (200 ml) grown aerobicallyin Luria medium. Before cell disruption with a Frenchpressure vessel, the cells were suspended in ice-cold 100 mMpotassium phosphate buffer (pH 7.5) with 10 mM mercapto-ethanol, and the supernatant was retained on ice for theBHBD assay after centrifugation for 1 h at 145,000 x g.

Protein concentrations in the cell extracts were determinedby the biuret method (20).BHBD activity assays. BHBD activity was assayed in the

physiological direction spectrophotometrically by measuringthe decrease in A340 due to the dehydrogenation of NADH(5). A standard assay mixture contained 100 mM potassiumphosphate buffer (pH 7.5), 10 mM mercaptoethanol, 0.1 mMNADH, and 20 ,uM acetoacetyl-CoA (substrate). The assaymixture was allowed to equilibrate for 5 min at 20°C beforethe assay was started with the addition of enzyme. The ratesthus measured were corrected for nonspecific NADH-de-pendent dehydrogenase activity. E. coli HB101(pCADH100)cell extracts were diluted to a concentration of less than 0.5jig/ml in 100 mM potassium phosphate buffer (pH 7.5) toobtain a suitable assay range.

RESULTS

Cloning and sequencing of the C. acetobutylicum hbd gene inE. coli. Youngleson et al. (54) reported the cloning in E. coliof the C. acetobutylicum adh-i gene on a 3.4-kilobase DNAfragment in pCADH100. Nucleotide sequencing of part of

the 3.4-kilobase C. acetobutylicum DNA insert located theadh-l gene of 1,164 base pairs (bp) at the 3' end of the insert(Youngleson et al., in press). Further nucleotide sequencingof the remainder of the C. acetobutylicum 3.4-kilobase DNAinsert revealed a second open reading frame (ORF) of 846 bplocated 354 bp upstream of the adh-i gene (Fig. 2). The ORFwas identified as the hbd gene, which codes for an NADH-dependent BHBD enzyme that catalyzes the conversion ofacetoacetyl-CoA to P-hydroxybutyryl-CoA (Fig. 1B). Thehbd gene encoded a BHBD enzyme of 282 amino acidresidues with a calculated Mr of 31,435. Upstream of the hbdgene a third ORF was detected (Fig. 2). This ORF of 435 bpwas incomplete and is separated from the hbd gene by 182bp.The production of an NADH-dependent BHBD enzyme

activity by E. coli(pCADH100) cells was investigated. E. colicells harboring pCADH100 produced approximately 160-foldhigher levels of NADH-dependent BHBD than did controlE. coli cells (4,330 versus 27 gxmol/min per mg of protein).The BHBD enzyme coded for by pCADH100 in E. colishowed a specific requirement for NADH, and no NADPH-dependent BHBD activity was detected.The C. acetobutylicum hbd gene was preceded by a

putative ribosome-binding site with the sequence AGGAGG(30) located 8 bp upstream of the ATG start codon (Fig. 2b).The hbd gene was terminated by a single TAA stop codon.The 354-bp downstream intergenic region between the hbdand adh-i genes did not contain a transcription terminationsequence (44). A putative procaryotic Rho-independent ter-mination sequence was identified 34 bp downstream of theadh-i gene (Youngleson et al., in press). Previously, Youn-gleson et al. (54) showed that the C. acetobutylicum adh-igene was expressed from nucleotide sequences upstream ofthe neighboring hbd gene.

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6802 YOUNGLESON ET AL.

a

pCADH100 -VEPOR:

P EC.R2MU1 (3.4

C. oo./obufy//cum DNA (3.2 kb)= > > = ->=

IC C6 - 0c-X w E °. Eoa E &C

I 1'WI X w X IXwm x u) II

ORF 3 hA Gone (843 bp) &&* Gone (I 1 4 bp)

-~~

i

kb)

X n'I-150

GAATAATTTCAGTTTTAATAATATATTATTTTCTATTTGAAAAGAAAAAGTTAATAATATAACAATAT0313*

-100 -50I E RV

AAATAATTAATTAACAATATTAATAATTTTTTAACAAGTTGuATTG m'TTAATCCTTAGTTAAAGTAATGCTATTTTAACTAAGGATATCTATAAAGAAATGAT.LTTTGAGGAGGAfAT.DCTS.D.

1 ATG GAA AAG ATT TTT GTA CTT GGA GCA GGA ACT ATG GGT, GCT GGT ATT GTT CAA GCA TTC GCA CAA AAA GGT TAT GAA GTA ATC GTA AGAM E K I F V L G A G T M G A G I V Q A F A Q K G Y E V I V R

91 GAT ATT AAA GAT GAA TTT GTT GAA AGA GGA ATT GCT GGA ATC AAC AAA GGA TTA ACT AAG CAA GTT GCT AAG GGA AAA ATG ACT GAA GAAD I K D E F V E R G I A G I N K G L T K Q V A K G K M T E E

181 GAT AAG GAA GCA ATA CTT TCT AGA ATT TCA GGA ACA ACT GAT ATG AAA TTA GCT GCT GAT TGC GAT TTA GTA GTT GAA GCT GCA ATT GAAD K E A I L S R I S G T T D M K L A A D C D L V V E A A I E

271 AAT ATG AAA ATT AAA AAA GAA ATT TTT GCT GAA TTA GAT GGC ATT TGT AAA GAA TCA ACT ATT TTA GCT TCA AAC ACT TCA TCA TTA TCAN M K I K K E I F A E L D G I C K E S T I L A S N T S S L S

jindIII361 ATA ACA GAA GTT GCT TCA GCA ACA AAG AGA CCT GAT AAA GTT ATC GGA ATG CAT TTC TTT AAC CCA GCT CCA GTA ATG AAG CTT GTT GAA

I T E V A S A T K R P D K V I G M H F F N P A P V N K L V E

451 ATT ATA AGG GGA ATA GCT ACT TCA CAA GAA ACT TTT GAT GCT GTT AAA CAA TTG TCA GTA GCA ATC GGA AAA GAA CCA GTA GAA GTT GCAI I R G I A T S Q E T F D A V K Q L S V A I G K E P V E V A

541 GAA GCT CCA GGA TTT GTT GTA AAC AGA ATC TTA ATC CCA ATG ATT AAT GAA GCT ACA TTC ATT TAT CAA GAA GGA ATT GCT TCA GTT GAAE A P G F V V N R I L I P M I N E A T F I .Y Q E G I A S V E

EcRV IglII631 GAT ATC GAT GCA GCT ATG AAA TAT GGT GCT AAT CAC CCA ATG GGA CCT TTA GCT TTA GGA GAT CTT ATT GGA TTA GAT GTT TGT CTA OCT

D I D A A M K Y G A N H P N G P L A L G D L I G L D V C L A

721 ATC ATG GAT GTT TTA TTT AAT GAA ACA GGA GAC AGC AAG TAC AGA GCT AGC AGC ATT TTA AGA AAA TAT GTT AGA GCT GGA TGG CTT GGAI N D V L F N E T G D S K Y R A S S I L R K Y V R A G W L G

a11 AGA AAA TCA GGA AAA GGA TTC TAT GAT TAT TCA AAA TAAR K S G K G F Y D Y S K *

FIG. 2. Molecular characterization of the C. acetobutylicum hbd gene, encoding BHBD. (a) Plasmid restriction map of pCADH100indicating the C. acetobutylicum adh-l and hbd genes, encoding alcohol dehydrogenase (ADH-1) and BHBD, respectively. A third ORF(referred to as ORF3) of unknown length or function is indicated upstream of the hbd gene. Arrows indicate regions and directions subjectedto sequence analysis. (b) Nucleotide and deduced amino acid sequences (in single-letter code) of the entire hbd gene and the upstreamintergenic region. A putative ribosome-binding site (S.D.) is undelined 8 bp upstream of the starting Met. The hbd gene is terminated by asingle TAA stop codon. The sequence of the downstream region containing the adh-1 gene has been described previously (Youngleson et al.,in press).

Codon usage. C. acetobutylicum DNA has a G+C contentof 28% (12). Our laboratory has reported the nucleotidesequences of five C. acetobutylicum genes: adh-l (Youngle-son et al., in press), hbd (this study), an endoglucanase gene(55), a xylanase gene (H. Zappe et al., manuscript inpreparation), and glnA (27), with G+C contents of 32.6,34,0, 33.8, 37.7, and 31.8%, respectively. The codon usageof these five genes was analyzed, and all of the genes showeda bias toward the use of codons in which A and U predom-inate (Table 1). In the hbd gene, only 30 of the possible 61codons were used more than once, and 20 codons were not

used at all. Of these 20 codons, two (CCC and CGG) werenot used in any of the five genes, and another eight codons(GGG, CAC, CGC, CUG, CCG, AGG, UCC, and UCG)were not used more than once in any one of the five genes.In E. coli, the following codons are regarded as minorcodons and are rarely utilized: GGA (Gly), UUA (Leu),AAU (Asn), CCA (Pro), AGA (Arg), UCA (Ser), and ACA(Thr) (1). In all of the five C. acetobutylicum genes, theseminor codons are utilized preferentially and at high frequen-cies in comparison with the other codons. Codon preferencehas been suggested to represent a hinR for abundant tRNAs,

b

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TABLE 1. Comparison of G+C content and codon usage in C. acetobutylicum and E. coli genes

Codon usage

Amino acid C. acetobutylicumb(codon)a mol% E. colic

Mean% (mol%)adh-l hbd xyl eng ginA

Ala (GCfU)(GCC)(GCA)(GCG)

Cys (UGU)(UGC)

Asp (Q1D(GAC)

Glu (GAA)(GAG)

Phe (UUU)(UUC)

Gly (GGU)(GGC)(G)d

(GGG)dHis (CAU)

(CAC)Ile (AIU)

(AUC)(AUA)d

Met (AUlG)Lys (AAA)

Leu QJW.A)d(UUG)d(CUU)d(CUC)d(CUA)d(CUG)

Asn (AAIDd(AAC)

Pro (CCU)d(CCC),(CC )(CCG)

Gln (CA)(CAG)

Arg (CGU)(CGC)(CGA)d(CGG)d

(AGG)dSer (UCU)

(UCC)

(UCG)d(AGU)d(AGC)

Thr (AMI)(ACC)(A)d(ACG)

Val (ULM)(GUC)(QUA)(GUG)

Trp (UGG)Tyr (UAU)

(UAC)

G+C (%)

4.60.33.11.01.00.34.60.57.70.53.91.02.10.83.60.32.60.34.10.52.34.15.23.65.90.30.50.01.60.03.40.30.80.03.90.32.30.00.00.00.00.02.30.00.50.03.40.30.50.02.10.04.10.01.60.33.10.50.53.60.3

32.6

7.50.03.20.00.70.45.30.47.80.02.51.41.40.47.10.00.40.45.72.81.43.96.02.54.30.41.80.00.40.01.41.40.70.02.10.02.10.00.00.00.00.03.20.40.40.04.30.00.01.12.80.01.80.05.00.02.80.00.42.10.4

34.0

1.20.43.10.40.80.41.91.52.70.83.10.81.93.53.50.40.40.42.71.22.32.35.01.92.31.21.50.00.00.05.02.71.20.01.50.01.52.30.40.40.80.01.50.00.40.42.30.05.01.93.81.54.61.52.31.21.50.42.75.01.2

37.7

4.70.51.10.00.50.54.71.22.60.52.90.21.91.43.50.01.20.21.90.25.42.64.51.92.60.50.90.50.00.26.11.02.10.01.40.02.61.40.50.00.00.00.70.03.10.34.20.22.80.96.10.54.70.02.60.52.10.02.43.81.4

33.8

3.40.05.40.21.10.25.90.97.00.73.61.11.10.75.20.21.80.03.40.70.72.95.61.67.20.20.20.00.90.26.61.40.70.03.80.02.50.00.00.00.00.04.30.01.40.00.90.20.70.01.80.02.70.02.70.23.00.20.92.90.9

31.8

4.260.223.230.330.820.344.490.895.560.493.170.921.681.334.570.171.130.243.551.082.423.175.252.294.460.491.000.090.560.094.481.331.080.002.560.052.210.740.170.080.150.002.410.071.130.123.000.141.800.783.330.403.580.312.820.422.500.231.363.480.81

29.9

3.112.092.553.400.320.452.692.585.041.831.602.083.553.030.340.510.651.022.323.880.032.914.551.380.550.780.830.630.176.640.882.750.370.250.822.681.153.183.091.860.120.170.060.021.321.280.420.570.321.081.172.490.290.972.800.951.712.000.741.061.55

51.0

6803

a C. acetobutylicum preferred codons are underlined.b Gene designations: adh-1, alcohol dehydrogenase (Youngleson et al., in press); hbd, BHBD (this study); xyl, xylanase (Zappe et al., in preparation); eng,

endoglucanase (55); glnA, glutamine synthetase (27).1 Fifty-two E. coli proteins combined (1).d E. coli rare codon.

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6804 YOUNGLESON ET AL. J. BACTERIOL.

PHAD 1 M A E Y L R L PG S L A M I R L C N P P V N A V S P T V I R E V R N G L Q K A G S D H T VD A I V I C G A N G N F C A G

PRAD 61 A D I H G F S A F T P G L A L G S L V D E I Q R Y Q R P V L A A I Q G V A L G G G L E L A L G C H Y R I A N A K A R V G

PRAD 121 L P E V T L G I L P G A R G T Q L L P R V V G V P V A L D L I T S G K Y L S A D E A L R L G I L D A V V K S D P V E E A

P}iAD 181 I K F A Q K I I D K P I E P R R I F N K P V P S L P N M D S V F A E A I A K V R K Q Y P G V L A P E T C V R S I Q A S V

PHAD 241 K H P Y E V G I K E E E K L F M Y L R A S G Q A A L Q Y A F F A E K S A N K W S TPMYG A S W KTES A Q PVS SMGlEAD 1 SSISTAAASLAJKKILWKH TDIED 1 M E K I FCRYS 1 A S P A A G D WL

PIAD 301 V L G L G T M G R G IAIS |FARV GI SVASAEDM[KfL ND A AKGJ-TLFT T A RAH7NGQ A S - - -

lEAD 20 VWGI G GM G I A V A A A T G H TV V LV D Q TED IJ A KS K KG I E@S L R K-VA K K K F A EN P K A [J DBEHBD 6 VLGAGTM G AG IAV F A Q K G Y E VII VRDIK DEFV E R GIA G I N K G V A K|G K M TMEE D K - _CRYS 10 1W S G1VGi RJ S WTJALML F A S G GF R KMYD I EMR[- - - - I T LLMN I K E M K S L Q ]S G SL UK -GMS

PRAD 358 - --A1TK P K Lff]1F ISSST- - ELS-____DL V V - -

_

F _jj~ L K K_ V F A V A L IrC KIPMEAD 79 E F V T L SMI S V H S T D L V V E A I V E Q L K V G E N L K K F IL K F AE

}D 63 - - - lEAI L S R I S GTDM KlA_AD C - - - D L V V E A - - - A I E NI1IIKKE I F A E L D GII C K ElCRtYS 65 L S A E Q L SC_ E H Q E C - MP E S

PHAD 402 G A F L C T S N V D D IA S STD RPTLV I G T H F S P A H V IPR T-f I A TEJM S(SllaD 136 H T N L V E V T SIKTLE S L V D FD 109 S TI L A S N T S S S E K K M H F F_N P AP I K L VEQ[GI AI S TE TFDA V

CRYS 115 R V L P S KIF I G L A H V K Q ALHPV NP P Y I V E LV HPMET SPE A TVDR T H AU

PEHAD 462 S K K I G K I G VV - G N C G V A Y G F F E-GLE - S K G L E E F F - - -

IIAD 194 S L KH S C - D TR G F N R L V G E D I D|T M - _BPBD 169 KGJEVPVIE - ALP G F V V I N T G V E D I DAA MKY A H - - -CRYS 175 M RK SIP VR L I DF Y AI3W L D L R Y A F I

PHA 516 G R V SI AG L D G W K R - Q|z1L|T GPL PIP T P V R K R G N SRL LGD E R QlED 250 G P F ELLDVG L DT K F I - WHDEG- MID SQ NLUtLF - - - - - - - - A M N A K KKBHB 225 G IL GL I G L D C L A M L F NIE T G D S Y - - - - - - - - - -

- E I L RL R| W RKCRYS 235 MN A E G M L S Y S D R Y SEM KR V LKS FMGS - - - - - - - - IE F S(A T V E K VNQ A M C K

PAD 575 G Y P L G R I H K P D P W L S T F L S Q Y R E V H H I E Q R T I S K E E I L E R C L Y S L I N E A F R I LlEAD 300 G ]G F Y K Y -|KIDD 274 GKGFYDYS!JCRYS 287 jPADPE L A A R R E W R D E C L K R E L A K L K R Q M Q P Q

PHAD 635 E E G M A A R P E H I D V I Y L H G Y G W P R H K G G P M F Y A A S V G L P T V L E K L QK Y Y R Q N P D I P Q L E P S

PHAD 695 D Y L R R L V A Q G S P P L K E W Q S L A G P H G S K L

FIG. 3. Comparison of the polypeptide sequences of C. acetobutylicum BHBD, pig heart muscle MHAD, the HAD part of the ratperoxisomal bifunctional enzyme (PHAD), and rabbit lens X-crystallin (CRYS). Identical amino acid residues are boxed. Symbols: *,N-terminal end of exon VII of the rat bifunctional enzyme; -, gaps introduced in the polypeptide sequences to optimize alignment.

thereby facilitating the biosynthesis of high levels of en-zymes utilizing these codons (14). It is interesting thatdespite the utilization of a high percentage of E. coli rarecodons, high levels of enzymes were produced by these fivegenes cloned in E. coli (27, 54, 55; Zappe et al., in prepara-tion; Youngleson et al., in press).BHBD amino acid sequence similarity. The deduced amino

acid sequence of the C. acetobutylicum hbd gene wascompared with the sequence of the unifunctional MHADenzyme (7) and part of the sequence of the bifunctionalenzyme that extended from amino acid residues 301 to 583(26, 41) (Fig. 3). The best alignment of the C. acetobutylicumBHBD sequence with that of the other two related proteinsshowed the following percentage similarities with respect toidentical amino acids or identical and conservatively relatedresidues (in parentheses): MHAD, 45.9% (66.5%); and HADpart of the bifunctional enzyme, 38.4% (55.2%). The simi-larity between the three enzymes extended over the entirelength of the BHBD polypeptide, although several regionscontaining more highly conserved blocks of amino acidscould be identified (Fig. 3). A six-residue block with the

sequence DLVVEA from positions 82 to 87 in the C.acetobutylicum BHBD was found to be conserved withoutany changes between all three enzymes. The significance ofthis highly conserved block of amino acid residues is not yetknown.There are three Cys residues in the C. acetobutylicum

BHBD, at positions 81, 106, and 237 (Fig. 3). Only one Cysresidue occurs in the pig MHAD, at position 204. The C.acetobutylicum BHBD contains two His residues, at posi-tions 137 and 221. Only His-137 is conserved in BHBD,MHAD, and the bifunctional enzyme. Because of their smallsize, Gly residues are frequently conserved in dehydroge-nases and other enzymes at positions where space con-straints preclude alternative residues (29). Of the 24 Glyresidues in the C. acetobutylicum BHBD, 14 are conservedbetween these three polypeptides (Fig. 3).The bifunctional enzyme contains an additional 295 and

139 amino acid residues at the N- and C-terminal ends,respectively. These extended regions of the bifunctionalenzyme have been shown by amino acid similarity withmitochondrial enoyl-CoA hydratase to correspond to the

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BHBD HOMOLOGIES 6805

TABLE 2. Alignment of a putative NAD-binding site from the C. acetobutylicum BHBD polypeptidewith that of seven dinucleotide-binding enzymes

Proteina Nucleotide A Secondary structurebcoenzyme PA B O

A * * * * * * * * 0LADH NAD 194 T C A V F G L G G V G L S V I M G C K A A G A - A R I I G V D I N 225PLDH NAD 22 K I T V V G V G A V G M A D A I S V L M K D L A D E V A L V D V M 54LGPD NAD 2 K I G I N G F G R I G R L V L R A A L S R G A - Q V V A V N D L T 33HGLR FAD 22 D Y L V I G G G S G G L A S A R R A A E L G A - - R A A V V E S H 52PHBH FAD 4 Q V A I I G A G P S G L L L G Q L L H K A G I - - D N V I L E R Q 34PHAD NAD 298 S V G V L G L G T M G R G I A I S F A R V G I - - S V V A V E S D 328MHAD NAD 17 H V T V I G G G L M G A G I A Q V A A A T G H - - T V V L V D Q T 47BHBD NAD 3 K I F V L G A G T M G A G I V Q A F A Q K G Y - - E V I V R D I K 33

a Abbreviations not given in text: LADH, Horse liver alcohol dehydrogenase; PLDH, pig lactate dehydrogenase; LGPD, lobster glyceraldehyde 3-phosphatedehydrogenase; HGLR, human erythocyte glutathione reductase; PHBH, p-hydroxybenzoate hydroxylase of Pseudomonasfluorescens; PHAD, rat peroxisomalbifunctional enzyme.

b Gaps in the sequence were allowed for alignment. The amino acid residues in bold type indicate direct homologies with the BHBD enzyme. Numbers indicatethe extent of amino acid residue positions used for the comparison. Sequence data and alignment of LADH, PLDH, LGPD, HGLR, and PHBH are from Wierengaand Hol (52). Symbols: *, conserved glycine; *, neutral or hydrophobic groups forming the hydrophobic core of the ,B-a-BP unit; A, the invariant negative chargeinvolved in hydrogen bonding to a ribose hydroxyl group; 0, invariant hydrophilic residue.

c FAD, Flavin adenine dinucleotide.

equivalent function in the bifunctional enzyme (N. Ishii etal., unpublished results). It is interesting that amino acidresidues 303 to 722, encoding the HAD part of the bifunc-tional enzyme, are derived from a single unusually long exon(exon VII) of 1,259 bp (26). The average length of exonsencoding proteins is approximately 140 bp (38), and exons of>300 bp are rare (24). The fact that the HAD part of thebifunctional enzyme is encoded by a single unusually longexon of 1,259 bp suggests that this exon may have beenacquired as a single unit.No significant amino acid homology (10%) was detected

between the C. acetobutylicum BHBD and the peroxisomaltrifunctional enzyme from Candida tropicalis (39).An unusual protein, X-crystallin, has recently been iso-

lated from rabbit lens (37). DNA sequence of cDNA clonesencoding the 35-kilodalton rabbit X-crystallin protein re-

vealed 30% amino acid similarity with MHAD and 26%similarity with the HAD part of the bifunctional enzyme

(Fig. 3). Low levels of non-lens expression and the presence

of a putative P-a-,B nucleotide-binding fold (45) have ledMulders et al. (37) to speculate that X-crystallin, or a highlyrelated sequence in non-lens tissue, may have an enzymaticfunction. The rabbit X-crystallin showed only 24% aminoacid similarity with the C. acetobutylicum BHBD, indicatinga distant phylogenetic relationship between the structuralX-crystallin protein and the bacterial enzyme.

Secondary structure analysis of C. acetobutylicum BHBD.Dehydrogenases are characterized by two major domainsinvolved with either coenzyme binding or substrate speci-ficity (16, 45). A putative dinucleotide-binding site at theN-terminal end of the C. acetobutylicum BHBD showedconsiderable amino acid similarity with the dinucleotide-binding sites of other NAD-dependent enzymes (Table 2)(46, 52).The best amino acid similarity over the nucleotide-binding

protein fragment exists between BHBD and the two HADenzymes, each with 13 identical residues out of 31 positions.Of the remaining sequences compared in Table 2, pig lactatedehydrogenase and lobster glyceraldehyde 3-phosphate de-hydrogenase both had 10 residues of 31 positions identical tothe BHBD sequence, supporting a general conservation ofthe ,-a-, supersecondary structure of the NAD-binding site(50).A theoretical prediction of the secondary structure of the

BHBD from C. acetobutylicum was made by using thecomputer programs of Genepro (10) and Microgenie (18) andthe GGBSM program (19) (Fig. 4). Predicted secondarystructure similarity was observed between the C. acetobu-tylicum BHBD and the pig MHAD for which the tertiarystructure has been determined (6). Although similarity withthe HAD part of the rat peroxisomal bifunctional enzymewas also observed, the primary amino acid sequence for thisenzyme includes approximately 400 residues responsible forthe enoyl-CoA hydratase activity, and therefore this enzymemay have a tertiary structure different from that reported forthe unifunctional MHAD (6). The C. acetobutylicum BHBDshowed a preference to form a-helical structures, averaging46%, and may therefore be classed as an a-helix-rich protein(18). The C. acetobutylicum BHBD showed 22% predictedP-pleated sheets, 7% ,1 turns, and 25% random coil struc-tures. Hydropathy profiles (31) also showed close similaritybetween the C. acetobutylicum BHBD and the pig MHAD(data not shown).

Phylogenetic relationship between HADs. Comparison ofpolypeptide sequences may be used to estimate the evolu-tionary distance between enzymes belonging to the samefunctional class. Fitch and Margoliash (17) have defined themutation distance between two proteins as the minimalnumber of nucleotides that would need to be altered in orderfor the gene for one protein to code for the other, and it isdetermined by making a pairwise comparison of homologousamino acids. Evolutionary relationships among the pigMHAD, the HAD part of the rat bifunctional enzyme, andthe C. acetobutylicum BHBD were estimated by usingFelsenstein's PHYLIP (Phylogeny Inference Package) pro-gram (version 2.7). The calculated percentage sequencedivergence between MHAD and the HAD part of the bifunc-tional enzyme was 66%, and that between BHBD andMHAD or the HAD part of the bifunctional enzyme was 54or 64%, respectively.

DISCUSSION

The amino acid sequences of the BHBD enzyme from thecentral pathway for the synthesis of butyrate and butanol inthe gram-positive anaerobic bacterium C. acetobutylicumwere compared with the sequences of the pig MHAD and theHAD part of the rat bifunctional enzyme, which form part of

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60 60 100fHAD

, PG GG G 2n

GG G G G

PHIAD282O0000

BHBD

PHAD

G G G20IMHAD = _-

G G G C -

BHBD _G G G

PHAD Atv\ AA

G gGG0000000 -\-307

G GG262

C G GG000~~~~

FIG. 4. Comparison of the predicted secondary structures (algorithm of Gascuel and Golmard [19]) of pig MHAD, C. acetobutylicumBHBD, and the HAD part of the rat bifunctional enzyme (PHAD). The pig MHAD is numbered at every 20 amino acid residues; dots arepositioned at every 20 residues on the MHAD, BHBD, and PHAD structures. The conserved Gly and His residues are indicted to assist inalignment of the three polypeptides. On the basis of the amino acid sequence alignment given in Fig. 3, gaps have been introduced to allowfor insertions or deletions in the polypeptide sequences. Symbols for secondary structures: QQu , a helix; , P-pleated sheet; - , coil.

the fatty acid P-oxidation pathways of vertebrate mitochon-dria and peroxisomes, respectively. Fatty acid p oxidationwas thought to occur only in the mitochondrial matrix inanimal tissues until the discovery of a p-oxidation system inrat liver peroxisomes (32). The peroxisomal P-oxidationenzymes differ from those of the mitochondrial 13-oxidationsystem with respect to their molecular and catalytic proper-ties (23, 32). The similarity between the clostridial fermen-tation and vertebrate fatty acid p-oxidation pathways andenzymes was noted previously (4, 51). Amino acid sequencecomparisons between the BHBD enzyme of the butyrate andbutanol biosynthetic pathway of C. acetobutylicum and theequivalent mitochondrial and peroxisomal fatty acid P-oxi-dation enzymes shows the relatively close relationship be-tween the bacterial BHBD and the vertebrate MHAD andthe HAD part of the bifunctional enzyme. The phylogeneticrelationship between these enzymes supports a commonevolutionary origin for the fatty acid p-oxidation pathway ofvertebrate mitochondria and peroxisomes and the bacterialacidogenic fermentation pathway used to synthesize bu-tyrate and butanol and provides evidence for an independentevolutionary origin for both of these eucaryotic fatty acidp-oxidation pathways.The homology between the C. acetobutylicum BHBD

enzyme and the equivalent mitochondrial and peroxisomalfatty acid p-oxidation enzymes has implications for thehypothesis postulating an endosymbiotic origin for a groupof eucaryotic organelles that includes peroxisomes, glyoxy-somes, and glycosomes (microbodies) (8, 15, 33, 40). Thissuggestion is based on the observation that microbodiescannot be formed de novo but arise from existing organellesby growth and division in the same way as do mitochondriaand chloroplasts (8, 33). The hypothesis postulating anendosymbiotic origin for mitochondria and chloroplasts hasgained general acceptance in recent years. Similarity be-tween genes and gene products of mitochondria and chloro-plasts with those of procaryotes has provided evidence forthe endosymbiotic hypothesis (21). Similarity studies onmicrobody fatty acid p-oxidation constituents with theequivalent bacterial enzymes have not been done.Microbodies differ in a number of major respects from

mitochondria and chloroplasts in that they are bound by asingle membrane, do not contain DNA, and are unable tosynthesize proteins (8, 32). Microbody proteins are made onfree polysomes and are translocated into the microbody after

synthesis. It has been proposed that the single membrane ofmicrobodies indicates that these organeiles were derivedfrom procaryotes that have a single membrane (gram posi-tive like), whereas mitochondria and chloroplasts were de-rived from procaryotes that possess a double membrane(gram negative like) (9).

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