Selection and characterization of a mutant of the cloned gene for mandelate racemase that confers resistance to an affinity label by greatly enhanced production of enzyme

The plasmid pSCR1 containing the gene for mandelate racemase (EC 5.1.2.2) from Pseudomonas putida (ATCC 12633) allows Pseudomonas aeruginosa (ATCC 15692) to grow on (R)-mandelate as its sole carbon source [Ransom, S. C., Gerlt, J. A., Powers, V. M., & Kenyon, G. L. (1988) Biochemistry 27, 540]; the chromosome of the P. aeruginosa host apparently does not contain the gene for mandelate racemase but does contain genes for the remaining enzymes in the mandelate pathway and enables growth on (S)-mandelate as carbon source. However, in the presence of alpha-phenylglycidate, an active-site-directed irreversible inhibitor (affinity label) of mandelate racemase, P. aeruginosa transformed with pSCR1 can utilize (S)-mandelate but not (R)-mandelate as carbon source. This inhibition of growth on (R)-mandelate provides a metabolic selection for mutants that are resistant to alpha-phenylglycidate. When (R)-mandelate is used as carbon source and alpha-phenylglycidate is present, a few colonies of P. aeruginosa transformed with pSCR1 grow slowly and appear on plates after several days. The plasmid isolated from these cells confers resistance to alpha-phenylglycidate on newly transformed cells of P. aeruginosa. This resistance to the affinity label is not due to a mutation within the primary structure of the enzyme. A single base change (C----A) located 87 bp upstream of the initiation codon for the gene for mandelate racemase was detected in three independent isolates of alpha-phenylglycidate-resistant colonies and appears responsible for a 30-fold increase in the amount of mandelate racemase encoded by the gene contained in the plasmid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mandelate pathway of Pseudomonas putida: sequence relationships involving mandelate racemase, (S)-mandelate dehydrogenase, and benzoylformate decarboxylase and expression of benzoylformate decarboxylase in Escherichia coli

The genes that encode the five known enzymes of the mandelate pathway of Pseudomonas putida (ATCC 12633), mandelate racemase (mdlA), (S)-mandelate dehydrogenase (mdlB), benzoylformate decarboxylase (mdlC), NAD(+)-dependent benzaldehyde dehydrogenase (mdlD), and NADP(+)-dependent benzaldehyde dehydrogenase (mdlE), have been cloned. The genes for (S)-mandelate dehydrogenase and benzoylformate decarboxylase have been sequenced; these genes and that for mandelate racemase [Ransom, S. C., Gerlt, J. A., Powers, V. M., & Kenyon, G. L. (1988) Biochemistry 27, 540] are organized in an operon (mdlCBA). Mandelate racemase has regions of sequence similarity to muconate lactonizing enzymes I and II from P. putida. (S)-Mandelate dehydrogenase is predicted to be 393 amino acids in length and to have a molecular weight of 43,352; it has regions of sequence similarity to glycolate oxidase from spinach and ferricytochrome b2 lactate dehydrogenase from yeast. Benzoylformate decarboxylase is predicted to be 499 amino acids in length and to have a molecular weight of 53,621; it has regions of sequence similarity to enzymes that decarboxylate pyruvate with thiamin pyrophosphate as cofactor. These observations support the hypothesis that the mandelate pathway evolved by recruitment of enzymes from preexisting metabolic pathways. The gene for benzoylformate decarboxylase has been expressed in Escherichia coli with the trc promoter, and homogeneous enzyme has been isolated from induced cells.     

Recruitment of a chromosomally encoded maleylacetate reductase for degradation of 2,4-dichlorophenoxyacetic acid by plasmid pJP4.

When Pseudomonas aeruginosa PAO1c or P. putida PPO200 or PPO300 carry plasmid pJP4, which encodes enzymes for the degradation of 2,4-dichlorophenoxyacetic acid (TFD) to 2-chloromaleylacetate, cells do not grow on TFD and UV-absorbing material with spectral characteristics of chloromaleylacetate accumulates in the culture medium. Using plasmid pRO1727, we cloned from the chromosome of a nonfluorescent pseudomonad, Pseudomonas sp. strain PKO1, 6- and 0.5-kilobase BamHI DNA fragments which contain the gene for maleylacetate reductase. When carrying either of the recombinant plasmids, pRO1944 or pRO1945, together with pJP4, cells of P. aeruginosa or P. putida were able to utilize TFD as a sole carbon source for growth. A novel polypeptide with an estimated molecular weight of 18,000 was detected in cell extracts of P. aeruginosa carrying either plasmid pRO1944 or plasmid pRO1945. Maleylacetate reductase activity was induced in cells of P. aeruginosa or P. putida carrying plasmid pRO1945, as well as in cells of Pseudomonas strain PKO1, when grown on L-tyrosine, suggesting that the tyrosine catabolic pathway might be the source from which maleylacetate reductase is recruited for the degradation of TFD in pJP4-bearing cells of Pseudomonas sp. strain PKO1.     

恶臭假单胞菌丙氨酸消旋酶基因的克隆、序列分析及表达

从恶臭假单胞菌(Pseudomonas putida)200的基因组出发,用PCR方法克隆到两个独立作用的丙氨酸消旋酶基因,称之为dadX和alr。DadX编码357个氨基酸长的多肽,计算分子量为38.82kDa,alr编码409个氨基酸长的多肽,计算分子量为44.182kDa。序列分析显示,DadX的氨基酸序列与Pseudomonas putidaKT2440,铜绿假单胞菌(Pseudomonas aeruginosa),鼠伤寒沙门氏菌(Salmonella typhimurium)和大肠杆菌(Escherichia coli)的DadX比较,相似性分别为96.64%、71.99%、44.88%和47.37%。Alr的氨基酸序列与Pseudomonas putidaKT2440比较,同源性为94.38%,而与铜绿假单胞菌(P.aeruginosa)、鼠伤寒沙门氏菌(S.typhimurium)和大肠杆菌(E.coli)的Alr比较,同源性均较低,分别为22.89%、25.72%和26.44%。在P.putida200的DadX和Alr氨基酸序列中部发现有对于酶活性至关重要的保守区域,如磷酸吡哆醛(PLP)结合位点。DadX和alr在大肠杆菌中得到表达,DadX丙氨酸消旋酶只对丙氨酸有消旋作用,而Alr丙氨酸消旋酶可以作用于丙氨酸和丝氨酸两种底物,且对丝氨酸特异性更高。Alr的表达不依赖于外源启动子,说明在其结构基因上游存在启动子结构。     

Identification and characterization of a mandelamide hydrolase and an NAD(P)+-dependent benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633

The enzymes of the mandelate metabolic pathway permit Pseudomonas putida ATCC 12633 to utilize either or both enantiomers of mandelate as the sole carbon source. The genes encoding the mandelate pathway were found to lie on a single 10.5-kb restriction fragment. Part of that fragment was shown to contain the genes coding for mandelate racemase, mandelate dehydrogenase, and benzoylformate decarboxylase arranged in an operon. Here we report the sequencing of the remainder of the restriction fragment, which revealed three further open reading frames, denoted mdlX, mdlY, and mdlD. All were transcribed in the opposite direction from the genes of the mdlABC operon. Sequence alignments suggested that the open reading frames encoded a regulatory protein (mdlX), a member of the amidase signature family (mdlY), and an NAD(P)(+)-dependent dehydrogenase (mdlD). The mdlY and mdlD genes were isolated and expressed in Escherichia coli, and the purified gene products were characterized as a mandelamide hydrolase and an NAD(P)(+)-dependent benzaldehyde dehydrogenase, respectively.     

Cloning, sequencing, and expression of the gene for NADH-sensitive citrate synthase of Pseudomonas aeruginosa

The structural gene for the allosteric citrate synthase of Pseudomonas aeruginosa has been cloned from a genomic library by using the Escherichia coli citrate synthase gene as a hybridization probe under conditions of reduced stringency. Subcloning of portions of the original 10-kilobase-pair (kbp) clone led to isolation of the structural gene, with its promoter, within a 2,083-bp length of DNA flanked by sites for KpnI and BamHI. The nucleotide sequence of this fragment is presented; the inferred amino acid sequence was 70 and 76% identical, respectively, with the citrate synthase sequences from E. coli and Acinetobacter anitratum, two other gram-negative bacteria. DEAE-cellulose chromatography of P. aeruginosa citrate synthase from an E. coli host harboring the cloned P. aeruginosa gene gave three peaks of activity. All three enzyme peaks had subunit molecular weights of 48,000; the proteins were identical by immunological criteria and very similar in kinetics of substrate saturation and NADH inhibition. Because the cloned gene contained only one open reading frame large enough to encode a polypeptide of such a size, the three peaks must represent different forms of the same protein. A portion of the cloned P. aeruginosa gene was used as a hybridization probe under stringent conditions to identify highly homologous sequences in genomic DNA of a second strain classified as P. aeruginosa and isolates of P. putida, P. stutzeri, and P. alcaligenes. When crude extracts of each of these four isolates were mixed with antiserum raised against purified P. aeruginosa citrate synthase, however, only the P. alcaligenes extract cross-reacted.     

Molecular cloning of salicylate hydroxylase genes from Pseudomonas cepacia and Pseudomonas putida

The sal gene encoding Pseudomonas cepacia salicylate hydroxylase was cloned and the sal encoding Pseudomonas putida salicylate hydroxylase was subcloned into plasmid vector pRO2317 to generate recombinant plasmids pTK3 and pTK1, respectively. Both cloned genes were expressed in the host Pseudomonas aeruginosa PAO1. The parental strain can utilize catechol, a product of the salicylate hydroxylase-catalyzed reaction, but not salicylate as the sole carbon source for growth due to a natural deficiency of salicylate hydroxylase. The pTK1- or pTK3-transformed P. aeruginosa PAO1, however, can be grown on salicylate as the sole carbon source and exhibited activities for the cloned salicylate hydroxylase in crude cell lysates. In wild-type P. cepacia as well as in pTK1- or pTK3-transformed P. aeruginosa PAO1, the presence of glucose in addition to salicylate in media resulted in lower efficiencies of sal expression P. cepacia apparently can degrade salicylate via the meta cleavage pathway which, unlike the plasmid-encoded pathway in P. putida, appears to be encoded on chromosome. As revealed by DNA cross hybridizations, the P. cepacia hsd and ht genes showed significant homology with the corresponding plasmid-borne genes of P. putida but the P. cepacia sal was not homologous to the P. putida sal. Furthermore, polyclonal antibodies developed against purified P. cepacia salicylate hydroxylase inactivated the cloned P. cepacia salicylate hydroxylase but not the cloned P. putida salicylate hydroxylase in P. aeruginosa PAO1. It appears that P. cepacia and P. putida salicylate hydroxylases, being structurally distinct, were probably derived through convergent evolution.     

Cloning and sequencing of rpoH and identification of ftsE-ftsX in Pseudomonas putida PpG1.

The rpoH gene encoding the heat-shock sigma factor of Pseudomonas putida was cloned by using its ability to complement the temperature-sensitive growth of the Escherichia coli rpoH mutant. The cloned DNA contained an open reading frame for a 284 amino acid sequence exhibiting high homology to the sigmaH proteins of P. aeruginosa and E. coli. Moreover, homologs to the cell division genes ftsX and ftsE were found immediately upstream of the rpoH gene.     

Isolation and characterization of a benzoylformate decarboxylase and a NAD+/NADP+-dependent benzaldehyde dehydrogenase involved in D-phenylglycine metabolism in Pseudomonas stutzeri ST-201

Following induction with D-phenylglycine both d-phenylglycine aminotransferase activity and benzoylformate decarboxylase activity were observed in cultures of Pseudomonas stutzeri ST-201. Induction with benzoylformate, on the other hand, induced only benzoylformate decarboxylase activity. Purification of the benzoylformate decarboxylase, followed by N-terminal sequencing, enabled the design of probes for hybridization with P. stutzeri ST-201 genomic DNA libraries. Sequencing of two overlapping genomic DNA restriction fragments revealed two open reading frames which were denoted dpgB and dpgC. Sequence alignments suggested that the genes encoded a thiamin-diphosphate-dependent decarboxylase and an aldehyde dehydrogenase, respectively. Both genes were isolated and expressed in Escherichia coli. The dpgB gene product was confirmed as a benzoylformate decarboxylase while the dpgC gene product was characterized as a NAD+/NADP+-dependent benzaldehyde dehydrogenase. In keeping with their high sequence identities (both greater than 85%) the kinetic properties of the two enzymes were similar to those of the homologous enzymes in the mandelate pathway of Pseudomonas putida ATCC 12633. However, Pseudomonas stutzeri ST-201 was unable to grow on either isomer of mandelate, and sequencing indicated that the dpgB gene did not form part of an operon. Thus it appears that the two enzymes form part of a d-phenylglycine, rather than mandelate, degrading pathway.     

Cloning, sequencing, and overexpression of a [2Fe-2S] ferredoxin gene from Escherichia coli.

Escherichia coli contains a soluble, [2Fe-2S] ferredoxin of unknown function (Knoell, H.-E., and Knappe, J. (1974) Eur. J. Biochem. 50, 245-252). Using antiserum to the purified protein to screen E. coli genomic expression libraries, we have cloned a gene (designated fdx) encoding this protein. The DNA sequence of the gene predicts a polypeptide of 110 residues after removal of the initiator methionine (polypeptide M(r) = 12,186, holoprotein M(r) = 12,358). The deduced amino acid sequence is strikingly similar to those of the ferredoxins found in animal mitochondria which function with cytochrome P450 enzymes and to the ferredoxin from Pseudomonas putida which functions with P450cam. The overall sequence identity is approximately 36% when compared with human mitochondrial and P. putida ferredoxins, and the identities include 4 cysteine residues proposed to coordinate the iron cluster. The protein was overproduced approximately 500-fold using an expression plasmid, and the holoprotein was assembled and accumulated in amounts exceeding 30% of the total cell protein. The overexpressed ferredoxin exhibits absorption, circular dichroism, and electron paramagnetic resonance spectra closely resembling those of the animal ferredoxins and P. putida ferredoxin.     

Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. I. Synthesis of enzymes by the wild type

Hegeman, G. D. (University of California, Berkeley). Synthesis of the enzymes of the mandelate pathway by Pseudomonas putida. I. Synthesis of enzymes by the wild type. J. Bacteriol. 91:1140-1154. 1966.-The control of synthesis of the five enzymes responsible for the conversion of d(-)-mandelate to benzoate by Pseudomonas putida was investigated. The first three compounds occurring in the pathway, d(-)-mandelate, l(+)-mandelate, and benzoylformate, are equipotent inducers of all five enzymes. A nonmetabolizable inducer, phenoxyacetate, also induces synthesis of these enzymes; but, unlike the metabolizable inducer-substrates, it does not elicit synthesis of enzymes that mediate steps in the pathway beyond benzoate. Under conditions of semigratuity, dl-mandelate elicits immediate synthesis at a steady rate of the first two enzymes of the pathway, but two enzymes which act below the level of benzoate are synthesized only after a considerable lag. Succinate and asparagine do not significantly repress the synthesis of the enzymes responsible for mandelate oxidation.     

Molecular cloning, characterization, and regulation of a Pseudomonas pickettii PKO1 gene encoding phenol hydroxylase and expression of the gene in Pseudomonas aeruginosa PAO1c.

A 26-kilobase BamHI restriction endonuclease DNA fragment was cloned from Pseudomonas pickettii PKO1, a strain isolated from a soil microcosm that had been amended with benzene, toluene, and xylene. This DNA fragment, cloned into vector plasmid pRO1727 and designated pRO1957, allowed Pseudomonas aeruginosa PAO1c to grow on phenol as the sole source of carbon. Physical and functional restriction endonuclease maps have been derived for the cloned DNA fragment. Two DNA fragments carried in trans and derived from subclones of pRO1957 show phenol hydroxylase activity in cell extracts of P. aeruginosa. Deletion and subcloning analyses of these fragments indicated that the gene encoding phenol hydroxylase is positively regulated. Phenol and m-cresol were shown to be inducers of the enzyme. o-Cresol and p-cresol did not induce enzymatic activity but could be metabolized by cells that had been previously exposed to phenol or m-cresol; moreover, the enzyme exhibited a rather broad substrate specificity and was sensitive to thiol-inhibiting reagents. A novel polypeptide with an estimated molecular mass of 80,000 daltons was detected in extracts of phenol-induced cells of P. aeruginosa carrying plasmid pRO1959.     

Cloning,DNA sequencing and heterologous expression of the gene for thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60 in Escherichia coli

The gene encoding the novel enzyme N-acylamino acid racemase (AAR) was cloned in recombinant phage -4 from the DNA library of Amycolatopsis sp. TS-1-60, a rare actinomycete, using antiserum against the enzyme. The cloned gene was subcloned and transformed in Escherichia coli JM105 using pUC118 as a vector. The AAR gene consists of an open-reading frame of 1104 nucleotides, which specifies a 368-amino-acid protein with a molecular mass of 39411Da. The molecular mass deduced from the AAR gene is in good agreement with the subunit molecular mass (40kDa) of AAR from Amycolatopsis sp. TS-1-60. The guanosine plus cytosine content of the AAR gene was about 70%. Although the AAR gene uses the unusual initiation codon GTG, the gene was expressed in Escherichia coli using the lac promoter of pUC118. The amount of the enzyme produced by the transformant was 16 times that produced by Amycolatopsis sp. TS-1-60. When the unusual initiation codon GTG was changed to ATG, the enzyme productivity of the transformant increased to more than 37 times that of Amycolatopsis sp. TS-1-60. In the comparison of the DNA sequence and the deduced amino acid sequence of AAR with those of known racemases and epimerases in data bases, no significant sequence homology was found. However, AAR resembles mandelate racemase in that requires metal ions for enzyme activity. Comparison of the deduced amino acid sequences of mandelate racemase and AAR revealed amino acid sequences in AAR similar to those of both the catalytic and metal-ion-binding sites of mandelate racemase.     

Molecular cloning of the nahG gene encoding salicylate hydroxylase from Pseudomonas fluorescens

A gene encoding the salicylate hydroxylase was cloned from the genomic DNA of Pseudomonas fluorescens SME11. The DNA fragment containing the nahG gene for the salicylate hydroxylase was mapped with restriction endonucleases and sequenced. The DNA fragment contained an ORF of 1,305 bp encoding a polypeptide of 434 amino acid residues. The nucleotide and amino acid sequences of the salicylate hydroxylase revealed several conserved regions with those of the enzyme encoded in P. putida PpG7: The homology of the nucleotide sequence is 83% and that of amino acid sequence is 72%. We found large conserved regions of the amino acid sequence at FAD and NADH binding regions. The FAD binding site is located at the amino terminal region and a lysine residue functions as a NADH-binding site.     

Conservation of the gene for outer membrane protein OprF in the family Pseudomonadaceae: sequence of the Pseudomonas syringae oprF gene.

The conservation of the oprF gene for the major outer membrane protein OprF was determined by restriction mapping and Southern blot hybridization with the Pseudomonas aeruginosa oprF gene as a probe. The restriction map was highly conserved among 16 of the 17 serotype strains and 42 clinical isolates of P. aeruginosa. Only the serotype 12 isolate and one clinical isolate showed small differences in restriction pattern. Southern probing of PstI chromosomal digests of 14 species from the family Pseudomonadaceae revealed that only the nine members of rRNA homology group I hybridized with the oprF gene. To reveal the actual extent of homology, the oprF gene and its product were characterized in Pseudomonas syringae. Nine strains of P. syringae from seven different pathovars hybridized with the P. aeruginosa gene to produce five different but related restriction maps. All produced an OprF protein in their outer membranes with the same apparent molecular weight as that of P.aeruginosa OprF. In each case the protein reacted with monoclonal antibody MA4-10 and was similarly heat and 2-mercaptoethanol modifiable. The purified OprF protein of the type strain P. syringae pv. syringae ATCC 19310 reconstituted small channels in lipid bilayer membranes. The oprF gene from this latter strain was cloned and sequenced. Despite the low level of DNA hybridization between P. aeruginosa and P. syringae DNA, the OprF gene was highly conserved between the species with 72% DNA sequence identity and 68% amino acid sequence identity overall. The carboxy terminus-encoding region of P. syringae oprF showed 85 and 33% identity, respectively, with the same regions of the P. aeruginosa oprF and Escherichia coli ompA genes.     

Nucleotide sequence analysis of the phosphomannose isomerase gene (pmi) of Pseudomonas aeruginosa and comparison with the corresponding Escherichia coli gene manA

Phosphomannose isomerase (PMI) has been proposed to catalyze the first step of the alginic acid biosynthetic pathway in Pseudomonas aeruginosa. The nucleotide sequence of the P. aeruginosa pmi gene contained on a 2.0-kb BamHI-SstI DNA fragment has been determined. The gene was defined by the start and stop codons and by in vitro disruption of an open reading frame of 1440 bp corresponding to a polypeptide product with a predicted Mr of 52 860. This polypeptide displayed an apparent Mr of approx. 56 000 upon electrophoresis of a maxicell extract on sodium dodecyl sulfate-polyacrylamide gels. The codon utilization of the pmi gene was distinct in the wobble base preference and influenced by the high G + C content (66 mol%) of the P. aeruginosa DNA. Computer assisted matching analysis failed to demonstrate any significant homology at the nucleotide level between the P. aeruginosa pmi and Escherichia coli manA (pmi) genes. However, sequences homologous to the P. aeruginosa pmi gene were found in other Pseudomonas species, such as P. putida and P. mendocina, and in Azotobacter vinelandii, all capable of producing alginic acid.     

Molecular cloning, coding nucleotides and the deduced amino acid sequence of P-450BM-1 from Bacillus megaterium

The gene encoding barbiturate-inducible cytochrome P-450BM-1 from Bacillus megaterium ATCC 14581 has been cloned and sequenced. An open reading frame in the 1.9 kb of cloned DNA correctly predicted the NH2-terminal sequence of P-450BM-1 previously determined by protein sequencing, and, in toto, predicted a polypeptide of 410 amino acid residues with an Mr of 47,439. The sequence is most, but less than 27%, similar to that of P-450CAM from Pseudomonas putida, so that P-450BM-1 clearly belongs to a new P-450-gene family, distinct especially from that of the P-450 domain of P-450BM-3, a barbiturate-inducible single polypeptide cytochrome P-450:NADPH-P-450 reductase from the same strain of B. megaterium (Ruettinger, R.T., Wen, L.-P. and Fulco, A.J. (1989) J. Biol. Chem. 264, 10987-10995).     

Sequence analysis of the hutH gene encoding histidine ammonia-lyase in Pseudomonas putida.

The complete nucleotide sequence of the hutH gene, encoding histidine ammonia-lyase (histidase), in Pseudomonas putida ATCC 12633 has been determined from the appropriate portions of the hut region that had been cloned into Escherichia coli. The resulting DNA sequence revealed an open reading frame of 1,530 base pairs, corresponding to a protein subunit of approximate molecular weight 53,600, in the location previously identified for the histidase gene by Tn1000 mutagenesis. Translation began at a GTG codon, but direct protein sequencing revealed that the initiating amino acid was removed posttranslationally to provide an N-terminal threonine; 11 additional residues completely agreed with the predicted amino acid sequence. This sequence excluded the possibility that a dehydroalanine unit, the postulated coenzyme for histidase, is attached at the N terminus of histidase subunits. Comparison of the P. putida histidase gene sequence with that of a Bacillus subtilis region encoding histidase revealed 42% identity at the protein level. Although the hutU (urocanase) and hutH (histidase) genes are induced by urocanate and normally are transcribed as a unit beginning with hutU, analysis of the region immediately upstream of the histidase gene revealed a potential weak promoter that may possibly be used to maintain a basal level of histidase for the generation of inducer (urocanate) when histidine levels are elevated.     

Cloning, nucleotide sequence, and overexpression of the gene coding for delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B.

The structural gene coding for the delta 5-3-ketosteroid isomerase (KSI) of Pseudomonas putida biotype B has been cloned, and its entire nucleotide sequence has been determined by a dideoxynucleotide chain termination method. A 2.1-kb DNA fragment containing the ksi gene was cloned from a P. putida biotype B genomic library in lambda gt11. The open reading frame of ksi encodes 393 nucleotides, and the amino acid sequence deduced from the nucleotide sequence agrees with the directly determined amino acid sequence (K. Linden and W. F. Benisek, J. Biol. Chem. 261:6454-6460, 1986). A putative purine-rich ribosome binding site was found 8 bp upstream of the ATG start codon. Escherichia coli BL21(DE3) transformed with the pKK-KSI plasmid containing the ksi gene expressed a high level of isomerase activity when induced by isopropyl-beta-D-thiogalactopyranoside. KSI was purified to homogeneity by a simple and rapid procedure utilizing fractional precipitation and an affinity column of deoxycholate-ethylenediamine-agarose as a major chromatographic step. The molecular weight of KSI was 14,535 (calculated, 14,536) as determined by electrospray mass spectrometry. The purified KSI showed a specific activity (39,807 mumol min-1 mg-1) and a Km (60 microM) which are close to those of KSI originally obtained from P. putida biotype B.