Identification and genetic characterization of the Pseudomonas aeruginosaleuB gene encoding 3-isopropylmalate dehydrogenase

The Pseudomonas aeruginosa leuB gene, encoding 3-isopropylmalate dehydrogenase, was identified upstream of asd, encoding aspartate-β-semialdehyde dehydrogenase. Genetic analysis indicated that leuB is identical to the previously mapped gene defined by the leu-10 allele. The chromosomal leuB locus was inactivated by gene replacement. The insertions had no adverse effect on expression of the downstream asd gene but resulted in leucine auxotrophy. The leuB gene encodes a protein containing 360 amino acids (with a molecular weight of 39153), which was expressed in Escherichia coli as a M, 42000 protein. The results suggested that, in contrast to the situation in other bacteria (E. coli, Salmonella typhimurium and Bacillus subtilis) the P. aeruginosa leuB gene is physically separated from the genes encoding the other enzymes of the isopropylmalate pathway. Received: 15 August 1996 / Accepted: 23 October 1996     

Isolation of a gene encoding 3-isopropylmalate dehydrogenase from rice

3-Isopropylmalate dehydrogenase (IPMDH, EC 1.1.1.85) is a key enzyme in the biosynthesis of leucine and glucosinolates in plants. IPMDH is a bifunctional dimeric enzyme that catalyzes dehydrogenation and decarboxylation reactions in the presence of NAD⁺. The leuB gene encoding IPMDH has been identified in a variety of bacteria and some plants. In this study, we analyze the gene for IPMDH from Oryza sativa (OsIPMDH). The analysis of an EST sequence and rice genome revealed a full-length open reading frame encoding 389 amino acids that correspond to a protein of approximately 41.2 kD. The predicted amino acid sequence of OsIPMDH was highly homologous with the IPMDHs from plants and bacteria. The OsIPMDH expression analysis in a leuB mutant of Escherichia coli revealed that OsIPMDH was capable of functionally complementing the leuB mutant. These results indicate that OsIPMDH encodes for a protein of 3-isopropylmalate dehydrogenase in rice.     

Cloning and expression of the 3-isopropylmalate dehydrogenase gene from Candida albicans

Abstract A variety of Saccharomyces cerevisiae genes e.g. HIS3, LEU2, TRP1, URA3 , are expressed in Escherichia coli and have been isolated by complementation of mutations in the corresponding E. coli genes [1]. The LEU2 gene was one of the first S. cerevisiae genes to be isolated in this way [2], and its isolation led to the development of transformation systems for S. cerevisiae [3,4]. The leuB gene in E. coli [5] and the LEU2 gene in S. cerevisiae [6] both code for 3-isopropylmalate dehydrogenase (3-IMDH; EC 1.1.1.85) which is essential for the biosynthesis of leucine in both organisms. This paper describes the cloning of a fragment of C. albicans DNA carrying the gene for 3-IMDH which will be useful in the development of transformation methods in C. albicans .     

Molecular cloning and nucleotide sequence of the 3-isopropylmalate dehydrogenase gene of Candida utilis

A 3-isopropylmalate dehydrogenase (3-IMDH, EC 1.1.1.85) gene was cloned from a gene library of Candida utilis. One of the plasmids, pYKL30, could complement Escherichia coli leuB and Saccharomyces cerevisiae leu2 auxotrophs; a 2.2 kb HindIII fragment subcloned in pBR322 could still complement the leuB mutation. Southern hybridization confirmed that this fragment was derived from C. utilis. An open reading frame of 1089 bp that corresponded to a polypeptide of 363 amino acids, one residue shorter than the 3-IMDH of S. cerevisiae, was found in the cloned fragment. The homology between the 3-IMDHs of C. utilis and S. cerevisiae was 76.2% in nucleotides and 85.4% in amino acids. In contrast, the homology between the 3-IMDHs of C. utilis and Thermus thermophilus was much smaller and was restricted to some regions of the gene.     

Cold adaptation of the thermophilic enzyme 3-isopropylmalate dehydrogenase

We have performed random mutagenesis coupled with selection to isolate mutant enzymes with high catalytic activities at low temperature from thermophilic 3-isopropylmalate dehydrogenase (IPMDH) originally isolated from Thermus thermophilus. Five cold-adapted mutant IPMDHs with single-amino-acid substitutions were obtained and analyzed. Kinetic analysis revealed that there are two types of cold-adapted mutant IPMDH: k(cat)-improved (improved in k(cat)) and K(m)-improved (improved in k(cat)/K(m)) types. To determine the mechanisms of cold adaptation of these mutants, thermodynamic parameters were estimated and compared with those of the Escherichia coli wild-type IPMDH. The Delta G(m) values for Michaelis intermediate formation of the k(cat)-improved-type enzymes were larger than that of the T. thermophilus wild-type IPMDH and similar to that of the E. coli wild-type IPMDH. The Delta G(m) values of K(m)-improved-type enzymes were smaller than that of the T. thermophilus wild-type IPMDH. Fitting of NAD(+) binding was improved in the K(m)-improved-type enzymes. The two types of cold-adapted mutants employed one of the two strategies of E. coli wild-type IPMDH: relative destabilization of the Michaelis complex in k(cat)-improved-type, and destabilization of the rate-limiting step in K(m)-improved type mutants. Some cold-adapted mutant IPMDHs retained thermostability similar to that of the T. thermophilus wild-type IPMDH.     

Cloning and nucleotide sequence of the glpD gene encoding sn-glycerol-3-phosphate dehydrogenase of Pseudomonas aeruginosa.

Nitrosoguanidine-induced Pseudomonas aeruginosa mutants which were unable to utilize glycerol as a carbon source were isolated. By utilizing PAO104, a mutant defective in glycerol transport and sn-glycerol-3-phosphate dehydrogenase (glpD), the glpD gene was cloned by a phage mini-D3112-based in vivo cloning method. The cloned gene was able to complement an Escherichia coli glpD mutant. Restriction analysis and recloning of DNA fragments located the glpD gene to a 1.6-kb EcoRI-SphI DNA fragment. In E. coli, a single 56,000-Da protein was expressed from the cloned DNA fragments. An in-frame glpD'-'lacZ translational fusion was isolated and used to determine the reading frame of glpD by sequencing across the fusion junction. The nucleotide sequence of a 1,792-bp fragment containing the glpD region was determined. The glpD gene encodes a protein containing 510 amino acids and with a predicted molecular weight of 56,150. Compared with the aerobic sn-glycerol-3-phosphate dehydrogenase from E. coli, P. aeruginosa GlpD is 56% identical and 69% similar. A similar comparison with GlpD from Bacillus subtilis reveals 21% identity and 40% similarity. A flavin-binding domain near the amino terminus which shared the consensus sequence reported for other bacterial flavoproteins was identified.     

Isolation and characterization of the yeast gene encoding the MDH3 isozyme of malate dehydrogenase.

The MDH3 isozyme of Saccharomyces cerevisiae was purified from a haploid strain containing disruptions in genomic loci encoding the mitochondrial MDH1 and nonmitochondrial MDH2 isozymes. Partial amino acid sequence analysis of the purified enzyme was conducted and used to plan polymerase chain reaction techniques to clone the MDH3 gene. The isolated gene was found to encode a 343-residue polypeptide with a molecular weight of 37,200. The deduced amino acid sequence was closely related to those of MDH1 (50% residue identity) and of MDH2 (43% residue identity). The MDH3 sequence was found to contain a carboxyl-terminal SKL tripeptide, characteristic of many peroxisomal enzymes, and immunochemical analysis was used to confirm organellar localization of the MDH3 isozyme. Levels of MDH3 were determined to be elevated in cells grown with acetate as a carbon source, and under these conditions, MDH3 contributed approximately 10% of the total cellular malate dehydrogenase activity. Disruption of the chromosomal MDH3 locus produced a reduction in cellular growth rates on acetate, consistent with the presumed function of this isozyme in the glyoxylate pathway of yeast. Combined disruption of MDH1, MDH2, and MDH3 loci in a haploid strain resulted in the absence of detectable cellular malate dehydrogenase activity.     

Restriction mapping and partial characterization of β-isopropylmalate dehydrogenase gene cloned from Citrobacter freundii

The β-isopropylmalate dehydrogenase gene of Citrobacter freundii was cloned in both Escherichia coli and Bacillus subtilis using pLS353 as a vector and PstI as an enzyme. The molecular weight of pCBL 1–7 containing β-isopropylmalate dehydrogenase gene was 3.4–7.7 megadalton (Md) and the restriction enzyme patterns of the plasmids were analyzed with enzymes such as BaII, BgII, EcoRI and SstII. The enzyme activities in both E. coli and B. subtilis transformants were 4–8-fold higher than those in the present strains.     

Identification of the Pseudomonas aeruginosa glmM gene, encoding phosphoglucosamine mutase

A search for a potential algC homologue within the Pseudomonas aeruginosa PAO1 genome database has revealed an open reading frame (ORF) of unknown function, ORF540 in contig 54 (July 1999 Pseudomonas genome release), that theoretically coded for a 445-amino-acid-residue polypeptide (I. M. Tavares, J. H. Leit?o, A. M. Fialho, and I. Sá-Correia, Res. Microbiol. 150:105-116, 1999). The product of this gene is here identified as the phosphoglucosamine mutase (GlmM) which catalyzes the conversion of glucosamine-6-phosphate to glucosamine-1-phosphate, an essential step in the formation of the cell wall precursor UDP-N-acetylglucosamine. The P. aeruginosa gene has been cloned into expression vectors and shown to restore normal peptidoglycan biosynthesis and cell growth of a glmM Escherichia coli mutant strain. The GlmM enzyme from P. aeruginosa has been overproduced to high levels and purified to homogeneity in a six-histidine-tagged form. Beside its phosphoglucosamine mutase activity, the P. aeruginosa enzyme is shown to exhibit phosphomannomutase and phosphoglucomutase activities, which represent about 20 and 2% of its GlmM activity, respectively.     

Cloning of a cDNA for rape chloroplast 3-isopropylmalate dehydrogenase by genetic complementation in yeast

Both insect and mammalian genes have previously been cloned by genetic complementation in yeast. In the present report, we show that the method can be applied also to plants. Thus, we have cloned a rape cDNA for 3-isopropylmalate dehydrogenase (IMDH) by complementation of a yeast leu2 mutation. The cDNA encodes a 52 kDA protein which has a putative chloroplast transit peptide. The in vitro made protein is imported into chloroplasts, concomitantly with a proteolytic cleavage. We conclude that the rape cDNA encodes a chloroplast IMDH. However, Southern analysis revealed that the corresponding gene is nuclear. In a comparison of IMDH sequences from various species, we found that the rape IMDH is more similar to bacterial than to eukaryotic proteins. This suggests that the rape gene could be of chloroplast origin, but has moved to the nucleus during evolution.     

Purification and characterization of 3-isopropylmalate dehydrogenase from a thermoacidophilic archaebacterium Sulfolobus sp. strain 7

Abstract 3-Isopropylmalate dehydrogenase was purified (about 2000-fold) to homogeneity for the first time from an archaebacterium, Sulfolobus sp. strain 7. The enzyme showed an apparent molecular mass of about 110 kDa by gel filtration and a single 36-kDa polypeptide band on SDS-PAGE, suggesting tri- or tetrameric structure. The p I value was 6.9. The N-terminal amino acid sequence was similar to enzymes from other sources. The enzyme activity was greatly stimulated by the presence of Mn²⁺, Cd²⁺, Mg²⁺, or Co²⁺. In contrast to 3-isopropylmalate dehydrogenase from other sources, monovalent cations such as K²⁺ and Na²⁺ were neither essential for activity nor stability of the protein. The enzyme was extraordinarily thermostable.     

Isolation and characterization of a gene encoding meso-diaminopimelate dehydrogenase fromGlycine max

A detailed characterization of the lysine biosynthetic pathway in plants is yet to be completed. It is, however, assumed that the diaminopimelic acid pathway exists in the plant kingdom, as commonly described forEscherichia coli. Modification and refinement of lytic complementation, a technique previously utilized in bacterial systems, facilitated the isolation of a functional Diaminopimelate Dehydrogenase gene from aGlycine max nuclear gene library. The isolated gene codes for the enzyme meso-diaminopimelate dehydrogenase. The coding capacity for the enzyme was originally contained on a 6.6kb fragment in a Charon 4a soybean gene bank. Subcloning of the 6.6kb fragment resulted in the recombinant plasmid pMW75. Subsequent subcloning resulted in a 4.05 kb fragment contained in pLW14. One region of homology was observed upon hybridization to EcoR1 digested soybean DNA. Homologous sequences were also observed in Triticum DNA. Meso-diaminopimelate dehydrogenase activity was demonstrated inGlycine max embryos. Maximum enzymatic activity of the cloned enzyme was observed at a pH of 8.0. The enzyme encoded by the soybean gene has an apparent molecular weight of 67 000.