Molecular Cloning, Nucleotide Sequencing, and Expression of Genes Encoding Alcohol Dehydrogenases From the ThermophileThermoanaerobacter brockiiand the MesophileClostridium beijerinckii

Anaerobe (1997) 3, 259–270

PHYSIOLOGY/STRUCTURAL BIOLOGY/BIOCHEMISTRY

Molecular Cloning, Nucleotide Sequencing, and Expression of Genes Encoding Alcohol Dehydrogenases From the Thermophile Thermoanaerobacter brockii and the Mesophile Clostridium beijerinckii Moshe Peretz1, Oren Bogin1, Shoshana Tel-Or1, Aliza Cohen2, Guangshan Li3, Jiann-Shin Chen3 and Yigal Burstein1 1

Department of Organic Chemistry, The Weizmann Institute of Science, 76100 Rehovot, Israel; 2Kimron Veterinary Institute, Beit Dagan, Israel; 3Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0308, U.S.A. (Received 20 December 1996, accepted in revised form 27 March 1997) Key Words: alcohol dehydrogenase, DNA sequence, overexpression, thermal stability, thermophiles

Proteins play a pivotal role in thermophily bacteria. Comparing the molecular properties of homologous proteins from thermophilic and mesophilic bacteria is important for understanding the mechanisms of microbial adaptation to extreme environments. The thermophile Thermoanaerobacter (Thermoanaerobium) brockii and the mesophile Clostridium beijerinckii contain an NADP(H)-linked, zinc-containing secondary alcohol dehydrogenase (TBADH and CBADH) showing a similarly broad substrate range. The structural genes encoding the TBADH and the CBADH were cloned, sequenced, and highly expressed in Escherichia coli. The coding sequences of the TB adh and the CB adh genes are, respectively, 1056 and 1053 nucleotides long. The TB adh gene encoded an amino acid sequence identical to that of the purified TBADH. Alignment of the deduced amino acid sequences of the TB and CB adh genes showed a 76% identity and a 86% similarity, and the two genes had a similar preference for codons with A or T in the third position. Multiple sequence alignment of ADHs from different sources revealed that two (Cys-46 and His-67) of the three ligands for the catalytic Zn atom of the horse-liver ADH are preserved in TBADH and CBADH. Both the TBADH and CBADH were homotetramers. The substrate specificities and thermostabilities of the TBADH and CBADH expressed in E. coli were identical to those of the enzymes isolated from T. brockii and C. beijerinckii, respectively. A comparison of the amino acid composition of the two ADHs suggests that the presence of eight additional proline residues in TBADH than in CBADH and the exchange of hydrophilic and large hydrophobic residues in CBADH for the small hydrophobic amino acids Pro, Ala, and Val in TBADH might contribute to the higher thermostability of the T. brockii enzyme. © 1997 Academic Press

Address correspondence to: Y. Burstein, Department of Organic Chemistry, The Weizmann Institute of Science, 76100 Rehovot, Israel. E-mail: [email protected].

1075-9964/97/040259 + 12 $25.00/0/an970083

© 1997 Academic Press

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Introduction The molecular basis for thermophily remains an enigma, but it has been shown that proteins, besides their structural and catalytic roles, may also regulate the stability and topology of DNA in thermophilic bacteria [1]. Elucidation of the mechanisms of thermophily will depend heavily on an understanding of the nature of differences in thermostability of proteins. In this pursuit, studies focused on any individual thermostable protein will be of limited value because a protein may have achieved thermostability via a unique route, and the properties of a select thermostable protein provides at best only a specific set of parameters to explain protein thermostability. On the other hand, analyses of families of universal, oligomeric enzymes with different thermostabilities should allow a more general conclusion on the principles governing thermophily. With the long-term goal of identifying structural parameters that contribute to thermostability in enzymes, we are studying families of closely related proteins from mesophilic and thermophilic microorganisms. Comparing the structure of these proteins could shed light on the principles governing protein thermostability as well as on the mechanisms of microbial adaptation to extreme conditions. Alcohol dehydrogenase (ADH) is a ubiquitous enzyme found in bacteria [2] and in eukaryotes ranging from yeast to man [2–6]. Multiple ADHs are present in these organisms, but it is often difficult to determine the role of a specific ADH. ADH requires either NAD(H) or NADP(H) as a coenzyme and reacts with primary and secondary, linear and branchedchain, aliphatic and aromatic alcohols and with their corresponding aldehydes and ketones. Most ADHs contain zinc at the active site, and the Zn-ADHs are either dimers, usually found in higher plants and mammals, or tetramers, such as those present in yeast and bacteria. The primary-secondary ADH (hereafter referred to as the secondary ADH) of the mesophile Clostridium beijerinckii NRRL B593 is clearly responsible for the production of butanol and 2-propanol [7]. While the role of the secondary ADH of the thermophile Thermoanaerobacter brockii HTD4 is unclear [8], a similar ADH of the thermophile Thermoanaerobacter ethanolicus JW200 has been postulated for ethanol formation [9]. Results from previous independent studies would suggest that the secondary ADHs from T. brockii [10–12] and C. beijerinckii [7,13] are closely related. Both the T. brockii secondary ADH (TBADH) and the C. beijerinckii secondary ADH (CBADH) are medium-chain, zinc-containing enzymes with similarly broad substrate ranges. The two ADHs are NADP(H)-linked, but have a low level of NAD(H)-

linked activity [7,12]. TBADH reversibly catalyses the oxidation of secondary alcohols to the corresponding ketones with a T1/260 min of 93°C (temperature at which 50% of the enzymatic activity is lost after 60 min) [14]. Although C. beijerinckii is classified as a mesophile, CBADH has a T1/260 min of 67°C [7,14]. TBADH was first isolated from T. brockii strain HTD4 [10]. In previous research, we purified TBADH to homogeneity and determined its primary structure [11]. TBADH is a 152-kDa tetramer comprising four identical subunits, each containing 352 amino-acid residues corresponding to a molecular weight of 37652. CBADH was purified to homogeneity from C. beijerinckii strain NRRL B593 and chemically characterized as an oligomeric protein comprising 38-kDa subunits [7]. The quaternary structure of CBADH was not determined. Both enzymes have been crystallized [15,16] and their three-dimensional structures are being determined. It has been argued that mechanisms of thermophilic adaptation may be detectable and the results are least ambiguous when homologous amino acid sequences of one specific protein from phylogenetically and taxonomically closely related organisms are compared [17,18]. The two members of an enzyme family being compared for this purpose should not only have a high degree of amino-acid sequence identity but also similar three-dimensional structures [19]. We believe that CBADH and TBADH are ideal candidates for comparative molecular studies because the enzymic properties of the two ADHs are remarkably similar [7,8,12,20] and because a comparison of the aminoterminal sequences (first 21 residues) of CBADH and TBADH revealed a high degree of sequence identity [7]. As a first step of the molecular characterization of TBADH and CBADH to correlate with their different thermostabilities to specific structural features, we report here the molecular cloning, complete nucleotide sequences, and high-level expression in Escherichia coli of the cloned genes encoding the two ADHs. Preliminary reports on the successful cloning, highlevel expression, sequence analysis, and site-directed mutagenesis of the TB adh gene as well as crystallization of the recombinant TBADH were published [16,21]. A gene encoding a secondary ADH with an amino acid sequence highly similar to those of TBADH and CBADH, but not to the other ADHs, was found in the anaerobic, pathogenic protozoan Entamoeba histolytica [22]. Very recently, the cloning and expression of the gene encoding a secondary ADH from T. ethanolicus 39E were reported [23]. The deduced amino acid sequence of the secondary ADH of T. ethanolicus is also highly similar to those of TBADH and CBADH, suggesting that these four secondary ADHs belong to a distinct family.

Cloning of T. brockii and C. beijerinckii ADH Genes

Materials and Methods Materials Acetone was from Fisher (Fairlawn, N.J.). 2-Propanol and 2-butanol were from BDH (Poole, U.K.). 2-Pentanol was from Fluka (Buchs, Switzerland). DEAEcellulose (DE-52) was from Whatman (Maidstone, U.K.). Red Sepharose-CL-6B was from Pharmacia (Uppsala, Sweden). The dye-binding protein assay kit was from Bio-Rad Laboratories. (Richmond, CA). [γ-32P]ATP and [α-35S]dATP were from Amersham (Amersham, U.K) or DuPont NEN (Boston, MA). Enzymes for DNA cloning, sequencing, and amplification were from Amersham., GIBCO-BRL (Uxbridge, U.K.), New England Biolabs (Beverly, MA), or Promega (Madison, WI). Oligonucleotides for the cloning and sequencing the TB adh gene were synthesized by the WIS Chemical Synthesis Laboratory (Rehovot, Israel).

Bacteria strains, plasmids, and growth conditions Thermoanaerobacter brockii strain HTD4 was maintained and cultured as described by Zeikus et al. [24]. Clostridium beijerinckii strain NRRL B593 was cultured as previously described [25]. Escherichia coli strains were maintained and manipulated using standard techniques [26]. Plasmid selection was generally performed on Luria–Bertani medium supplemented with ampicillin (100 µg/mL).

Cloning of the T. brockii adh gene After electrophoretic fractionation on 1% agarose gels, the restriction fragments of the genomic DNA of T. brockii were transferred to a GeneScreen membrane and probed by a mixture of three 32P-labeled oligonucleotides by the method of Southern [27]. The three degenerate oligonucleotide probes (18, 29, and 20 bases each) corresponded to the coding sequences for, respectively, amino acid residues 1–6 (N-terminal region), 123–132 (middle section), and 337–343 (C-terminal region) of the TBADH polypeptide [11]. The TB adh gene was located on an EcoR1 fragment (2700 bp), and the fragment was first cloned into the EcoR1 site of pBluescript II. XbaI digestion of a positive clone produced a smaller (1673 bp) DNA fragment containing the entire TB adh gene, which was ligated to XbaI-digested pBluescript II to form the plasmid pBS-M105/2. The insert was composed of the DNA encoding the 352 amino acid residues of TBADH and flanking regions of 249 nucleotides upstream of the initiation codon and 342 nucleotides downstream of

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(and including) the termination codon. pBS-p89TBADH was a deletion mutant in which the upstream region was limited to 89 bases preceding the initiation codon for the TB adh gene, and the shortened fragment was cloned into the SacI-XbaI sites of pBluescript II SK( + ) (with the adh gene in the same orientation as the lacZ gene). The ligation mixture was used to transform E. coli JM109 or TG1 cells, and transformants were scored on Luria–Bertani agar containing ampicillin (100 µg/mL), IPTG (isopropylD-thiogalactopyranoside, 40 µg/mL), and x-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 20 µg/mL). A PCR-generated DNA fragment encompassing the coding region of the TB adh gene was also cloned into the pET-11a expression vector. To adapt the N-terminus-encoding sequence to the vector, the forward primer (28 bases long) corresponded to the coding sequence for amino acid residues 1 through 8 of TBADH and had an NdeI site added to the 5' end. The reverse primer (29 bases long) corresponded to the stop codon and the next 20 bases of the complementary strand, and a BamH1 site was added to the 3' end. The 1078-nucleotide NdeI-BamH1 fragment was cloned into the vector pET-11a to form the plasmid pET-11aTBADH. This recombinant plasmid was used to transform E. coli BL21(DE3) cells, and the TBADH expressed in E. coli was isolated and crystallized as previously reported [16].

Cloning of the C. beijerinckii adh gene High molecular weight DNA was isolated from C. beijerinckii NRRL B593 using a modified Marmur procedure [28]. Based on the N-terminal amino acid sequence MKGFAMLGINKLGWIEKERP determined from the purified enzyme, two oligonucleotide probes were designed for detecting the adh gene on restriction fragments of C. beijerinckii DNA. The two probes were a 17-mer (32-fold degeneracy) corresponding to the coding strand for FAMLGI and a 20-mer (24-fold degeneracy) corresponding to the non-coding strand for WIEKERP. These degenerate probes were inadequate for the detection of the adh gene. However, they were useful as PCR primers for the amplification of the intervening DNA sequence. The PCR product was cloned into the SmaI site of pUC19, and DNA sequence analysis confirmed that the 50-bp insert encoded the expected amino acid sequence of CBADH. The 50-bp fragment was a useful probe and detected a single band (size in parentheses) with C. beijerinckii DNA digested with each of the following restriction enzymes: Bgl II ( ~ 1.1 kb), HindIII ( ~ 1.8 kb), and EcoR1 ( ~ 2.5 kb). The 2.5-kb adh-containing EcoR1 fragment was cloned as follows. Fragments of EcoR1 digested C. beijerinckii DNA

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were ligated onto the lambda gt10 arms (GIBCO BRL) and packaged in vitro [26]. Escherichia coli strain C600hfl (GIBCO BRL) was infected with the lambda phage, and recombinant plaques were screened for the presence of the adh gene. Five positive clones were identified by using the 32P-labeled 50-bp fragment as a probe, and the size of the insert in these clones was about 2.5 kb. Because three of the recombinant lambda phages were unstable during propagation, the inserts in the original clones were preserved by PCR, using the lambda gt10 forward and reverse primers (GIBCO BRL), to allow further studies. The DraI and Sau3A fragments of a PCR-amplified insert as well as additional fragments from genomic DNA were subcloned into M13mp19 for DNA sequence analysis. After the nucleotide sequence of the adh gene on the 2.5-kb EcoR1 fragment was determined, the adh gene was cloned again as a smaller piece of DNA. The coding region of the adh gene and its 5' intergenic region were amplified from genomic DNA by PCR, with restriction sites introduced to the ends as needed. The PCR product was cloned into either pUC or pBluescript vectors. pGL89 (in pUC18) and pGL99 (in pUC19) had the insert cloned between the BamH1 and the EcoR1 sites. pBS-P200CBADH (in pBluescript II) had the insert cloned into the Sma I site. The inserts in pGL99 and pBS-P200CBADH were sequenced to confirm the preliminary sequence determined from the insert in the lambda vector.

NADPH per min under initial velocity conditions. Measurement of kinetic parameters was performed with a Beckman DU-7500 spectrophotometer equipped with multicomponent/SCA/kinetics software package. The kinetics parameters, Km and Vmax were calculated from initial velocities of the enzymatic reactions performed with various concentrations of NADP (20-100 µM) and C3 to C5 secondary alcohols (1.0–10 mM). Secondary ADH activities in E. coli DH5α cells transformed with pGL89 (pUC18 containing CB adh) or pGL99 (pUC19 containing CB adh) were measured by two methods: (1) to measure activities in whole cells, acetone or 2-propanol (50 to 100 mM) was added to 14–20-h old cultures (5–50 mL) of transformed E. coli cells grown at 37°C in the Luria–Bertani medium containing 100 µg/mL ampicillin, with or without IPTG (0.4 mM). After an incubation period up to 3 h, culture supernatant (1 µL) was assayed for 2-propanol and acetone by gas chromatography [30]; (2) and the ADH activity in cell-free extracts was measured in a reaction mixture (1 mL) containing NADPH (0.2 mM), acetone (6.7 mM), Tris-HCl (50 mM, pH 7.5), and cellfree extracts. Cells were broken by passing through a French pressure cell at 16000 psi and cell debris was removed by centrifugation at 16 000 3 g for 20 min at 4°C. Escherichia coli DH5α transformed with pUC18 served as a control.

Purification of recombinant TBADH and CBADH DNA sequencing The sequence of cloned DNA was determined by the Sanger chain-termination method [29] using a Sequenase (T7 DNA polymerase) kit and with either singlestranded (from M13) or denatured double-stranded (from pBluescript) templates. Both strands of overlapping restriction fragments of the TB and CB adh genes were sequenced by the ladder-climbing method with synthetic oligonucleotide primers (Figure 1). Manual sequencing results were verified by automated sequencing with an Applied Biosystems Inc. Model 373A or a DuPont Genesis 2000 DNA Sequencer.

Escherichia coli TG1 cells carrying the recombinant plasmids pBS-P89TBADH or pBS-P200CBADH were grown aerobicaly for 16 h at 37°C in the Luria–Bertani medium containing ampicillin (100 µg/mL). Approximately 10 g of cells were collected from 2.5 L by

(a) X

N

C

A

X

(b)

Enzymatic assay and determination of kinetic parameters of recombinant TBADH and CBADH ADH activity was routinely measured by following the absorbance increase at 340 nm (e340 = 6.2 mM/cm) for the formation of NADPH from NADP in a reaction mixture containing NADP (0.5 mM), 2-butanol (150 mM), and Tris-HCl (100 mM, pH 8.0) at 40°C. One unit of ADH activity is defined as the amount of enzyme that catalyses the formation of 1 µmol of

B

H

Figure 1. Restriction map and sequence strategy for the (a) T. brockii and (b) C. beijerinckii secondary adh genes and their flanking regions. The open boxes represent coding regions. The horizontal arrows indicate the direction and the extent of individual sequencing runs. The vertical arrow heads mark the restriction sites: X, XbaI; N, NarI; C, AccI; A, AatII; B, BglII; H, HincII.

Cloning of T. brockii and C. beijerinckii ADH Genes centrifugation (7000 3 g for 10 min at 4°C) and resuspended in 100 mL of 25 mM Tris-HCl (pH 7.3) containing 0.1 mM DTT, 0.1 mM EDTA, 0.02% sodium azide and 0.1 M NaCl (Buffer A) plus 0.1 mM PMSF, 10 µM leupeptin and 0.5 mM benzamidine. The suspended cells were disrupted by sonication at 4°C, and cell debris was removed by ultracentrifugation (100 000 3 g for 20 min at 4°C). The supernatant was exposed to heat (70°C for 5 min) and then recentrifuged at 30 000 3 g for 10 min. The clear supernatant was passed through a DE-52 column (3 3 10 cm), with the effluent flowing directly onto a Red Sepharose CL-6B column (3 3 30 cm). The Red Sepharose column was first washed with 150 mL of Buffer A and then eluted at 4°C using a linear gradient of 600 mL of NaCl (0.1-0.8 M for TBADH and 0.5–1.5 M for CBADH) in Buffer A at a flow rate of 1 mL/min. The column effluent was monitored for ADH activity. The active fractions (8 mL each) were combined, and the purity of the preparation was assessed by SDSPAGE.

Analytical procedures Protein concentrations were determined by the dyebinding method of Bradford [31] with bovine immunoglobulin G as a standard or by monitoring the molar absorption of the proteins at 280 nm (TBADH: e280 = 33/mM/cm; CBADH: e280 = 44/mM/cm). To determine the amino acid composition of TBADH and CBADH, proteins were hydrolysed for 24 h in 6 M HCl at 110°C in a Waters Pico-Tag station and analysed with a Dionex D-400 amino acid analyser. The purity of proteins was assessed by SDS-PAGE performed according to Laemmli [32], using a 12% slab gel and a 5% stacking gel. The gels were stained with coomassie brilliant blue.

Results and Discussion Nucleotide sequences and the deduced amino acid sequences The entire TB adh gene was located on a 1673-bp Xba I fragment subcloned from the 2.7-kbp EcoR1 fragment. The nucleotide sequence of the Xba I fragment is shown in Figure 2. The 1056-bp open-reading frame encoded a 352-amino acid polypeptide which was identical to the amino acid sequence determined from the purified TBADH [11]. The 2.5-kbp EcoR1 fragment containing the CB adh gene was manually sequenced after subcloning into M13mp19. The CB adh gene was identified as a

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1053-bp open-reading frame on the 2557-bp EcoR1 fragment. The adh gene codes for a polypeptide of 351 amino acid residues, and the first 20 amino acid residues had an exact match with the sequence determined from the purified CBADH [7]. With a 352-bp intergenic region, the CB adh gene was preceded by a truncated open-reading frame (999 bp cloned), which may code for a polypeptide with an internal 240-amino acid region related to the NtrCtype of transcription activator (data not shown). Investigation on this ntrC-like gene is in progress. The Cb adh gene was subsequently recloned from amplified genomic DNA into pBluescript II SK, pUC18, and pUC19 in separate experiments to give, respectively, pBS-P200CBADH, pGL89, and pGL99. The insert in pBS-P200CBADH was completely sequenced with the strategy shown in Figure 1, and part of the insert in pGL99 was sequenced to confirm the sequence of three codons. The nucleotide sequence of the Hind III-EcoR1 fragment encompassing the CB adh gene was aligned against that of the Xba I fragment of T. brockii (Figure 2). The TBADH and CBADH were highly similar at both the DNA and the protein levels. In the coding region, the nucleotide sequence was 72% identical, with the first 500 nucleotides (76% identity) being more similar than the second 500 nucleotides (67% identity). The deduced amino acid sequence of the T. ethanolicus 39E secondary ADH (TEADH), recently reported by Burdette et al. [23], shows a 99.1% identity and a 99.4% similarity with TBADH. The coding regions of the TB and TE adh genes show a 99.5% identity and differ by only five (out of 1056) bases. Moreover, the 5' (273 bp) and the 3' (68 bp) untranslated regions of the TB and TE adh genes show, respectively, 98.5% and 98.4% identity. This high degree of sequence identity suggests that the three genes are homologous, and this relationship may aid investigation on the propagation and evolution of the adh genes among these anaerobic species. The phylogenetic relationship between these three species has been studied [33,34]. Structural homology between TBADH and CBADH is discussed in a later section. A possible ribosome-binding site (AGGAGG) was present in the –11 to –16 region of both the TB adh and the CB adh coding regions. However, putative promoter elements were identified further upstream in the –85 and –108 regions (relative to the start codon) of the TB adh gene but not the CB adh gene. The nucleotide sequences in the 5' upstream region showed little similarity between the two genes, which is not surprising because the TBADH appears constitutively expressed [35], whereas expression of the CBADH is subject to the solventogenic switch [30]. This is consistent with the observation that the CBADH was expressed from the cloned gene on pUC19 (as pGL99 in E. coli DH5α) but not on pUC18

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Cloning of T. brockii and C. beijerinckii ADH Genes (as pGL89 in E. coli DH5α): Secondary ADH activities were 27 and 0.8 µmol/min/mg, respectively, in cellfree extracts of E. coli DH5α (pGL99) grown in the presence and absence of IPTG, whereas secondary ADH activities were not detected in extracts of E. coli DH5α(pGL89) or E. coli DH5α(pUC18). Secondary ADH activities measured in whole cells of E. coli transformants (data not shown) paralleled the activities present in cell-free extracts. The results indicate that transcription did not start from the clostridial DNA in E. coli. However, transcription of the TB adh gene appears to have started from the thermoanaerobacter DNA in E. coli (data not shown). It is interesting to note that the nucleotide sequence of the CB adh gene on pBS-P200CBADH, as shown in Figure 2, differed in three places from the sequence of the amplified insert from the lambda gt10 clone. In Figure 2, codons 154, 234, and 295 were, respectively, ACT (Thr), AAA (Lys), and TGT (Cys), whereas the corresponding positions were TCT (Ser), GAA (Glu), and TGC (Cys) in the lambda gt10 clone. To determine which sequence is the correct one, the pertinent regions in pGL99 were sequenced, and the results agreed with that shown in Figure 2. To further investigate the problem, a 0.9 kbp BglII-EcoR1 fragment that covers the regions in question was cloned from genomic DNA, without involving PCR, in the LITMUS-29 vector (New England Biolabs). The singlestranded templates from four independent clones Figure 2. Nucleotide and deduced amino acid sequences of the T. brockii (TB) and C. beijerinckii (CB) adh genes. The nucleotide sequence of the TB adh gene is given above that of the CB adh gene. Identical nucleotides are connected by a short vertical line. The deduced amino acid residue is shown in the single-letter code above (TBADH) or below (CBADH) the first nucleotide of each codon. Every tenth amino acid residue is numbered. For CBADH, only amino acid residues that differ from those of TBADH are shown. A possible ribosome-binding site, putative promoter elements (see text), the termination codon, and a long palindromic sequence centered at nucleotide 1071 of the TB adh gene are indicated.

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were sequenced, and the results also agreed with that shown in Figure 2. Therefore, we believe that the sequence of the CB adh gene reported here is that present in the genome, but it remains a puzzle as to how the three base changes observed in the lambda gt10 clone arose. While the differences in codons 154 (Thr vs. Ser) and 295 (same amino acid) may be inconsequential, the change in codon 234 (Lys vs. Glu) may affect the property of the ADH. Further study is needed to show how the enzymic properties of ADH with Glu-234 or Lys-234 compare with those of the characterized enzyme from C. beijerinckii NRRL B593.

Codon usage The codon usage pattern for the adh genes from T. brockii and C. beijerinckii is shown in Table 1. The codon usage for the T. ethanolicus adh gene [23] is essentially identical to that of TBADH. For comparison, we also calculated from the published sequences the codon usage for the adh genes from Alcaligenes eutrophus [36] and Entamoeba histolytica [37], whose amino acid sequences are related to those of the TBADH and CBADH (Figure 3). The codon-usage pattern for the T. brockii adh gene seems less biased than those of the other three adh genes. Of the 61 sense codons, only four were not used at all in the TB adh gene, whereas nine, 13, and 21 were not used, respectively in the C. beijerinckii, A. eutrophus, and E. histolytica adh genes. However, when the more frequently used codons (codons used more than ten times or about 3%) for each gene were counted, a different picture emerged. The 13 or 14 most frequently used codons accounted for 59–64% of the amino acid residues of the ADHs of T. brockii, C. beijerinckii, and A. eutrophus, except for the E. histolytica adh gene, in which the 15 most frequently

Table 1. Codon usage of sADHs of T. brockii (Tb), C. beijerinckii (Cb), A. eutrophus (Ae) and E. histolytica (Eh) Tb

Cb

Ae

Eh

TTT-Phe TTC-Phe TTA-Leu TTG-Leu

13 1 3 5

10 1 15 2

1 8 0 0

4 4 13 1

TCT-Ser TCC-Ser TCA-Ser TCG-Ser

CTT-Leu CTC-Leu CTA-Leu CTG-Leu

3 3 5 4

4 1 5 1

0 4 1 25

ATT-Ile 16 ATC-Ile 6 ATA-Ile 4 ATG-Met 15

13 1 12 18

GTT-Val GTC-Val GTA-Val GTG-Val

19 0 13 3

16 4 14 3

Tb

Cb

Ae

Eh

Tb

Cb

Ae

Eh

2 1 0 2

5 1 5 0

0 3 1 5

1 0 11 1

17 0 0 0

CCT-Pro 13 CCC-Pro 2 CCA-Pro 6 CCG-Pro 0

4 0 7 2

1 8 1 6

0 0 15 0

2 26 0 10

26 3 0 12

ACT-Thr ACC-Thr ACA-Thr ACG-Thr

6 5 1 1

5 0 6 1

0 19 0 5

1 7 0 20

23 4 4 0

GCT-Ala 17 GCC-Ala 7 GCA-Ala 10 GCG-Ala 1

10 0 14 3

1 18 4 18

Tb

Cb

Ae

Eh

TAT-Tyr TAC-Tyr TAA-*** TAG-***

5 1 1 0

7 0 1 0

1 8 0 0

5 0 1 0

TGT-Cys TGC-Cys TGA-*** TGC-Trp

1 3 0 4

5 0 0 4

0 9 1 1

5 0 0 5

CAT-His CAC-His CAA-Gln CAG-Gln

5 5 1 2

8 3 5 1

1 7 0 12

14 1 4 0

CGT-Arg CGC-Arg CGA-Arg CGG-Arg

4 1 0 2

4 2 0 0

1 10 0 6

0 0 0 0

6 2 7 0

AAT-Asn 8 AAC-Asn 2 AAA-Lys 19 AAG-Lys 5

12 1 16 6

2 11 1 13

10 1 21 4

AGT-Ser AGC-Ser AGA-Arg AGG-Arg

4 0 7 0

4 1 5 3

0 5 0 1

1 0 13 0

5 1 20 1

GAT-Asp 14 GAC-Asp 6 GAA-Glu 18 GAG-Glu 3

16 5 15 2

5 19 6 9

25 2 19 0

GGT-Gly GGC-Gly GGA-Gly GGG-Gly

19 12 11 1

15 4 23 3

2 34 2 5

6 1 40 0

The data of A. eutrophus and E. histolytica are calculated from the nucleotide sequences in [36] and [37], respectively.

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used codons accounted for 78% of the amino acid residues. Thus, the codon usage pattern is biased in these adh genes, and these 13 to 15 frequently used codons may reflect the abundant tRNA species in these four organisms. The DNA base composition of these four organisms spans a wide range, with the mol% G + C values of E. histolytica (22–27% [38]), C. beijerinckii (26–28% [39]), and T. brockii (30–31% [24]) near the minimum and that of A. eutrophus (66–68% [40]) near the maximum for all organisms. The G + C value of the coding

region of the adh genes may be poorly related to the G + C value of the genome because the first and second positions of the codons are interrelated to the amino acid composition of each protein. Proteins having a similar amino acid composition must have a similar G + C value for the first and second positions of the codons, which tends to dampen any asymmetry in codon usage based on the third position of the codon. Thus, the mol% G + C values for the coding region were 44, 38, 36, and 68% for the adh genes of T. brockii, C. beijerinckii, E. histolytica, and A. eutrophus,

Figure 3. Alignment of amino acid sequences of ADHs. HLADH is the horse liver ADH, EE enzyme [57]; AEADH is the ADH of Alcaligenes eutrophus [36], and EHADH is the ADH-1 of Entamoeba histolytica [37]. Gaps were introduced to optimize alignment between the sequences. Amino acid residues are numbered above the HLADH and below the TBADH sequences. Identical amino acid residues between TBADH and at least one other ADH are printed in boldface. #, ligands of catalytic Zn of HLADH [44]; *, strictly conserved residues in different ADHs [5], ‡, strictly conserved Asp in NAD-linked ADHs or conserved Gly in NADP-linked ADHs.

Cloning of T. brockii and C. beijerinckii ADH Genes respectively, where only the AE adh gene had a G + C content similar to that of the genome. On the other hand, the choice of alternative codons in the adh genes is expected to reflect the DNA base composition (and the tRNA population) of these organisms if these enzymes are abundantly expressed proteins. The third base position in the codons of the adh genes of T. brockii, C. beijerinckii, and E. histolytica had a distinctive preference for A or T, showing respective mol% A + T values of 74, 86, and 92%, whereas the A. eutrophus adh gene showed an A + T value of 10% in the third base. Although the G + C content of the T. brockii adh gene was slightly higher than those of the C. beijerinckii and E. histolytica adh genes, the TB adh gene is not expected to have a greater intrinsic thermostability than the CB or EH adh gene. In fact, hyperthermophiles within the genus Pyrococcus have an optimal growth temperature of 100°C, but their genomes have a mol% G + C value of only 38% [41], suggesting that in thermophilic organisms the DNA is not inherently more thermostable but is stabilized by other factors, such as a multitude of DNA-binding proteins [1].

Amino acid sequence alignment of TBADH and CBADH with ADHs from different sources As shown in Figure 3, the amino acid sequence of the ADH from the mesophile C. beijerinckii (37°C optimal growth temperature) had a 76% identity and 86% similarity (allowing for conservative replacements) with the sequence of the ADH from the thermophile T. brockii (76°C optimal growth temperature). Such a high degree of identity is often seen between isozymes but is exceptional for ADHs isolated from different organisms [2]. The data in Figure 3 indicate that the E. histolytica ADH (66% identity and 77% similarity to TBADH) belongs to this closely related group, which lacks a stretch of 18 amino acids (residues 96–113 of HLADH, located in the variable region V2I identified by Danielsson et al. [4] that form a loop and contain the four cysteinyl ligands of the structural Zn of HLADH. Not shown in Figure 3 are the N-terminal amino acid sequences of the NADP-linked secondary ADHs of the two methanogenic archaebacteria Methanobacterium palustre and Methanocorpusculum parvum [42]; the ADHs of these two anaerobes probably also belong to this distinctive group [7]. In contrast, the A. eutrophus and horse liver ADHs show, respectively, only 38% and 27% identities with TBADH, approaching the low values of identity that are common among other microbial group I ADHs. Figure 3 also shows that both CBADH and TBADH retain all nine structurally important amino acid residues (eight glycines and one valine) that are conserved throughout all ADHs [43]. Numbering of residues in Figure 3 is based on

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that of the horse liver ADH [44]. Additional highly conserved amino acids in the stretch of residues 66 to 80 (GHEXXGXXXXXGXXV), namely His-67 and Glu68, and the conserved residues of the microbial ADHs (such as Asp-49 and Ala-211) are also conserved in CBADH and TBADH. Two (Cys-46 and His-67) of the three ligands for the catalytic Zn of HLADH are preserved in all members of this group. That the third ligand (Cys-174) for the catalytic Zn of HLADH may have been replaced by an Asp in TBADH and AEADH [21,11] and in the other ADHs has been suggested (reviewed by Reid and Fewson [2]). Asp-150 could also function as the third ligand for Zn in CBADH. Three Gly residues (Gly-199, Gly-201, and Gly-204) that form the NADP-binding β-α-β fold [45,46] are preserved in both CBADH and TBADH. In both TBADH [11,47] and CBADH (and also in other NADPlinked ADHs), Asp-223 of the horse liver ADH (and all other NAD-linked ADHs) has been replaced by Gly.

Amino acid composition The amino acid composition of homologous proteins from mesophiles and thermophiles presumably reflects the mechanisms of thermal adaptation. Although the general ‘traffic rules of stabilization’, in terms of amino-acid changes accompanying a thermophilic shift, have not yet been deduced [48], several investigators have suggested that certain amino acid exchanges increase the thermostability of proteins [18]. Because the highly homologous TBADH and CBADH differ in their thermostability (the respective T1/260 min values for TBADH and CBADH are 93°C and 67°C [14]), we examined the differences between their amino acid compositions (Table 2). The most striking differences in TBADH relative to CBADH are an increase in TBADH in the number of hydrophobic amino acids Ala and Pro (eight additional residues each), and to a lesser extent Phe and Val ( + 3 and + 2), as well as in the charged amino acid Glu ( + 4). Also noteworthy is the decrease in the number of the hydrophilic amino acids Ser, Asn, and Gln (–7, –3, and –3, respectively) as well as the large hydrophobic amino acids Leu and Met (–5 and –3) in TBADH. Similar trends, such as an increase in Phe, Val, and Glu and discrimination against Asp and Asn, have been reported for the glyceraldehyde-3-phosphate dehydrogenases [18,49]. Other typical trends for glyceraldehyde-3-phosphate dehydrogenases (such as an increase in Ile and Tyr), however, were not observed in the ADHs. Several investigators have reported that proline contributes to thermostability by decreasing the entropy of the unfolded state [50-54]. For example, Delboni et al. [55] claim that a high number of prolines

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contribute to the high thermostability of triosephosphate isomerase from the thermophile B. stearothermophilus and Watanabe et al. [56] suggest the same for oligo-1,6-glycosidases. Thus, the additional eight prolines of TBADH could be related to the ADH’s higher thermostability. Furthermore, the exchange of hydrophilic and large hydrophobic residues in CBADH for the small hydrophobic amino acids Pro, Ala, and Val in TBADH (Table 2) may also contribute to the higher thermostability of TBADH by locking the enzyme in a conformation with a higher density of packing and decreased structural flexibility [18], which could also lead to lower enzymatic activity at lower temperatures. Indeed, the specific activity of CBADH at 40°C is 142 units/mg versus only 63 units/mg protein for the thermophilic TBADH under the same assay conditions (Table 3).

Table 2. Differences in the amino acid compositions of the sADHs of T. brockii and C. beijerinckiia No. of residues in CBADH

TBADH

∆

Aromatic: Phe

11

14

+3

Hydrophobic: Val Leu Met

35 28 18

37 23 15

+2 –5 –3

Small hydrophobic: Ala Pro Gly

27 13 45

35 21 43

+8 +8 –2

Charged: Glu Lys

17 22

21 24

+4 +2

Hydrophilic: Ser Asn Gln

16 13 6

9 10 3

–7 –3 –3

Amino acid

a The

number of the following amino acids Trp (4), Ile (26), and Arg (14) was identical in the two ADHs. The number (with that of CBADH preceding that of TBADH) of the following amino acids differed by only one residue: Tyr (7, 6), Asp (21, 20), His (11, 10), Thr (12, 13), and Cys (5, 4).

Over-expression of TBADH and CBADH in E. coli The cloned TB adh gene on plasmids pBS-M105/2 and pBS-P89TBADH was expressed in E. coli strain TG1. The level of TBADH expressed (under aerobic growth conditions) was about 30% of the total extractable protein (Table 3), which was 30-fold higher than in the native bacterium T. brockii [11]. IPTG induced expression of the cloned adh gene on pET-11aTBADH in E. coli B21 (DE3) cells, yielding similar levels of the enzyme. The cloned CB adh gene on the plasmid pBSP200CBADH was also over-expressed in E. coli TG1 cells, and the level of CBADH expressed was approximately 10% of the total extractable protein, which was 50-fold higher than in the native bacterium C. beijerinckii NRRL B593 [7]. The CBADH was expressed to 1% of the total extractable protein from the cloned adh gene on pUC19 (as pGL99 with the adh gene in the same orientation as the lacZ gene) in E. coli DH5α and the level of expression increased to 40% in the presence of IPTG. In contrast, the TEADH was expressed to 1–5% of the total extractable protein either in the native organism or from the cloned adh gene on pBluescript II KS ( + ) (as pADHB25 with the adh gene in the same orientation as the lacZ gene) in E. coli DH5α, whether or not IPTG was present [23]. The transcriptional start sites for these similar adh genes in E. coli will need to be determined when the promoter regions for these genes are studied in the native organisms. The recombinant TBADH and CBADH were purified from E. coli sonicates using two major steps, a heat treatment and salt-elution affinity chromatography, as summarized in Table 3. The kinetic properties with C3 to C5 substrates and the thermostability of the purified recombinant ADHs were equivalent to those of the enzymes purified from the respective native organisms (data not shown). The TBADH and CBADH purified from E. coli could thus replace the enzymes from the native sources, and the higher level of ADHs produced in E. coli has facilitated preparation of crystals for the determination of the three-dimensional structure of this distinct class of ADH.

Table 3. Purification of cloned TBADH and CBADH from E. coli strain TG1 Step no.

Fraction

Protein (mg)

Activity (units)

Sp. act. (units/mg)

Yield (%)

Purification (×fold)

TBADH 1 2 3

Crude extract Heat treatment Red Sepharose

985 556 315

22655 20570 19825

23 37 63

100 91 87

1 1.6 2.7

CBADH 1 2 3

Crude extract Heat treatment Red Sepharose

640 110 46

8448 7950 6530

13 72 142

100 94 77

1 5.5 11

Cloning of T. brockii and C. beijerinckii ADH Genes Acknowledgement This work was supported by grants from the Joseph and Ceil Mazer Center for Structural Biology (to Y.B.) and from U.S. Department of Energy (grant DE-FG05-85-ER13368 to JSC). JSC has been supported by the Cooperative State Research Service, U.S. Department of Agriculture, under project number 6129960, and thanks M. Rifaat for technical assistance and Katherine Chen and Julianna Toth for assistance with DNA sequence analysis. Y.B. is the Maynard I. and Elaine Wishner Professor for Bioorganic Chemistry. We thank Dr. Virginia Buchner for helpful suggestions during the preparation of this manuscript.

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Appendix Abbreviations ADH, alcohol dehydrogenase; CBADH, C. beijerinckii ADH; HLADH, horse liver ADH; TBADH, T. brockii ADH; IPTG, isopropyl-D-thiogalactopyranoside; X-gal, 5-bromo-4-chloro-3-indolyl-β-D-galactopyrano side. Note. The nucleotide sequence of the T. brockii adh gene has been submitted to the EMBL database and is available under the accession number X64841. The nucleotide sequence of the C. beijerinckii adh gene has been submitted to the Gen Bank database and is available under the accession number M84723.