Gene, 117 (1992) 125-130 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00

125

GENE 06544

Cloning, sequencing and heterologous expression of a Klebsiella pneurnoniae gene encoding an FAD-independent acetolactate synthase (Acetohydroxy acid synthase; acetoin; acetolactate decarboxylase; diacetyl; Enterobacter; pyruvate decarboxylase; 2,3butanediol biosynthesis; Voges-Proskauer test)

Hwei-Ling Peng a, Pei-Yu Wang a, Chiou-Mei Wu b, Der-Chian Hwang b and Hwan-You Chang b "Department of Microbiologyand hnmunology, and b Department of Molecular and CellularBiology, Chang.GungMedical College. Kwei-San,(Taiwan ROC)

Received by G.N. Godson: 24 September 1991; Revised/Accepted: 6 February/3 March 1992;Received at publishers: 6 April 1992

SUMMARY The gene encoding the valine-resistant and FAD-independent acetolactate synthase of Klebsiella pneumoniae was isolated and expressed in Escherichia coll. The nucleotide sequence of this gene was determined and it exhibited an open reading frame of 1680 bp in length. In vivo expression of the acetolactate synthase-encoding gene in E. coli revealed a single 60kDa protein which is consistent with the molecular weight calculated from the deduced amino acid sequence of the gene product. The gene product shares about 20-30% homology with the acetolactate synthases ofE. coli, yeast and higher plants.

INTRODUCTION The synthesis of acetolactate, a key intermediate in valine biosynthesis in Escherichia coli and other organisms is catalyzed by acetolactate synthase (ALS; EC 4.1.3.18, Fig. 1) which is also known as acetohydroxy acid synthase (AHAS). There are three isoforms of acetolactate synthase, ALS I, ALS II and ALS Ill encoded by ilvBN, ilvGMEDA and ilvlH operons, respectively, in E. coll. Biochemical analysis has shown that all three ALS isozymes are composed of two different polypeptides, a large 60-kDa subunit

Correspondence to: Dr. H.Y. Chang, Department of Molecular Cellular Biology, Chang-Gung Medical College, Kwei-San, (Taiwan ROC) Tel. (886-3)328-1200; Fax (886-3)328-3031.

Abbreviations: aa, aminoacid(s); ADC,acetolactate decarboxylase;ALS, acetolactate synthase; Ap, ampicillin; AR, acetoin reductase; bp, base pair(s); DH, dihydroxy acid dehydrase; En., Enterobacter; FAD, ravine adenine dinucleotide; IPTG, isopropyl-/?-D-thiogalactopyranoside; IR, acetohydroxy acid isomeroreductase; K., Klebsiella; kb, kilobase(s) or 1000 bp; LB, Luria-Bertani broth; nt, nucleotide(s); ORF, open reading frame; PA, polyacrylamide;PDC, pyruvatedecarboxylase;TrB, transaminase B; VP, Voges-Proskauer (test); [ ], denotes plasmid-carrier state.

Pyruvate

Diaeetyl

Aeetolaetate "ADC~Aeetoin

Valine

2,3.butanedlol

Fig. 1. Main steps of the biosynthetic pathway of valine and 2,3butanedioi (adopted and modified from Goelling and Stahl, 1988). The enzymesinvolvedare abbreiviated as follows:ALS,acetolactate synthase, IR, acetohydroxyacid isomeroreductase; DH, dihydroxyacid dehydrase; TrB, transaminase B (for a review see Lawther et al., 1987); ADC, acetolactate decarboxylase (Loken and Stormer, 1970); AR, acetoin reductase (Stzrmer, 1974). and a smaller 10-17-kDa subunit (Eoyang and Silverman, 1984; Lawther et al., 1987; Squires et al., 1983). ALS I and ALS III are feedback inhibited by valine (Guardiola et al., 1977). ALS II is cryptic in the E. coli K-12 cells because

126 of a frameshift mutation in the ilvG gene which encodes the large subunit ofthis enzyme (Lawther et al., 1981). Genetic and biochemical evidence indicates that both the large and small subunits are catalytically essential (Lu and Umbarger, 1987; Squires et al., 1981). The small subunit has also been shown to confer valine sensitivity on the enzyme (DeFelice et al., 1974). In addition to being used as the precursor of valine biosynthesis, acetolactate can be converted into acetoin and to 2,3-butanediol ill some bacterial species (Fig. 1). Since the 2,3-butanediol pathway is very active and is not regulated by valine, an ALS distinct from those ALS isoenzymes of E. coil has been proposed. In Enterobacter aerogenes, two ALS isozymes have been reported (Stormer, 1975). One is valine sensitive and FAD dependent. The other, the so-called pH 6 acetolactate-forming enzyme, is valine resistant and is thought to be the major enzyme for the acetolactate synthesis in the ~.,~-butanediol pathway. We intended to analyze the molecular genetics of the three enzymes (Fig. 1) involved in the 2,3-butanediol pathway. In this paper, we describe the cloning, sequencing and heterologous expression in E. coil of a gene encoding an FAD-independent ALS of K. pneumoniat.. Based on the enzymatic properties characterized, we believe this enzyme is equivalent to the pH6 acetolactate-tbrming enzyme of the close related En. aerogenes.

EXPERIMENTAL AND DISCUSSION

(a) Isolation of a VP-positive Escherichia coil cosmid clone CD64 Initially, we attempted to isolate the gene encoding the ADC of K. pneumoniae and anticipated it may convert E. coli into VP-positive. A cosmid library was constructed using Sau3AI partially digested genomic DNA of K. pneumoniae strain CG21 (wild type) and BamHI site of vector pBTI-I (Boehringer Mannheim Biochemicals). After transformation into E. coil strain DH1, colonies resistant to Ap were transferred individually to 96-well dishes containing VP semi-solid agar (Tanabe Seiyaku Co. Ltd., Osaka, Japan) and incubated at 37°C overnight. VP reaction of each cosmid clone was carried out as described (Krampitz, 1957). One of the two VP-positive cosmid clones was designated as CD64 and was further characterized as described in section b. (b) The VP reaction of CD64 clone was conferred by ALS Since the VP reaction is positive not only with acetoin but also with diacetyl, the VP-positive cosmid clones may carry either the ADC-encoding gene or a gene encoding ALS (Fig. 1). In order to distinguish these two possibilities, the crude cell lysates of the VP-positive clones were tested

for their ability to form acetolactate from pyruvate as described (Stormer, 1975). In the presence of valine, we did not detect any ALS activity in the crude lysate of E. coil DH 1 cells. This was ex,ected since it has been shown that the ALS I and ALS III of E. coil K-12 strain were sensitive to valine and the ilvG gene which encoded the large subunit of ALS II was not functional because it suffered a frameshift mutation. In contrast, the CD64 cosmid clone exhibited a strong acetolactate-forming activity, suggesting that the CD64 cosmid clone contained a gene encoding for a vaiine-resistant ALS. In addition, the pH 6 optimum of the ALS also distinguished it from the ALS isoenzymes of E. coll. This ALS-encoding gene of K. pneumoniae was designated as ilvK in accordance with the nomenclature of other ALS-encoding genes in bacteria and yeast. Its functional role in the isoleucine-leucine-valine biosynthesis, however, remained to be investigated.

(c) Localization of the ilvK gene in p(~D64 The cosmid DNA of CD64 was prepared and shortened by using appropriate restriction enzymes. One of the smallest subclones which was still capable of producing a functional ALS, designated pTL8, was constructed by inserting a Taql partially digested DNA fragment into the Accl site of vector pUC 18. The 2.1-kb insert on pTL8 was first mapped with a variety of restriction enzymes. Deletion studies were then carried out and showed that K. pneumoniae gene ilvK should expand over at least 1.5-kb. Furthermore, the orientation of this insert DNA relative to the adjacent vector DNA did not influence the production of a functional ALS which suggested that the gene is under the control of its own promoter. (d) Nucleotide sequence of ilvK gene The nt sequence of the 2. l-kb DNA insert of pTL8 encoding ALS was determined by the dideoxy-chain termination method of Sanger et al. (1977) using the Sequenase kit (US Biochemicals, Cleveland, OH). The complete 2055 nt sequence and the deduced aa sequence of the major ORF are shown in Fig. 2. The only ORF which is consistent with the deletion-functional assay result should encode for a polypeptide of 559 aa residues with a calculated Mr of 60 328. The predicted number is perfectly matched with that of the large subunit of other bacterial ALS a:, well as the pH 6 acetolactate-forming enzyme of En. aerogenes (Stormer, 1974). A potential -10 concensus s~.quence GATAAT was found 48 nt upstream from the first in frame ATG codon. No sequences were evident which were strongly homologous to the consensus -35 region proposed for E. coil (Doi and Wang, 1986). It has been shown that the genes encoding the small subunit of each of the three ALS isozymes are located immediately downstream from the gene encoding the large subunit (Friden et al.,

Ala Ser GIy GCC AGC GGG

Ala Lys Leu GCG AAG CTT

Set Gln Pro Glu AGC CAG CCG GAA

Pro Val Thr CCA GTC ACC

Phe TTC

Glu GAA

Phe TTC

Pro CCG

Leu CTG

Ala GCG

Leu CTG

Met Ala AT(] G C C

Set TCT

Ala GCG

Gln Val CAG GTG

Ile ATT

Met ATG

His CAT

Phe TTC

Leu CTG

Thr ACC

Ser AGC

Val GTG

Tyr ~ ~r Pro TAC AGC CCG

Leu Val lie Cys Ile Gly CTG GTG ATC TGC ATC GGC

Gin CAG

Glu GAA

Asp GAC

Asp GAT

Thr ACC

Gly GGC

Ile ATC

Lys AAA

Gly GGC

Ala GCG

Val GTG

Gly GGC

Ile ATC

Tyr TAC

Arg CGT

Ash AAC

Set AGC

Leu CTG

Asp GAC

Val GTG

Set AGC

Leu CTG

Ala GCG

Ala GCG

Thr ACC

GIy GGC

"

Asn GIy Gln AAC GGC CAG

Val Asn GTC

Arg CGC Met ATG His CAT ser TCC

Leu CTG Gin CAG Leu CTT Tyr TAT Glu GAA Thr ACC

Arg CGC Ala GCC Phe TTC Ile ATC Trp TGG Leu CTG His CAT Lys AAA Ala GCC Pro CCG

Val Asp GTG GAT

Leu TEN CTG TAA GTCATCACAATAAGGAAAGAAAAATGAAAAAAGTCGCACTTGTTACCGGCGCCGGCCAGGGG ATTGGTAAAGCTATCGCCCTTCGTCTGG.Y~AAGGATGGATTTGCCGTGGCCATTGCCGATTATAACGACG CCACCGCCAAAGCC~TCGCCTCCGAAATCAACCAGGCCGGCGGCCGCGCCATGGCGGTGAAAGTGGATGT TTCTGACCGCGACCAGGTATTTGC(.GCCGTCCA

216 648 234

~52

270

72

864

288

810

756

702

594

198

540

180

486

162

432

144

128 378

324

108

270

~0

216

Leu CTC

Ile ATC

Pro Glu CCT GAG

Ser Gly Asp Gly TCC GGC GAC GGC

Gly GGC

Set Ala Glu Ala AGC GCC GAG GCG

Leu Glu CTG GAG

Tyr TAT

Lys AAA

Leu CTG

Phe TTC

Ala GCC

Met ATG

Set AGC

Arg CGC

Asp GAC

Set TCC

Ile ATC

Leu CTG

Tyr Arg Asp TAT CGC GAT

Asn AAC

Pro Leu Leu Leu Met Gly Gln His Leu CCG CTG CTG ATG GGC CAG CTG CAT CTG

Set Gln Ile AGT CAG ATT

Arg Ala Ala Met Asp Val Asp Gly Pro Ala Val VaI Ala Ile Pro CGC GCG GCG ATG GAC GTC GAC GGC CCG GCG GTA GTG GCC ATC CCG

Lys GIy Phe Ala Val Glu AAA GGG TTT GCC GTG GAA

Ala

Phe Gly TTC

Glu GAG Val Glu Phe Gly "Pro Met Asp Phe Lys Ala GTC GAG TTT GGG CCG ATG GAT TTT AAA GCC

Ash Gly Tyr Ash Met Val Ala Ile Gln Glu AAC GGC TAC AAC ATG GTC GCT ATC CAG GAA

Ser

Leu CTG

Set Val TCC GTC

Val GTG Ile Gly ATC GGC

Gln CAG

Gly GGC

Val GTT

Leu CTG

Leu CTC

Leu Glu Thr Ala Val Arg Leu Lys Ala ASh Val CTG GAG ACC GCC GTC CGC CTG AAA GCC AAC GTG

Arg Lys Val Val CGC AAA GTG GTC

TCC

GCG

Arg Ala Arg CGC GCC CGT

Met Gly Val Ala Leu Pro Trp Ala ATG GGC GTC GCC CTG CCC TGG GCT

Arg Tyr Leu Tyr Thr Phe CGC TAC CTG TAC ACG TTC

Ser Gly TCC GGC

GGC

Leu CTG

Asn Set Asp Val Thr Leu Thr Val Asp Met AAC AGC GAC GTC ACG TTG ACC GTG GAC ATG

Gln Arg Leu CAG CGC CTG

Ile Trp Val Asp ATC TGG GTC GAT

Ser Set Met Glu TCG AGC ATG GAG

AAT

Gln Arg Glu CAG CGC GAG

His Arg Leu Val CAT CGG CTG GTG

Leu His Pro Leu Arg Ile CTG CAT CCC CTG CGC ATC

Arg Asp Arg Gln His CGC GAC CGC CAG CAC

Asn Gln Phe Ala AAC CAG TTT GCC

Leu CTC

Gln Thr CAG ACC

Ile Trp Ile Ala ATC TGG ATT GCC

Gln Asp Ile Val CAG GAT ATC GTC

Gly Ala Gln GGC GCG CAG

Ala Ala Glu GCG GCG GAG

Gln CAG

Leu Ala Gln Asn Ile Asp CTG GCG CAA AAT ATC GAT

Asp GAT

Pro CCG

54

162

Lys AAG

Gly GGC

Thr Leu Asn ACT CT~ AAC

Tyr Thr Pro Asp Val Glu Leu Val TAC ACC CCG GAT GTC GAG CTG GTG

GIy GGC

Asn AAC

Ala GCC

36

108

Arg CGC

Tyr GI~, Glu TAT GAA GAG

Val GTG

Pro Ala CCC GCC

54

Set Gly Asn Ala Thr Leu Val His Ile Asp AGC GGC AAC GCG ACG CTG GTG CAC ATC GAC

558 16"/4

540 1620

522 1566

1512

504

1458

486

468 1404

450 1350

432 1296

414 1242

396 1188

378 1134

360 1080

342 1026

324 972

306 918

Fig. 2. Nucleotide sequence of the ilvK gene from K. pneumoniae CG21. Translation begins at the first in-flame Met codon. A potential -10 concensus sequence is underlined. TER, stop codon. The sequence has been assigned the GenBank accession No. M73842.

Asp GAC

Ala Val Asn GCG GTG AAT

Leu Glu CTGGAG

Asn Gln A]a GIy A A C CA(] G C C G G G

Tyr Gln Ala Ala GIy TAT CAG GCC GCC GGA

Arg Leu CGTTTG

Leu CTC

Arg Val Gly Leu Phe Asn CGG GTT GGG CTG TTT AAC

Thr ACC

Asn Ser Lys Ala Leu Arg AAC AGC AAG GCG CTG CGC

Pro Ile Phe Leu CCG ATC TTC CTG

Pro Asp Asp Ala CCG GAT GAT GCC

Pro Gin Met GIy Ala Ala CCG CAG ATG GGC GCC GCG

Gln Ala Lys Asn CAG GCG AAG AAC

Pro Val Set CCG GTC AGC

GIy GGC

Pro CCG

Phe Arg Ala Ala GIu Gln GIy Arg TTC CGC GCC GCC GAG CAG GGC CGG

Gln Asp Val Va] Asp Gly CAG GAT GTG GTC GAT GGC

Ile Ala ATC GCC

Ala GCC

Pro CCG

Phe Ala Gly TTC GCC GGC

Gln Leu Ala CAG CTC GCC

Arg CGC

Val Ser Leu GTT AGC CTG

Asp GAP

Thr Ala Pro ACG GCG CCG

Ile Glu Val ATC GAG GTG

Lys Tyr Ala AAA TAC GCC

Val Val Ser Asn Ala GTG GTC TCC AAC GCC

Ser Pro Val AGC CCG GTC

Thr ACG

Set Met Asp AGT ATG GAT

G]y GGC

Pro Val Val Ala Leu CCG GTG GTG GCC CTG

Lys Gln Val His Gln AAG CAG GTC CAC CAG

Ser Glu GIy Asp AGC GAA GGC GAC

Arg Ala Asp Lys Ala CGC GCC GAT AAA GCG

Asn AAC

Lys AAA

Thr Ala ACC GCG

Val GTA

Ala GCC

Met ATG

GIy GGC

Leu CTG

Cys Ser Asn TGT TCC AAC

Thr ACC

Phe Met Ala Ala Ala Val Gly Arg Ile TTT ATG GCC GCC GCC GTC GGA CGC ATT

Leu Val Thr Ser Gly Pro Gly CTG GTC ACC TCC GGT CCG GGC

Ala GCG

Lys AAA

GIy Val Ala GGC GTG GCG

Ala GCA

Val GTA

Lys AAA

Val GTC

}/is Glu Ala Asn Ala CAC GAA GCC AAC GCC

Ala GCC Pro CCG

Asp Ser Ser GAT TCC TCC

Gly GGC

Val GTC

Ile Arg Ile Ile ATT CGC ATT ATT

Set Leu Leu TCA CTG CTG

Pro CCC

Leu CTC

Arg CGC

Phe Asp TTT GAT

Phe GIy Ile TTC GGC ATC

Ala Asp GCC GAT

Asp Lys Val GAC AAG GTC

Gln GIy Val Arg Gln Val CAG GGA GTA CGC CAG GTG

His GIy CAC GGC

Ile ATC

Leu Glu Ala CTG GAA GCT

Arg Gln Trp Ala CGC CAG TGG GCG

Gln CAG

Pro Val CCG GTA

Set AGT

Gln Tyr CAG TAT

18

Lys AAA

Asp GAC

Met ATG

Asn AAC

Ala Met Trp GCG ATG TGG

Glu GAA

Pro CCG

-71 -1

TCGACCACGGGGTGCTGACC TTCGGCGAAATTCACAAGCTGATGATCGACCTGCCCGCCGACAGCGCGTT CCTGCAGGCTAATC TGCATCCCGATAATCTCGATGCCGCCATCCGTTCCGTAGAAAGTTAA~TCAC

-.J

128 1985; Law~er etal., 1987; Squires et al., 1983). We have observed multiple stop codons in all three reading frames v¢ithin the 240 bp downstream the stop codon of the gone which exclude the possibility to form a small subunit. This was not surprising since the pH 6 acetolactate-forming enzyme of En. aerogenes was shown to be a dimer of two 60-kDa subunits (Stzrmer, 1975). It is also interesting to note that a deletion of the last ten aa residues at the C terminus completely abolished ALS activity~ indicating their critical role in enzyme activity.

(e) Homology of the ilvK gone product with PDC of Zymomonas mobilis and ALS of other species When the nt sequence of ilvK and its deduced aa sequence were used to search for homologous sequences in the GenBank (updated July, 1991) using a DNASTAR program (DNASTAR Inc., Madison, Wl), significant homology of the ALS of K. pneumoniaewith ALS of other species was detected. These included ALS of bacteria, yeast and p l ~ t origins (Fig. 3). No homology was observed between the ilvK gene product and the ADC ofEn. aerogenes (Sone et al., 1989). The highest overall aa sequence homology was observed between the ALS of K. pneumoniae and the large subunit orALS II of E. coil, which reached 30.4 ~o. Extended regions with > 50% sequence identity were also observed. In addition to genes encoding ALS isozymes, a homology between a region of a 50 aa stretch beginning at aa 420 of the ALS polypeptide with the PDC of Zymomonas mobiiis (Conway etal., 1987; Reynen and Sahm, 1988) at the aa position 413 was observed (Fig. 4). This region was also conserved in all other ALS, Further experiments are

pH6 ALB ALSI ALB III ALS II ILV2

SuN PDG

420 422 421 40 0 523 508

413

~r-.'- -.~lS z

~

(f) Heterologous expression of the ilvK gone in Escherichia coli The 2.1-kb insert D N A fragment ofpTL8 was subcloned into expression vector p G E M 2 in an orientation placing the O R F of ilvK under the control of the T7 promoter. The resulting plasmid, pGEM-ALS, was transformed into JM 109(DE3) cells (Promega, Madison, WI). The JMI09 (DE3) contains an IPTG-inducible T7 RNA polymerase and can specifically transcribe any gene controlled by the T7 promoter at high level upon induction. The whole cell protein profile of IPTG-induced JM109(DE3)[pGEMALS] strain was analyzed and compared with those of JM109(DE3) and JM109(DE3)[pGEM2]. As shown in Fig. 4, a unique protein band about 60-kDa in size was observed only in the JM 109(DE3)[pGEM-ALS]. The size is consistent with the Mr of the ilvK gone product calculated from the deduced amino acid sequence. (g) Characterization of the Klebsiella pneumoniae acetolactate synthase To eliminate the possibility of contamination with the endogenous ALS activity, E. coil strain CU1147 which

I

2

3

(kDa), v AI'ff] wl~'£'d'~w ~v ~ ~n Kv VlS~ F OIL ~1~ IAIi~viva.K ~.A.U~fl~V VlCI.YJ F O I L P I ~ I ~ O A I 9 VA~l~lN DTIVVl O ! YOILPl AIATO AI q V AIClP~S LIVIL D ! O ILPl A IA.LO AI A V o Z~p~__~VLV.2]D X W S V ~ AIAIFI~I¥A V OAIP~A~N ~ I LM

qMNIqlE~SITAILqYe-LPV[VLJMqS

73

~ o D oJs ~ lvl ~ NV q l ~ - £ ] ~ ~

4472 4Sl

vDJoODOiSIFIZ M NVqlE.U~rrI~-KV I,-t~ LPlVlKZ ~;; VO[~.~JS~QLTAQIE[VAQM[VRLKILPIVII I F L

ssgS?~ 464

i~ ~ O I s L M M N I

needed to elucidate if the region is indeed a domain for pyruvate binding as previously speculated (Reynen and Sabra, 1988).

QIEI-~AITAISIIN O i I'L"MHHE

q L P ~ K Z .LI~ D[O DI-~'SJFJN M T LTIE LIS S_.l~lq A O T PIVIK I ILII

Fi},. 3, 3omparisonera conserved 50.aa stretchamongvariouspyruvate utilization enzymes.The position of the first aa in the polypeptideis indicated to the lef~of the selected part of the chains; the position of the last aa is indicated to the right. Boxed regions indicate positions identical with the aa sequence of K. pneumoniaeALS. The enzymecompared are abbreviated as follows: pH 6 ALS , acetolactate synthase of K. pneumoniae; ALS I, I! and Ill, the large subunits of ALS isozymesof Escherichia coli; PDC, pyruvate decarboxylase of Zymomonas mobilis (Conway etal., 1987; Reynen and Sahm, 1988); ILV2, ALS of Saccharomyces cerevisiae (Falco etal., 1985); SuR, ALS of Nicotiana tabacum (Lee etal., 1988)

66-

!

4534.7 Fig, 4. In rive expression of K. pneummziaegone ih,K in E. coil Lanes: 1, JMI09(DE3); 2, JMI09(DE3)[pGEM2]; 3, JMI09(DE3)[pGEMALS]. All the cellsweregrown in LB plus 0.5% glucosetill mid-logphase, induced with IPTG at a finalconcentrationof 100pg/ml for 4 h. The cells were pelleted and resuspended in 2 x sample buffer (Laemmli, 1970), boiled, and resolvedon 0.1% SDS- 10% PA gel.The markers used in this experiment were bovineserum albumin(66 kDa), chicken ovalbumin(45 kDa) and pepsin (34.7 kDa). The arrow on the right indicatesthe in rive synthesized pH 6 ALS.

129

lacks all three ALS isozymes was used as the recombinant host of pTL8 for the study. Several purification steps were carried out to obtain a partially purified ALS protein for further characterization. These steps included streptomycine sulfate and ammonium sulfate precipitation, DEAEcellulose (Whatmann, Masidstone, UK) chromatography and Sephacryl 300 (Pharmacia LKB Biotech., Uppsala, Sweden) gel filtration column. Only a single ALS peak was detected eluting from the column after the purification steps. The partially purified enzyme was then analyzed for several basic enzymatic properties. The possibility of direct conversion of pyruvate into diacetyl or acetoin by the enzyme preparation was ruled out since no detectable amount of these substances was present in the incubated reaction mixture prior to sulfuric acid decarboxylation (Table I). The enzyme apparently did not require FAD for full activity and its activity was not inhibited in the presence of 1 mM valine. The pH 6 optimum of this enzyme also suggests that it is equivalent to the pH 6 acetolactate-forming enzyme of En. aerogenes. Acetate activation, a property of the pH 6 acetolactate-forming enzyme was also tested. The enzyme activity was decreased to approx. 60~o when acetate buffer was substituted by the phosphate buffer of the same pH. To our surprise, the omission of manganese ion and thiamine pyrophosphate did not seem to affect the activity of the pH 6 ALS. The manganese ion requirement TABLE i Enzymatic properties of the pH 6 ALS obtained from Escherichia coli CU ! 147[pTL8] Conditions"

Complete reaction mix Minus H2SO 4 decarboxylation Minus ALS Minus pyruvate Minus thiamine pyrophosphate Minus MnCI2 Minus MnCI,+I mM EDTA Minus sodium acetate+phosphate buffer Complete mix+0.1 mM FAD Complete mix+l mM valine Complete mix, pH 7.0 Complete mix, pH 5.0

Acetolactate synthesizedb (#reel)

% of activity

3.36 0.11 0.04 0.00 3.13 3.21 3.17 2.11 3.27 3.48 1.96 0.94

100.0 3.3 0. I 0.0 93.1 95.5 94.3 62.8 97.3 103.6 58.3 27.9

" Complete reaction mix contained 40 mM N a'pyruvate/87/~M thiamine pyraphosphate/0.5 mM MnCl2/50 mM Na.acetate. The mixture was adjusted to pH 6.0 and ALS was added unless otherwise indicated. The reaction mixtures of various conditions at a final volume of 0.45 ml were incubated at 37°C for 20 min. b The acetolactate synthesized in each sample was decarboxylated to acetoin by incubating with 50/~1of 50% H2SO 4 at 37°C for 20 min. The acetoin was then determined colormetrically as described by Krampitz (1957). The reaction remains linear up to approx. 10/~mol/ml of acetolactate. Data are the averages of two trials.

was further tested by including EDTA to a final concentration of 1 mM in the reaction mixture. Again, no effect was observed. Although the absolute requirements of manganese and thiamine pyrophosphate have never been established for this particular enzyme, we consider that these properties may be distinct from those of the p H 6 A L S of Enterobacter, since these c o m p o n e n t s were routinely included in the assay mixture by earlier investigators. To solve the puzzle, b o t h the p H 6 ALS of En. cloacae and K. pneumoniae CG21 were purified as described by Stormer (1975) and their properties were c o m p a r e d with those of the recombinant A L S . Neither of the three enzymes required an exogenous supply o f manganese and thiamine p y r o p h o s p h a t e (not shown). It can, therefore be concluded that the iivK gene on p T L 8 encoded an A L S which was undistingushable from the p H 6 acetolactate-forming enzyme of Kiebsiella and Enterobacer in all properties tested.

ACKNOWLEDGEMENT

We thank Dr. H.E. Umbarger (Department of Biological Sciences, Purdue University) for providing the E. coil strain CUll47. We are also indebted to Drs. T. V. Wang and A. Herp for careful reading of the manuscript. This work was supported by Chang-Gung Medical College (grant CMRP278) to H.-L. Peng and National Science Council of Republic of China (grant NSC80-0412-B 18239) to H.-Y. Chang.

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