Dihydrolipoamide dehydrogenase from Haloferax volcanii: gene cloning, complete primary structure, and comparison to other dihydrolipoamide dehydrogenases.

Dihydrolipoamide dehydrogenase from Haloferax volcanii: gene cloning, complete primary structure, and comparison to other dihydrolipoamide dehydrogenases

'

NATARAJ N. VETTAKKORUMAKANKAV AND KENNETH J . STEVENSON

Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by UNIV OF NORTH CAROLINA AT on 11/12/14 For personal use only.

Department of Biological Sciences, University of Calgary, Calgary, Alta., Canada T2N IN4 Received March 29, 1992

VETTAKKORUMAKANKAV, N. N., and STEVENSON, K. J. 1992. Dihydrolipoamide dehydrogenase from Haloferax volcanii: gene cloning, complete primary structure, and comparison to other dihydrolipoamide dehydrogenases. Biochem. Cell Biol. 70: 656-663. We used the N-terminal amino acid sequence of dihydrolipoamide dehydrogenase from Haloferax volcanii, to design and synthesize two oligonucleotide probes that were used to identify and clone a 4.3 kilobase pair (kbp) fragment from MboI restriction endonuclease digestion of Hf. volcanii genomic DNA. The nucleotide sequence of a 1.5-kbp region of this clone was determined and this revealed an open reading frame that translated into a protein with good homology to dihydrolipoamide dehydrogenase from other sources. The first 48 amino acids were identical with the N-terminal sequence data obtained from the purified protein. The complete primary structure of the halophilic dihydrolipoamide dehydrogenase was analyzed in terms of its homologies to dihydrolipoamide dehydrogenases from other sources and its molecular adaptations to high intracellular ionic strength. Key words: Archaea, halophile, flavoenzyme, nucleotide sequence. VETTAKKORUMAKANKAV, N. N., et STEVENSON, K. J. 1992. Dihydrolipoamide dehydrogenase from Haloferax volcanii: gene cloning, complete primary structure, and comparison to other dihydrolipoamide dehydrogenases. Biochem. Cell Biol. 70 : 656-663. Nous avons utilise la sequence N-terminale des acides m i n t s de la dihydrolipoamide dthydrogknase de Haloferax volcanii pour construire et synthetiser deux sondes oligonucltotidiques qui nous ont servi A identifier et cloner un fragment de 4,3 kbp obtenu par digestion du DNA genomique de Hf.volcanii avec I'endonucltase de restriction MboI. Nous avons determine la sequence nucltotidique d'une region de 1.5 kbp de ce clone et cela nous a rev616 un cadre de lecture ouvert qui se traduit en une protkine montrant une forte homologie avec la dihydrolipoamide dtshydrogknase d'autres sources. Les premiers 48 acides amines sont identiques aux donnkes de la sequence N-terminale obtenue de la protkine purifite. La structure primaire complkte de la dihydrolipoamide dCshydrogCnase est analysee en termes de ses homologies avec les dihydrolipoamide deshydrogtnases d'autres sources et de ses adaptations molCculaires A une force ionique intracellulaire Clevte. Mots clPs : Archaea, halophile, flavoenzyme, sequence nucliotidique. [Traduit par la redaction]

Introduction Dihydrolipoamide dehydrogenase catalyses the reaction dihydrolipoamide + NAD -- lipoarnide + NADH + H (reviewed by Williams 1976). This reaction is part of the metabolic process catalysed by pyruvate and other 2-0x0 acid dehydrogenase multienzyme complexes (Perham et al. 1987). It is also an integral component of the glycine cleavage multienzyme complex (Kikuchi and Hiraga 1982; Freudenberg et al. 1989). Recent evidence has also implicated the possible role for dihydrolipoamide dehydrogenase in sugar transport (Richarme and Heine 1986). In the halophilic Archaea (Archaebacteria), pyruvate and other 2-0x0 acids are metabolized by oxidoreductases that employ ferredoxin rather than lipoic acid as the electron acceptor (Kersher and Oesterhelt 1982; Danson 1988). Thus, in the Archaea the 2-0x0 acid dehydrogenase multienzyme complexes are not present, nor is there evidence for the existence of a NAD+-dependent glycine cleavage system (Javor 1988). The finding of dihydrolipoamide dehydrogenase in the halophiles (Danson et al. 1984) and in other members of Archaea (Danson et al. 1991) was, therefore, unexpected

and suggested additional metabolic roles for dihydrolipoamide. To characterize an archaeal dihydrolipoamide dehydrogenase as a first stage in understanding possible roles of this enzyme, DHLipDH was purified to homogeneity from the halophile Halobacterium halobium (Danson et al. 1986). The elucidation of the amino-terminal sequence on this enzyme was unsuccessful owing to the apparent block of the N-terminal amino acid, which yielded no information upon Edman degradation. For this reason and the availability of shuttle vectors (Lam and Doolittle 1989), expression vectors (Nieuwlandt and Daniels 1990), and transformation protocols for another halophilic Archaea (Cline et al. 1989), DHLipDH from Haloferax volcanii was purified to homogeneity and characterized, and the assignment of 48 residues of the N-terminal sequence was obtained (Vettakkorumakankav et al. 1992). We now report the use of two sets of degenerate oligonucleotide probes to identify, clone, and sequence the gene encoding for DHLipDH from Hf. volcanii. This is the first report of the complete primary structure of a DHLipDH from an Archaea.

ABBREVIATIONS: kbp, kilobase pair(s); DHLipDH, dihydrolipoamide dehydrogenase; X-gal, 5-bromo-4-chloro3-indolyl-P-~galactoside;IPTG, isopropylthiogalactoside; TAE, Tris-acetate buffer containing EDTA; SSC, saline sodium citrate; SDS, sodium dodecyl sulfate; LB, Luria-Bertani broth; BrCH,CONHPhAsO, p-[(bromoacetyl)amino]phenyl arsenoxide. ' ~ u t h o rto whom all correspondence should be addressed.

Materials All restriction enzymes, ultrapure agarose, X-Gal, IPTG, ultrapure urea, Random primers labelling kit and other enzymes required for DNA manipulations, were purchased from Bethesda Research Laboratories, Gathiersburg, Md. Ampicillin was obtained from Boehringer-Mannheim Canada, Laval, Que. [Y-"PI~ATP (5000 Ci/mmol; 1 Ci = 37 GBq) for end labelling of

+

Printed in Canada / Imprim4 au Canada

+

Methods and materials

VETTAKKORUMAKANKAV AND STEVENSON

TABLE1. Oligonucleotide probes designed and synthesized based on the N-terminal sequence of dihydrolipoamide dehydrogenase from Hf. volcanii"


Probe no. 1 5'-GT(G/C) GT(G/C) GG(C/G/T) GAC ATC GC(G/C) ACI GG(C/G/T) ACI GA-3 ' Probe no. 2 5'-TAC GT(G/C) GC(G/C) GC(G/C) ATC CGI GC(G/C) GC(G/C) CAG AAC GG(C/G/T) ATC GAC-3 ' "The nucleotide sequence of Hf.volcanii dihydrofolate reductase gene (Zusman el al. 1989) and the H. cutirubrum superoxide dismutase gene (May and Dennis 1989) were used to represent the codon preference in Hf.volcanii. Probe I (29-mer) was derived from residues 1-10 and probe I1 (39-mer) was derived from residues 21-33 (Vettakkorumakankav el al. 1992).

oligonucleotide probes and [c~-~'s]~ATP (> 1000 Ci/mmol) for dideoxy sequence analysis were obtained from Amersham Canada Limited, Oakville, Ont. The plasmid vectors Bluescript KS + and KS - were purchased from Stratagene, La Jolla, Calif. The oligonucleotide probes were purchased from the Regional DNA Synthesis laboratory at The University of Calgary. Calgary, Alta. Bacterial strains and phages Haloferax volcanii (ATCC no. 29605) was obtained from the American Type Culture Collection, Rockville, Md. Libraryefficient Escherichia coli strain DH5a{F-~80dlacZAM15 (lacZYA-argF) U 169deoRrecA lendA 1hsd R 17 (rk - , mk +) supE44A - thi-lgyrA96relAl) and DH5aF ' (480dlacZAM 15 (IacZYA-argF) U 169deoRrecA 1endAl hsd R17 (rk- , mk ) supE44A-thi-lgyrA96relAl) were obtained from Bethesda Research Laboratories, Gaithersburg, Md. The helper phage VCS M13 was obtained from Stratagene, La Jolla, Calif. +

Growth of Hf. volcanii cells and isolation of genomic DNA Haloferax volcanii cells were cultured in 100-mL flasks with vigorous aeration in the rich medium prescribed in the American Type Culture Collection Handbook. Genomic DNA was isolated from 2-3 g of freshly cultured wet cells according to the procedure of Marmur (1961), except that cells were lysed by osmotic shock by resuspending the cells in 25 mL of 0.15 M NaCl containing 0.1 M EDTA (pH 8.0). Identification and cloning of the gene encoding dihydrolipoamide dehydrogenase The oligonucleotide probes employed are shown in Table 1, together with the amino acid sequence from which they were derived (Vettakkorumakankav et al. 1992). The probes were redundant with a bias for G or C in the third nucleotide position, based on the codon usage in the sequence of the dihydrofolate reductase gene (Zusman et al. 1989) from Hf. volcanii and the superoxide dismutase gene (May and Dennis 1989) from Halobacterium cutirubrum. Genomic DNA was digested to completion with a variety of restriction endonucleases in the appropriate buffer according to the supplier of the enzyme. The digests were subsequently electrophoretically separated on a 0.7% agarose gel cast in TAE buffer (Sambrook et al. 1989). The DNA fragments were transferred onto a Millipore nitrocellulose filter baked at 80°C in vacuo for 30 min and prehybridized for 2 h (Sambrook et al. 1989). Hybridization was carried out for 18 h with the oligonucleotide probes labelled with [ * I - ~ ~ P ] ~ A(Sambrook TP et al. 1989) at either 50-55°C for probe I or 58-62°C for probe 11. The filters were washed at room temperature with 1 x SSC (0.15 M NaCl and 0.015 M sodium citrate) containing 0.5% SDS and autoradiographed using a Kodak X-OMAT diagnostic film (Sarnbrook et al. 1989). The DNA fragments obtained by MboI restriction endonuclease digestion of Hf. volcanii genomic DNA in the range of 4.3 kbp

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1:0y

1300

1100

I

I

900 I

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700

500

300'

I

I

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Bases

FIG. 1. Partial restriction map of pNat82 indicating the sequencing strategy. The direction and length of the arrows indicate the direction and extent of the sequencing reactions. were ligated to the plasmid vector Bluescript KS + , which had been digested to completion with BamHI restriction endonuclease and dephosphorylated with calf intestinal alkaline phosphatase (Sambrook et al. 1989). The ligation mixture was used to transform competent E. coli DH5a library-efficient cells, which were screened for recombinant plasmids by plating on LB-agar plates (Sambrook et al. 1989) containing ampicillin (100 pg/mL) and X-gal (20 pg/mL). The white, ampicillin-resistant colonies (500 total) were applied in duplicate with a sterile toothpick onto gridded agar plates containing LB-ampicillin media. These recombinant cells were screened by isolation of the plasmid DNA and digestion with restriction endonucleases, and the location of the DHLipDH gene was identified using the DHLipDH-specific oligonucleotide probe (probe 11) as described below. Batches of recombinants were cultured (20 clones per batch) in 5 mL of LB-ampicillin (100 pg/mL). Plasmid DNA was isolated and digested with PstI and XbaI restriction endonucleases. The digests were separated by 0.7% agarose gel electrophoresis transferred onto nitrocellulose and hybridized to probe I1 as described above. Upon identification of a positive batch, the individual colonies therein were screened separately in an identical manner. Sequencing of dihydrolipoamide dehydrogenase gene Figure 1 illustrates the strategy adopted to sequence the DHLipDH open reading frame and the construction of the appropriate subclone. Single-stranded DNA template in Bluescript (KS + and KS - ) was obtained with the helper phage VCS M13 (Sambrook et al. 1989). Nucleotide sequence determinations on these singlestranded DNA templates were performed using the dideoxy chain termination method (Sanger et al. 1977) and T7 DNA polymerase as the catalytic enzyme (Pharmacia T7 sequencing kit), with appropriately designed synthetic primers. The new primers were designed from previous sequence information, such that the sequence information obtained from the previous primer overlaps with the sequence obtained with the newly designed primer. Regions of ambiguity (due to compressions) were resolved by electrophoresis at higher temperatures (60°C). Sequence alignments The sequences of DHLipDH from several sources were aligned using the program PCGene version 6.6 supplied by Intelligenetics Inc., Mountain View, Calif. (Fig. 3). Using this program, amino acid compositions of DHLipDH from human, Azotobacter vinelandii, E. coli, and Hf. volcanii were also calculated from their complete primary structures. Isolation of RNA and Northern blotting Total RNA from Hf. volcanii cells was isolated using the procedure described by Nieuwlandt and Daniels (1990). RNA (72 pg) was separated by electrophoresis on a formaldehyde-agarose gel

BIOCHEM.CELL BIOL.VOL. 70,1992 GACGGCGCGCAGGGCGTCGGTTCACGAACCGCGTGAAGGAACTGCTCGAAGACCCCAAACTGCTGGTGTTAGAATA

ATG GTC GTC GGA GAC ATC GCA ACC GGA ACC GAA CTG CTC GTC ATC GGC GCG GGA CCC GGC 1

M e t val v a l g l y asp i l e a l a t h r q l y t h r glu leu l e u v a l i l e


916

a s p g l y g l u q l u asn g l u t y r a r g a l a a s p l y s v a l l e u v a l a l a v a l g l y a r g ser p r o GTC ACC GAC ACG ATG GAC ATC GAG AAC GCC GGC CTC GAA GCC GAC GAC CGC GGC TTC CTC

281

856

g l u g l y a l a t h r g l y t r p a r q g l u g l u a s p a s p g l y i l e met v a l t h r t h r g l u t h r g l u GAC GGC GAG GAA AAC GAG TAC CGC GCC GAC AAG GTG CTC GTC GCC GTC GGG CGC TCG CCC

261

796

a s p v a l a l a a r q v a l v a l a r g l y s a r g a l a g l u g l u l e u g l y i l e a s p met h i s l e u g l y GAG GGC GCG ACG GGC TGG CGC GAG GAG GAC GAC GGC ATC ATG GTG ACG ACC GAG ACC GAA

241

736

l e u g l y a l a a s p v a l t h r v a l v a l g l u met l e u a s p a s p i l e l e u p r o g l y t y r g l u s e r GAC GTG GCC CGC GTC GTC CGC AAG CGC GCC GAA GAG CTC GGC ATC GAC ATG CAC CTC GGT

221

676

a r g l e u v a l v a l v a l q l y q l y g l y t y r i l e g l y met g l u l e u s e r t h r t h r phe a l a l y s CTC GGC GCG GAC GTG ACC GTC GTC GAG ATG CTC GAC GAC ATC CTG CCG GGC TAC GAG TCC

201

616

phe g l y a s p q l u p r o v a l t r p s e r s e r a r q a s p a l a l e u g l u a l a a s p t h r v a l p r o g l u CGA CTG GTC GTC GTC GGC GGC GGC TAC ATC GGG ATG GAG CTG TCC ACG ACG TTC GCC AAG

181

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q l u phe g l u h i s c y s i l e i l e a l a t h r q l y s e r a r g v a l i l e q l n i l e p r o q l y phe a s p TTC GGC GAC GAG CCG GTG TGG TCG TCG CGC GAC GCC CTC GAG GCC GAC ACC GTC CCG GAG

161

496

l y s asp g l u asn a l a v a l a r g i l e a l a h i s q l y g l y glu g l y g l n g l y s e r g l u t h r i l e GAG TTC GAG CAC TGC ATC ATC GCC ACC GGC TCG CGC GTC ATC CAG ATT CCC GGT TTC GAC

141

436

q l y v a l g l u l y s l e u c y s l y s a l a a s n q l y v a l a s n l e u v a l g l u g l y t h r a l a a r g phe AAG GAC GAG AAC GCC GTC CGC ATC GCC CAC GGC GGC GAG GGG CAG GGC TCG GAG ACC ATC

121

376

val val asp m e t s e r gln leu arg asp t r p lys s e r gly val v a l asp gln l e u t h r gly GGC GTC GAG AAG CTC TGT AAG GCG AAC GGC GTC AAC CTC GTC GAG GGA ACC GCC CGC TTC

101

316

g l y a l a a s n l e u a l a h i s g l u a l a g l y asn a l a g l u g l u m e t g l y i l e h i s a l a a s p p r o GTC GTG GAC ATG TCG CAA CTG CGC GAC TGG AAG AGC GGC GTC GTG GAC CAA CTC ACC GGC

81

256

a s p a l a t y r g l y g l y t h r ~ v lse u a s n t v r a l v c v s i l e p r o s e r l y s a l a l e u i l e t h r GGG GCG AAC CTC GCC CAC GAG GCG GGC AAC GCC GAG GAG ATG GGC ATC CAC GCC GAC CCC

61

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v a l t h r a s p t h r met a s p i l e g l u a s n a l a g l y l e u g l u a l a a s p a s p a r g g l y phe l e u TCG GTC GAC GAC CGC CGC CGC ACC GAC GTG GAG CAC ATC TAC GCC GTC GGC GAC GTG GTC 1036

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s e r val asp asp arq arg arq t h r asp val glu h i s i l e t y r a l a val gly asp v a l val GAG GAC ACG CCG ATG CTC GCC CAC GTC GCC TCG AAG GAG GGC ATC GTC GCC GCC GAG CAC 1096

321

g l u a s p t h r p r o met l e u a l a h i s v a l a l a ser l y s g l u g l y i l e v a l a l a a l a g l u h i s GTC GCC GGC GAA CCG GTC GCC TTC GAC AGT CAG GCC GTC CCC GCC GCG GTG TTC ACC GAC 1156

341

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FIG. 2. Nucleotide sequence and the translated amino acid sequence of DHLipDH from Hf. volcanii. The nucleotide numbers are indicated to the right of the sequence and the amino acid numbers to the left. The adenine-binding region of FAD (amino acids 16-21), the redox disulfide (amino acids 47-52), and the sequence encompassing the active site histidine (amino acids 449-454) are underlined. The translation termination codon is indicated by an asterisk.


CCC GAA ATC GGC ACG GTC GGC ATG ACC GAG GCC GAC GCC GAG GAG GCC GGC TTC ACG CCC 1216 361

p r o g l u i l e g l y t h r v a l g l y met t h r g l u a l a a s p a l a g l u g l u a l a g l y phe t h r p r o

GTC GTC GGG CAG ATG CCC TTC CGG GCG TCC GGC CGC GCG CTG ACG ACG AAC CAC GCC GAC 1276 381

v a l v a l g l y g l n met p r o phe a r g a l a s e r g l y a r g a l a l e u t h r t h r asn h i s a l a a s p


GGC TTC GTC CGC GTC GTC GCC GAC GAG GAG TCC GGC TTC GTC CTC GGG GCG CAA ATC GTC 1336 401

g l y phe v a l a r g v a l v a l a l a a s p g l u g l u s e r g l y phe v a l l e u g l y a l a g l n i l e v a l

GGC CCC GAG GCC TCC GAA CTC ATC GCC GAA CTC GCG TTC GCC ATC GAG ATG GGC GCG ACG 1396 421

g l y p r o g l u a l a s e r g l u l e u i l e a l a g l u l e u a l a phe a l a i l e g l u met g l y a l a t h r

CTC GAA GAC GTG GCC TCG ACC ATC CAC ACC CAC CCG ACG CTC GCG GAA GCG GTC ATG GAG 1456 441

l e u g l u a s p v a l a l a s e r t h r i l e h i s t h r h i s Dro t h r l e u a l a g l u a l a v a l met g l u

GCC GCC GAG AAC GCG CTC GGA CAG GCG ATT CAC ACC CTG AAT CGG TGA 461

a l a a l a g l u asn a l a l e u g l y g l n a l a i l e h i s t h r l e u asn a r g

GGAGCGTCCGAAA 1517

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FIG.2 (concluded)

and transferred onto a nitrocellulose filter using a Vacublotting instrument (Tyler Instrument Co., Edmonton, Alta.). The filter was prehybridized for 2 h and then hybridized for 18 h at 60°C to the 1.0-kbp San fragment (see Fig. 1) derived from the DHLipDH open reading frame. The fragment was labelled by random priming (Random primers labelling kit, Bethesda Research Laboratories). The filter was washed once with 1 x SSC at room temperature and twice at 60°C with 0.1 x SSC, and then subjected to autoradiography (Sambrook et al. 1989).

Results Identification and cloning of the gene The restriction endonuclease digests of Hf. volcanii genomic DNA when hybridized with either probe I or I1 revealed a 4.3-kbp MboI restriction endonuclease fragment and a 5.5-kbp XhoI fragment (data not shown). Screening of the recombinant clones by the plasmid isolation method was preferred over the colony hybridization method, because it eliminated the possibility that the probes hybridized to the dihydrolipoamide dehydrogenase gene of E. coli. To minimize the time and labor involved in screening a large number of colonies by the plasmid isolation procedure, recombinant colonies were cultured in batches each containing 20 colonies. One batch of the seven tested was positive for the hybridizing fragment of DNA. Screening of the individuals comprising the positive batch revealed one positive clone. Sequence analysis of the DHLipDH gene The sequencing strategy is illustrated in Fig. 1 in conjunction with the partial restriction map and the DHLipDH open reading frame. The sequence was verified entirely on both the strands of the DNA. The open reading frame had an additional methionine residue on its N-terminal sequence, but was otherwise identical to that of the N-terminal sequence obtained from the pure protein. The deduced nucleotide sequence contained 77 bases upstream of the initiating methionine codon and 72 bases downstream of the termination codon. The complete nucleotide sequence along with the translated amino acid sequence is illustrated in Fig. 2. The sequence involved in binding the flavin cofactor,

2. Comparison of amino acid composition of DHLipDH TABLE

from Hf. volcanii, A. vinelandii," human,b and E. colic sources Mol% Amino acid Ala Arg Asn ASP CYs Gln Glu GlY His Ile Leu LYs Met Phe Pro Ser Thr T~P TYr Val

Hf. volcanii

A. vinelandii

Human

E. coli

12.0 4.0 3.O 8.0 1.0 2.0 10.0 11.0 3O . 5O . 6.0 2.0 4.0 3.0 3.0 4.0 7.0 1.0 1.0 11.0

13.6 2.5 2.0 5.2 0.6 3.1 6.0 10.9 2.5 6.0 8.1 7.3 1.8 2.9 3.3 4.8 5.0 0.2 1.6 11.7

9.4 3.3 4.1 4.3 1.9 2.9 6.0 11.3 2.7 7.4 7.0 7.2 2.1 3.1 3.3 5.5 6.0 0.5 2.1 8.8

10.5 3.2 2.9 5.2 1.0 1.7 8.2 10.7 2.7 8.2 7.2 8.2 2.3 2.9 4.4 2.9 5.5 0.8 1.7 9.5

Westphal and deKoK (1988). b~tulakowskiand Robinson (1987). 'Stephens e t a / . (1983).

and the sequences around the active site cysteine residues and the active site histidine residue, are underlined.

Primary structure of DHLipDH The open reading frame has 475 amino acids and shows a high degree of homology upon alignment with the dihydrolipoamide dehydrogenase sequences from a variety of sources. The aligment is indicated in Fig. 3. The correctness of this sequence is supported by the amino acid sequence data of a peptide (residue 325-347) that we have obtained from cyanogen bromide cleavage of the pure pro-


BIOCHEM. CELL BIOL. VOL. 70, 1992

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304 292 305 341


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AAVFTDPEIGTVGMTEADAEEAGFTPVVGQMPFRASGRALTTNHADGFVR AVCFTDPEVVVVGKTPEQASQQGLDCIVAQFPFAANGRAMSLESKSGFVR AVIYTHPEIAGVGKTEQALKAEGVAINVGVFPFAASGRAMAANDTAGFVK SVIYTHPEVAWVGKSEEQLKEEGIEYKVGKFPFAANSRAKTNADTDGMVK

404 391 404 440


VVADEESGFVLGAQIVGPEASELIAELAFAIEMGATLEDVASTIHTHPTL VVARRDNHLILGWQAVGVAVSELSTAFAQSLEMGACLEDVAGTIHAHPTL VIADAKTDRVLGVHVIGPSAAELVQQGAIAMEFGTSAEDLGMMVFAHPAL ILGQKSTDRVLGAHILGPGAGEMVNEAALALEYGASCEDIARVCHAHPTL

454 441 454 490


AEAVMEAAENA-LGQAIHTLNR-GEAVQEAALRA-LG---HALHI-SEALHEAALAVS-GHAIHVANRKK SEAFREANLAASFGKSINF-----

... .. . * * .

.**

***

.

*

. * ..* * . **..

.* . . . * .

.

.**.*

475 459 477 509

FIG. 3. Alignment of amino acid sequences of dihydrolipoamide dehydrogenase from Hf.volcanii (HF2), Pseudomonas putida (PSEPU), Azotobacter vinelandii (AZOVI), and humans (HUMAN). Asterisks indicate perfectly conserved residues and dots indicate well-conserved residues. Dashes denote gaps introduced into the sequence to achieve maximum alignment. tein (N. Vettakkorumakankav and K.J. Stevenson, unpublished observations). Table 2 illustrates the amino acid composition of DHLipDH from various organisms. The most notable differences are the excess of aspartic acid and

glutamic acid and the marked reduction in the number of lysines in the Hf.volcanii enzyme. The numbers of the basic amino acids histidine and arginine are similar in DHLipDH from all organisms.


TABLE3. Codon usage in DHLipDH gene of Hf.volcanii"


TTT Phe F TTC Phe F TTA Leu L TTG Leu L

13

-

CTT Leu L CTCLeuL CTA Leu L CTG Leu L

-

ATT Ile I ATC Ile I ATA Ile I ATGMetM

2 24

GTT Val V GTC Val V GTA Val V GTGValV

-

22

8

13 40

11

TCT Ser S TCC Ser S TCA Ser S TCG Ser S

-

CCT Pro P CCCProP CCA Pro P CCG Pro P

-

ACT Thr T ACC Thr T ACA Thr T ACGThrT

-

GCT Ala A GCC Ala A GCA Ala A GCGAlaA

TATTyrY TAC Tyr Y TAA OCH Z TAG AMB Z

-

CAT His H CACHisH CAA Gln Q CAG Gln Q

-

AAT Asn N AAC Asn N AAALysK AAGLysK

1 11

-

- GAT Asp D

-

40 2 17

36 11 38

5

10

9 1 6 18 -

15

GAC Asp D GAA Glu E GAGGluE

7

12 3 6

10

TGT Cys C TGC Cys C TGA OPA Z TGG Trp W

2 2 1 3

CGT Arg R CGCArgR CGA Arg R CGG Arg R

-

AGT Ser S AGC Ser S AGA Arg R AGGArgR

1 1

-

GGT Gly G GGC Gly G GGA Gly G GGGGlyG

2 40 5 7

18 1 2

"OCH, AMB, and OPA are ochre, amber, and opal termination codons, respectively.

Putative regulatory elements Archaeal promoters are composed of two elements defined as box A and a weakly conserved box B (Zillig et al. 1988). The consensus for box A elements is TTTAATA (Thomm and Wich 1988). The sequence TGAAGGAA in the DHLipDH gene from Hf. volcanii is similar to the consensus box A element and is located 35 nucleotides upstream of the initiating methionine codon. Furthermore, the sequence AAGTTA (or a related sequence) is considered important in the binding of RNA polymerase (Dennis 1985). Even though the putative regulatory elements are not identical to the consensus sequence, Northern blot analysis revealed that the gene is transcribed from its own promoters (data not shown). Archaebacterial termination sequences are usually A-T rich and a stem and loop structure may or may not be present (Sutherland et al. 1990). In the DHLipDH sequence from Hf. volcanii, an A-T rich sequence is found immediately downstream of the stop codon. Discussion This is the first report of the complete primary structure of an archaeal dihydrolipoamide dehydrogenase. The catalytic mechanism of the enzyme entails the participation of a redox active disulfide formed by two cysteines separated by four residues (C-L-N-V-G-C) (Williams 1976; Carothers et al. 1989). Such a sequence (C-L-N-Y-G-C) is present in the halophilic archaeal enzyme as well (cf. Fig. 2, residues 47-52, underlined). We have also shown, through chemical modification with trivalent organoarsenical reagents, the involvement of a redox active disulfide in the catalytic mechanism of this enzyme (Vettakkorumakankav et al. 1992). The FAD- and NAD+-binding sequences in DHLipDH from Hf. volcanii are also well conserved when compared with the consensus motif G-X-G-X-X-G in glutathione reductase (Perham 1990). The catalytic mechanism involves the abstraction of a proton from the substrate dihydrolipoamide by a basic residue believed to be histidine (Williams 1976; Carothers et al. 1989), which has been shown to be histidine 444 in the E. coli DHLipDH through the inactivation of the pyruvate dehydrogenase multienzyrne complex

with a bifunctional trivalent organoarsenical reagent (BrCH2CONHPhAsO) (Adamson and Stevenson 1981). The sequence adjacent to this active site histidine has also been elucidated (Holmes and Stevenson 1986). The threedimensional structure of human glutathione reductase (Pai and Schulz 1983) and Azotobacter vinelandii DHLipDH (Schierbeek et al. 1989) reaffirmed the presence of this histidine in the active site. A similar conserved sequence is found in the identical region of the DHLipDH from Hf. volcanii, implicating the same catalytic mechanism (cf. Fig. 2, residues 449-454, underlined). The elucidated primary structure of DHLipDH from Hf. volcanii shows good homology with the enzyme from other sources (cf. Fig. 3). To understand the molecular basis of halotolerance of DHLipDH from the halophile Hf. volcanii, a comparison of the primary structures and amino acid compositions of DHLipDH from a variety of species was performed. The increase in the amount of glutamic acid and aspartic acid concomitant with a decrease in the lysine residues was evident in the halophilic DHLipDH. This is consistent with the observations made on other halophilic proteins (Lanyi 1974). The excess acidic residues are considered to compete effectively for the available water in the high salt environment, so as to maintain a sphere of hydration under the conditions of low water activity (Lanyi 1974). Another amino acid of interest in this context is threonine. It is hypothesized that halophilic enzymes show a marked reduction in the amount of hydrophobic residues and a concomitant increase in the number of serine and threonine residues, as an adaptive mechanism to prevent hydrophobic collapse of the protein in a high salt environment (Lanyi 1974). We observe an increase in the number of threonine residues when the amino acid composition of the halophilic DHLipDH is compared with that of the DHLipDH from nonhalophiles. The serine content of DHLipDH from the halophiles and the nonhalophiles is comparable (Table 2). The DHlipDH gene from Hf. volcanii shows a preference for G or C in the third nucleotide position, as is evident from Table 3. This observation is consistent with the codon usage found in the genes coding for superoxide dismutase from


662

BIOCHEM. CELL

Halobacterium cutirubrum (May a n d Dennis 1989) a n d the dihydrofolate reductase gene from Hf. volcanii (Zusman et a/. 1989). There is also a preference for C in the third position over G, which is characteristic of Hf.volcanii genes (Conover and Doolittle 1991). The gene for DHLipDH from Hf. volcanii has 476 codons, in which 297 (62%) have C in the third nucleotide position. It is suggested that this high occurrence o f C in the open reading frame may be due t o the availability of certain t R N A molecules in Hf. volcanii (Conover a n d Doolittle 1991; Gupta 1985). This is the first report of the complete primary structure of a dihydrolipoamide dehydrogenase from an Archaea. This study provides the basis for investigating the evolution o f the flavoprotein disulfide reductases a n d those adaptive features required by enzymes t o tolerate highly saline intracellular environments. Dihydrolipoamide dehydrogenase is a good model system t o study this because o f the availability o f primary structures f o r D H L i p D H from a variety of sources a n d the three-dimensional structure of D H L i p D H from A. vinelandii.

Acknowledgements This work was supported financially by the Natural Sciences a n d Engineering Research Council of Canada (K.J.S.). The authors acknowledge Dr. Sui-Lam Wong of the Department of Biological Sciences, University of Calgary, for his guidance in the work described in this paper a n d for reading the manuscript.

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Citrate synthase from Haloferax volcanii: enzyme purification and gene cloning.

The interaction of arsenical drugs with dihydrolipoamide and dihydrolipoamide dehydrogenase from arsenical resistant and sensitive strains of Trypanosoma brucei brucei.

A structural model for human dihydrolipoamide dehydrogenase.

Avian alcohol dehydrogenase: the chicken liver enzyme. Primary structure, cDNA-cloning, and relationships to other alcohol dehydrogenases.

Localization of the human dihydrolipoamide dehydrogenase gene (DLD) to 7q31----q32.

Purification and characterization of acetoin:2,6-dichlorophenolindophenol oxidoreductase, dihydrolipoamide dehydrogenase, and dihydrolipoamide acetyltransferase of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system.

Purification, characterization and function of dihydrolipoamide dehydrogenase from the cyanobacterium Anabaena sp. strain P.C.C. 7119.

Characterization of the transcriptional regulatory region of the human dihydrolipoamide dehydrogenase gene.

Dihydroxyacetone metabolism in Haloferax volcanii.

Recombinant expression, characterization and application of a dihydrolipoamide dehydrogenase with diaphorase activity from Bacillus sphaericus.

Inhibiting sperm pyruvate dehydrogenase complex and its E3 subunit, dihydrolipoamide dehydrogenase affects fertilization in Syrian hamsters.

Cytotoxic mechanism related to dihydrolipoamide dehydrogenase in Leydig cells exposed to heavy metals.

Primary structure and glycosylation of the S-layer protein of Haloferax volcanii.

A pH-dependent kinetic model of dihydrolipoamide dehydrogenase from multiple organisms.

Purification and comparative studies of dihydrolipoamide dehydrogenases from the anaerobic, glycine-utilizing bacteria Peptostreptococcus glycinophilus, Clostridium cylindrosporum, and Clostridium sporogenes.

Mitochondrial Dihydrolipoamide Dehydrogenase is Upregulated in Response to Intermittent Hypoxic Preconditioning.

The Adaptive Immune System of Haloferax volcanii.

Biochemical and structural characterization of the apicoplast dihydrolipoamide dehydrogenase of Plasmodium falciparum.

Inhibition of alpha-ketoglutarate dehydrogenase activity by a distinct population of autoantibodies recognizing dihydrolipoamide succinyltransferase in primary biliary cirrhosis.

Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle.

Archaeal membrane-associated proteases: insights on Haloferax volcanii and other haloarchaea.

Thermostable NAD(+)-dependent alcohol dehydrogenase from Sulfolobus solfataricus: gene and protein sequence determination and relationship to other alcohol dehydrogenases.

Generation and phenotyping of a collection of sRNA gene deletion mutants of the haloarchaeon Haloferax volcanii.

Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein-protein interactions by the MDM2 ligand nutlin-3.