Cloning and sequence analysis of the meso-diaminopimelate decarboxylase gene from Bacillus methanolicus MGA3 and comparison to other decarboxylase genes.

The lysA gene of Bacillus methanolicus MGA3 was cloned by complementation of an auxotrophic Escherichia coli lysA22 mutant with a genomic library of B. methanolicus MGA3 chromosomal DNA. Subcloning localized the B. methanolicus MGA3 lysA gene into a 2.3-kb SmaI-SstI fragment. Sequence analysis of the 2.3-kb fragment indicated an open reading frame encoding a protein of 48,223 Da, which was similar to the meso-diaminopimelate (DAP) decarboxylase amino acid sequences of Bacillus subtilis (62%) and Corynebacterium glutamicum (40%). Amino acid sequence analysis indicated several regions of conservation among bacterial DAP decarboxylases, eukaryotic ornithine decarboxylases, and arginine decarboxylases, suggesting a common structural arrangement for positioning of substrate and the cofactor pyridoxal 5'-phosphate. The B. methanolicus MGA3 DAP decarboxylase was shown to be a dimer (M(r) 86,000) with a subunit molecular mass of approximately 50,000 Da. This decarboxylase is inhibited by lysine (Ki = 0.93 mM) with a Km of 0.8 mM for DAP. The inhibition pattern suggests that the activity of this enzyme in lysine-overproducing strains of B. methanolicus MGA3 may limit lysine synthesis.     

S-Adenosylmethionine decarboxylase from the archaeon Methanococcus jannaschii: identification of a novel family of pyruvoyl enzymes

Polyamines are present in high concentrations in archaea, yet little is known about their synthesis, except by extrapolation from bacterial and eucaryal systems. S-Adenosylmethionine (AdoMet) decarboxylase, a pyruvoyl group-containing enzyme that is required for spermidine biosynthesis, has been previously identified in eucarya and Escherichia coli. Despite spermidine concentrations in the Methanococcales that are several times higher than in E. coli, no AdoMet decarboxylase gene was recognized in the complete genome sequence of Methanococcus jannaschii. The gene encoding AdoMet decarboxylase in this archaeon is identified herein as a highly diverged homolog of the E. coli speD gene (less than 11% identity). The M. jannaschii enzyme has been expressed in E. coli and purified to homogeneity. Mass spectrometry showed that the enzyme is composed of two subunits of 61 and 63 residues that are derived from a common proenzyme; these proteins associate in an (alphabeta)(2) complex. The pyruvoyl-containing subunit is less than one-half the size of that in previously reported AdoMet decarboxylases, but the holoenzyme has enzymatic activity comparable to that of other AdoMet decarboxylases. The sequence of the M. jannaschii enzyme is a prototype of a class of AdoMet decarboxylases that includes homologs in other archaea and diverse bacteria. The broad phylogenetic distribution of this group suggests that the canonical SpeD-type decarboxylase was derived from an archaeal enzyme within the gamma proteobacterial lineage. Both SpeD-type and archaeal-type enzymes have diverged widely in sequence and size from analogous eucaryal enzymes.     

Ribonucleotide reduction in Pseudomonas species: simultaneous presence of active enzymes from different classes.

Three separate classes of ribonucleotide reductases exist in nature. They differ widely in protein structure. Class I enzymes are found in aerobic bacteria and eukaryotes; class II enzymes are found in aerobic and anaerobic bacteria; class III enzymes are found in strict and facultative anaerobic bacteria. Usually, but not always, one organism contains only one or two (in facultative anaerobes) classes. Surprisingly, the genomic sequence of Pseudomonas aeruginosa contains sequences for each of the three classes. Here, we show by DNA hybridization that other species of Pseudomonas also contain the genes for three classes. Extracts from P. aeruginosa and P. stutzeri grown aerobically or microaerobically contain active class I and II enzymes, whereas we could not demonstrate class III activity. Unexpectedly, class I activity increased greatly during microaerobic conditions. The enzymes were separated, and the large proteins of the class I enzymes were obtained in close to homogeneous form. The catalytic properties of all enzymes are similar to those of other bacterial reductases. However, the Pseudomonas class I reductases required the continuous presence of oxygen during catalysis, unlike the corresponding Escherichia coli enzyme but similar to the mouse enzyme. In similarity searches, the amino acid sequence of the class I enzyme of P. aeruginosa was more related to that of eukaryotes than to that of E. coli or other proteobacteria, with the large protein showing 42% identity to that of the mouse, suggesting the possibility of a horizontal transfer of the gene. The results raise many questions concerning the physiological function and evolution of the three classes in Pseudomonas species.     

Identification of an l-dopa decarboxylase gene from Sorangium cellulosum So ce90

During a screening program intended to identify genes encoding enzymes typical for secondary metabolism in Sorangium cellulosum So ce90, an aromatic amino acid decarboxylase gene (ddc) was detected. Expression of ddc in Escherichia coli and subsequent enzyme assays with cell-free extracts confirmed the proposed function derived from amino acid sequence comparisons. In contrast to other aromatic amino acid decarboxylases of eukaryotic origin, the S. cellulosum Ddc converted only L-dihydroxy phenylalanine. This is the first report of a gene encoding an L-dihydroxy phenylalanine decarboxylase in bacteria.     

Cloning and nucleotide sequence of the phosphoenolpyruvate carboxylase-coding gene of Corynebacterium glutamicum ATCC13032

As a first step in determining the importance of the anaplerotic function of phosphoenolpyruvate carboxylase (PEPC) in amino acid biosynthesis, the ppc gene coding for PEPC of Corynebacterium glutamicum ATCC13032 has been cloned by complementation of an Escherichia coli ppc mutant strain. PEPC activity encoded by the cloned gene is not affected by acetyl-CoA under conditions where the E. coli enzyme is strongly activated, whereas acetyl-CoA is able to relieve inhibition by L-aspartate used singly or in combination with alpha-ketoglutarate. Amplification of the ppc gene in a C. glutamicum lysine-excreting strain resulted in increased PEPC-specific activity and lysine productivity. The nucleotide sequence of a DNA fragment of 4885 bp encompassing the ppc gene has been determined. At the amino acid level, PEPC from C. glutamicum presents overall a high degree of similarity with corresponding enzymes from three different organisms. The location of some strictly conserved regions may have important implications for PEPC activity and allostery.     

Purification and properties of pyridoxal-5''-phosphate-dependent histidine decarboxylases from Klebsiella planticola and Enterobacter aerogenes.

Histidine decarboxylases from Klebsiella planticola and Enterobacter aerogenes were purified to homogeneity and compared with the histidine decarboxylase from Morganella morganii. All three enzymes required pyridoxal 5'-phosphate as a coenzyme, showed optimal activity at pH 6.5, decarboxylated only histidine among the amino acids derived from protein, and were tetramers or dimers of identical subunits. Amino-terminal sequences of the three enzymes showed up to 81% homology through residue 33, but the enzymes differed sufficiently in amino acid composition and sequence so that no cross-reaction occurred between the K. planticola or E. aerogenes enzymes and antibodies to the decarboxylase from M. morganii. All three enzymes were inhibited by carbonyl reagents; by amino-, carboxyl-, and some methyl-substituted histidines; and by alpha-fluoromethylhistidine. These decarboxylases, all from gram-negative organisms, differed greatly in subunit structure, biogenesis, and other properties from the pyruvoyl-dependent histidine decarboxylases from gram-positive organisms described previously.     

Cloning of dapD, aroD and asd of Leptospira interrogans serovar icterohaemorrhagiae, and nucleotide sequence of the asd gene.

Metabolites such as diaminopimelate and some aromatic derivatives, not synthesized in mammalian cells, are essential for growth of bacteria. As a first step towards the design of a new human live vaccine that uses attenuated strains of Leptospira interrogans, the asd, aroD and dapD genes, encoding aspartate beta-semialdehyde dehydrogenase, 3-dehydroquinase and tetrahydrodipicolinate N-succinyltransferase, respectively, were cloned by complementation of Escherichia coli mutants. The complete nucleotide sequence of the asd gene was determined and found to contain an open reading frame capable of encoding a protein of 349 amino acids with a calculated Mr of 38,007. Comparison of this deduced L. interrogans aspartate beta-semialdehyde dehydrogenase amino acid sequence with those of the same enzyme from Saccharomyces cerevisiae and Corynebacterium glutamicum revealed 46% and 36% identity, respectively. By contrast, the identity between the L. interrogans enzyme and the Streptococcus mutans or E. coli enzymes was less than 31%. Highly conserved sequences within aspartate semialdehyde dehydrogenase from the five organisms were observed at the amino and carboxyl termini, and around the cysteine of the active site.     

Structural characterization of Escherichia coli phosphatidylserine decarboxylase

Phosphatidylserine decarboxylase of Escherichia coli is one of a small group of pyruvoyl-dependent enzymes (Satre, M., and Kennedy, E.P. (1978) J. Biol. Chem. 253, 479-483). The DNA sequence of the structural gene (psd) and partial protein sequence studies demonstrate that the enzyme contains two nonidentical subunits, alpha (Mr = 7,332) and beta (Mr = 28,579), which are derived from a single proenzyme. These two subunits are blocked at their respective amino termini. Reduction of the enzyme with NaCNBH3 in the presence of radiolabeled phosphatidylserine resulted in association of the label with the alpha subunit. Similar reduction in the presence of ammonium ions exposed a new amino terminus for the alpha subunit beginning with alanine. Therefore, the pyruvate prosthetic group is in amide linkage to the amino terminus of the alpha subunit. The amino terminus of the beta subunit was determined to be formylmethionine. The carboxyl terminus of the beta subunit was determined to be glycine as predicted by the DNA sequence. Comparison of the DNA sequence and protein sequence information revealed that the decarboxylase is made as a proenzyme (Mr = 35,893), and the predicted amino acid at the position of the pyruvate within the open reading frame of the proenzyme is serine. Therefore, as with other pyruvoyl-dependent decarboxylases, the prosthetic group is derived from serine through a post-translational cleavage of a proenzyme.     

Cyclohexadienyl dehydratase from Pseudomonas aeruginosa. Molecular cloning of the gene and characterization of the gene product.

The gene encoding cyclohexadienyl dehydratase (denoted pheC) was cloned from Pseudomonas aeruginosa by functional complementation of a pheA auxotroph of Escherichia coli. The gene was highly expressed in E. coli due to the use of the high-copy number vector pUC18. The P. aeruginosa cyclohexadienyl dehydratase expressed in E. coli was purified to electrophoretic homogeneity. The latter enzyme exhibited identical physical and biochemical properties as those obtained for cyclohexadienyl dehydratase purified from P. aeruginosa. The activity ratios of prephenate dehydratase to arogenate dehydratase remained constant (about 3.3-fold) throughout purification, thus demonstrating a single protein having broad substrate specificity. The cyclohexadienyl dehydratase exhibited Km values of 0.42 mM for prephenate and 0.22 mM for L-arogenate, respectively. The pheC gene was 807 base pairs in length, encoding a protein with a calculated molecular mass of 30,480 daltons. This compares with a molecular mass value of 29.5 kDa determined for the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since the native molecular mass determined by gel filtration was 72 kDa, the enzyme probably is a homodimer. Comparison of the deduced amino acid sequence of pheC from P. aeruginosa with those of the prephenate dehydratases of Corynebacterium glutamicum, Bacillus subtilis, E. coli, and Pseudomonas stutzeri by standard pairwise alignments did not establish obvious homology. However, a more detailed analysis revealed a conserved motif (containing a threonine residue known to be essential for catalysis) that was shared by all of the dehydratase proteins.     

Evolutionary relationships among gamma-carboxymuconolactone decarboxylases

gamma-Carboxymuconolactone decarboxylase (EC 4.1.1.44) from Azotobacter vinelandii resembled the isofunctional enzymes from Acinetobacter calcoaceticus and Pseudomonas putida. All three decarboxylases appeared to be hexamers formed by association of identical subunits of about 13,300 daltons. The A. vinelandii and P. putida decarboxylases cross-reacted immunologically with each other, and the NH2-terminal amino acid sequences of the enzymes differed in no more than 7 of the first 36 residues. In contrast, the A. calcoaceticus decarboxylase did not cross-react with the decarboxylase from A. vinelandii or P. putida; the NH2-terminal amino acid sequences of these enzymes diverged about 50% from the NH2-terminal amino acid sequence of the A. calcoaceticus decarboxylase.     

Isolation and expression of the Rhodobacter sphaeroides gene (pgsA) encoding phosphatidylglycerophosphate synthase.

The Rhodobacter sphaeroides pgsA gene (pgsARs), encoding phosphatidylglycerophosphate synthase (PgsARs), was cloned, sequenced, and expressed in both R. sphaeroides and Escherichia coli. As in E. coli, pgsARs is located immediately downstream of the uvrC gene. Comparison of the deduced amino acid sequences revealed 41% identity and 69% similarity to the pgsA gene of E. coli, with similar homology to the products of the putative pgsA genes of several other bacteria. Comparison of the amino acid sequences of a number of enzymes involved in CDP-diacylglycerol-dependent phosphatidyltransfer identified a highly conserved region also found in PgsARs. The pgsARs gene carried on multicopy plasmids was expressed in R. sphaeroides under the direction of its own promoter, the R. sphaeroides rrnB promoter, and the E. coli lac promoter, and this resulted in significant overproduction of PgsARs activity. Expression of PgsARs activity in E. coli occurred only with the E. coli lac promoter. PgsARs could functionally replace the E. coli enzyme in both a point mutant and a null mutant of E. coli pgsA. Overexpression of PgsARs in either E. coli or R. sphaeroides did not have dramatic effects on the phospholipid composition of the cells, suggesting regulation of the activity of this enzyme in both organisms.     

Pyruvate decarboxylase is like acetolactate synthase (ILV2) and not like the pyruvate dehydrogenase E1 subunit

Protein sequences of pyruvate decarboxylase (PDC) derived from cloned yeast (Saccharomyces cerevisiae) and bacterial (Zymomonas mobilis) genes were compared with each other and with sequence databases. Extensive sequence similarities were found between them and with two others: cytochrome-linked pyruvate oxidase from Escherichia coli and acetolactate synthase (ilvI in E. coli; ILV2 gene in S. cerevisiae). All catalyse decarboxylation of pyruvate using thiamine pyrophosphate (TPP) as cofactor. General overall similarity suggests common ancestry for these enzymes. None of the sequences was similar to the E1 component of pyruvate dehydrogenase from E. coli which also decarboxylates pyruvate with the help of TPP.     

Cloning, sequencing, and expression of the P-protein gene (pheA) of Pseudomonas stutzeri in Escherichia coli: implications for evolutionary relationships in phenylalanine biosynthesis

The pheA gene encoding the bifunctional P-protein (chorismate mutase:prephenate dehydratase) was cloned from Pseudomonas stutzeri and sequenced. This is the first gene of phenylalanine biosynthesis to be cloned and sequenced from Pseudomonas. The pheA gene was expressed in Escherichia coli, allowing complementation of an E. coli pheA auxotroph. The enzymic and physical properties of the P-protein from a recombinant E. coli auxotroph expressing the pheA gene were identical to those of the native enzyme from P. stutzeri. The nucleotide sequence of the P. stutzeri pheA gene was 1095 base pairs in length, predicting a 365-residue protein product with an Mr of 40,844. Codon usage in the P. stutzeri pheA gene was similar to that of Pseudomonas aeruginosa but unusual in that cytosine and guanine were used at nearly equal frequencies in the third codon position. The deduced P-protein product showed sequence homology with peptide sequences of the E. coli P-protein, the N-terminal portion of the E. coli T-protein (chorismate mutase:prephenate dehydrogenase), and the monofunctional prephenate dehydratases of Bacillus subtilis and Corynebacterium glutamicum. A narrow range of values (26-35%) for amino acid matches revealed by pairwise alignments of monofunctional and bifunctional proteins possessing activity for prephenate dehydratase suggests that extensive divergence has occurred between even the nearest phylogenetic lineages.     

The crystal structure of a bacterial class II ketol-acid reductoisomerase: domain conservation and evolution

Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) catalyzes two steps in the biosynthesis of branched-chain amino acids. Amino acid sequence comparisons across species reveal that there are two types of this enzyme: a short form (Class I) found in fungi and most bacteria, and a long form (Class II) typical of plants. Crystal structures of each have been reported previously. However, some bacteria such as Escherichia coli possess a long form, where the amino acid sequence differs appreciably from that found in plants. Here, we report the crystal structure of the E. coli enzyme at 2.6 A resolution, the first three-dimensional structure of any bacterial Class II KARI. The enzyme consists of two domains, one with mixed alpha/beta structure, which is similar to that found in other pyridine nucleotide-dependent dehydrogenases. The second domain is mainly alpha-helical and shows strong evidence of internal duplication. Comparison of the active sites between KARI of E. coli, Pseudomonas aeruginosa, and spinach shows that most residues occupy conserved positions in the active site. E. coli KARI was crystallized as a tetramer, the likely biologically active unit. This contrasts with P. aeruginosa KARI, which forms a dodecamer, and spinach KARI, a dimer. In the E. coli KARI tetramer, a novel subunit-to-subunit interacting surface is formed by a symmetrical pair of bulbous protrusions.     

A novel thermostable branching enzyme from an extremely thermophilic bacterial species, Rhodothermus obamensis

A branching enzyme (EC 2.4.1.18) gene was isolated from an extremely thermophilic bacterium, Rhodothermus obamensis. The predicted protein encodes a polypeptide of 621 amino acids with a predicted molecular mass of 72 kDa. The deduced amino acid sequence shares 42-50% similarity to known bacterial branching enzyme sequences. Similar to the Bacillus branching enzymes, the predicted protein has a shorter N-terminal amino acid extension than that of the Escherichia coli branching enzyme. The deduced amino acid sequence does not appear to contain a signal sequence, suggesting that it is an intracellular enzyme. The R. obamensis branching enzyme was successfully expressed both in E. coli and a filamentous fungus, Aspergillus oryzae. The enzyme showed optimum catalytic activity at pH 6.0-6.5 and 65 degrees C. The enzyme was stable after 30 min at 80 degrees C and retained 50% of activity at 80 degrees C after 16 h. Branching activity of the enzyme was higher toward amylose than toward amylopectin. This is the first thermostable branching enzyme isolated from an extreme thermophile.     

A SUICIDE VECTOR FOR CONSTRUCTING A LYSA INSERTION MUTATION IN ESCHERICHIA COLI

The objective of this study was to construct an insertion mutation in the lysA gene of Escherichia coli and investigate the effect of the insertion mutation on the growth rate response. The lysA gene encodes the last enzyme in the lysine biosynthetic pathway in most bacteria. A suicide plasmid pXL1 carrying a segment of the lysA sequence was transformed into E. coli SM10 that is lysogenic for λ pir. The plasmid was extracted from the transformant and confirmed to contain the segment of lysA by restriction enzyme digestion. After this confirmation, pXL1 was transferred by conjugation to an E. coli strain that cannot support replication of the plasmid. Maintenance of the selectable drug marker on the plasmid requires that the plasmid integrate into the chromosome at the lysA locus. The constructed mutant could grow in the absence of lysine supplementation although its growth rate was significantly (P < 0.05) depressed when compared to the parent strain.