Characterization of the blaCARB-3 gene encoding the carbenicillinase-3 beta-lactamase of Pseudomonas aeruginosa.

We have isolated the blaCARB-3 structural gene encoding the CARB-3 carbenicillinase of Pseudomonas aeruginosa strain Cilote, tested the specificity of blaCARB-3 DNA probes and determined the nucleotide sequence of blaCARB-3. Three restriction fragment probes internal or delimiting the blaCARB-3 structural gene were hybridized with purified plasmid DNA coding for 18 other beta-lactamases (Blas). Under high-stringency conditions, only blaPSE-1, blaPSE-4, and blaCARB-4 sequences cross-hybridized with blaCARB-3. Sequencing of blaCARB-3 identified the structural gene which encodes a polypeptide product of 268 amino acids with a calculated estimated Mr of 29,246 for the mature form of the protein. Homology studies and computer analysis of primary structures confirmed that CARB-3 is a class-A Bla. The CARB-3 carbenicillinase differs from PSE-4 at two positions: Phe (PSE-4) instead of Leu188 (CARB-3), and Glu (PSE-4) instead of Ala266 (CARB-3), which changes the isoelectric value from (PSE-4) 5.4 to 5.75 (CARB-3). The possible effects of these two mutations were examined by comparisons on the 2 A crystal structure of the Staphylococcus aureus penicillinase, and they were shown to be silent substitutions causing no changes in the phenotype. The nucleic acid hybridization studies and sequence data confirmed that carbenicillinase-encoding bla genes are closely related and that blaCARB-3 is a variant of blaPSE-4.     

Nucleotide sequence and characterization of a carbenicillin-hydrolyzing penicillinase gene from Proteus mirabilis.

The structural gene of a carbenicillinase was cloned from the chromosomal DNA of Proteus mirabilis GN79. This gene codes for a protein of 270 amino acids. Alignment of the amino acid sequence with those of known beta-lactamases revealed that the enzyme is a novel class A beta-lactamase with a unique conserved triad, RTG. By using a DNA fragment of the structural gene, a lack of cross hybridization was confirmed between the DNA probe and total DNAs from natural isolates of P. mirabilis, suggesting that the carbenicillinase may not be a species-specific beta-lactamase of P. mirabilis.     

Carbenicillin-hydrolyzing penicillinases of Proteus mirabilis and the PSE-type penicillinases of Pseudomonas aeruginosa

Three carbenicillin-hydrolyzing penicillinases were found in Proteus mirabilis strains, N-3, N-29, and GN79. The former two strains were isolated in 1978, but strain GN79 was one of our stock cultures isolated in 1965. These penicillinases closely resembled each other, and the PSE-1 and PSE-4 enzymes produced by Pseudomonas aeruginosa, in their substrate profiles and kinetic properties for hydrolyzing various beta-lactams. However, differences were found in their molecular weights and isoelectric points which ranged from 22,000 to 27,000 and from 6.0 to 6.9, respectively. The antiserum against the purified penicillinase of N-29 cross-reacted with the enzyme of N-3 and inhibited its activity by more than 80%. The antiserum also reacted with the PSE-1 and PSE-4 enzymes. The antiserum did not react with the penicillinase from strain GN79 and the PSE-2 and PSE-3 enzymes of P. aeruginosa. Enzyme production in N-3 and N-29 was mediated by R plasmids.     

Nucleotide sequence of the PSE-4 carbenicillinase gene and correlations with the Staphylococcus aureus PC1 beta-lactamase crystal structure

The nucleotide sequence of the PSE-4 beta-lactamase gene from Pseudomonas aeruginosa strain Dalgleish has been determined. The structural gene encodes a polypeptide product of 252 amino acids with an estimated molecular mass of 29,246 Da for the mature form of the protein. The PSE-4 gene has limited homology with other beta-lactamases at the DNA level. An alignment of all known class A beta-lactamases permitted as to identify specific residues important for enzyme structure and function. To confirm observations based on the linear sequences, we designed a new molecular model for PSE-4 beta-lactamase based on x-ray data from the Staphylococcus aureus PC1 beta-lactamase at 2.0-A resolution. The structural similarities between PSE-4 and class A beta-lactamases are more extensive than indicated by earlier biochemical studies. The combined structural and sequence information now available for a series of beta-lactamases identifies conserved residues in these molecules, giving insight of their divergence and ancestry. Analysis of the PSE-4 flanking DNA sequences revealed an integration site common to antibiotic resistance genes inserted into transposons of the Tn21 family with the target integration sequence AAGTT.     

Insights into the molecular basis for the carbenicillinase activity of PSE-4 beta-lactamase from crystallographic and kinetic studies

PSE-4 is a class A beta-lactamase produced by strains of Pseudomonas aeruginosa and is highly active for the penicillin derivative carbenicillin. The crystal structure of the wild-type PSE-4 carbenicillinase has been determined to 1.95 A resolution by molecular replacement and represents the first structure of a carbenicillinase published to date. A superposition of the PSE-4 structure with that of TEM-1 shows a rms deviation of 1.3 A for 263 Calpha atoms. Most carbenicillinases are unique among class A beta-lactamases in that residue 234 is an arginine (ABL standard numbering scheme), while in all other class A enzymes this residue is a lysine. Kinetic characterization of a R234K PSE-4 mutant reveals a 50-fold reduction in k(cat)/K(m) and confirms the importance of Arg 234 for carbenicillinase activity. A comparison of the structure of the R234K mutant refined to 1.75 A resolution with the wild-type structure shows that Arg 234 stabilizes an alternate conformation of the Ser 130 side chain, not seen in other class A beta-lactamase structures. Our molecular modeling studies suggest that the position of a bound carbenicillin would be shifted relative to that of a bound benzylpenicillin in order to avoid a steric clash between the carbenicillin alpha-carboxylate group and the conserved side chain of Asn 170. The alternate conformation of the catalytic Ser 130 in wild-type PSE-4 may be involved in accommodating this shift in the bound substrate position.     

The D-glucosaminate dehydratase alpha-subunit from Pseudomonas fluorescens exhibits thioredoxin reductase activity

The complete amino acid sequence of the D-glucosaminate dehydratase (GADH) alpha-subunit from Pseudomonas fluorescens was determined by PCR using genomic DNA from P. fluorescens as a template. The alpha-subunit comprises 320 amino acids and has a molecular mass of about 33.9 kDa. The primary structure of the alpha-subunit demonstrates a high similarity to the structures of thioredoxin reductase (TrxR) from many prokaryotes, especially Pseudomonas aeruginosa (identity 85%, positive 91%), Vibrio cholerae (identity 73%, positive 85%), and Escherichia coli (identity 71%, positive 83%). The purified glucosaminate dehydratase alpha(2)-enzyme exhibited NADPH-dependent TrxR activity, while TrxR from E. coli showed pyridoxal 5'-phosphate (PLP)-dependent GADH activity. The TrxR from E. coli suggests that there are three cofactor binding sites, FAD, NADPH, and PLP in the enzyme and that TrxR catalyzes the FAD- and NADPH-dependent oxidation-reduction reaction and the PLP-dependent alpha,beta-elimination reaction.     

Pseudomonas aeruginosa diaminopimelate decarboxylase: evolutionary relationship with other amino acid decarboxylases

The lysA gene encodes meso-diaminopimelate (DAP) decarboxylase (E.C.4.1.1.20), the last enzyme of the lysine biosynthetic pathway in bacteria. We have determined the nucleotide sequence of the lysA gene from Pseudomonas aeruginosa. Comparison of the deduced amino acid sequence of the lysA gene product revealed extensive similarity with the sequences of the functionally equivalent enzymes from Escherichia coli and Corynebacterium glutamicum. Even though both P. aeruginosa and E. coli are Gram-negative bacteria, sequence comparisons indicate a greater similarity between enzymes of P. aeruginosa and the Gram- positive bacterium C. glutamicum than between those of P. aeruginosa and E. coli enzymes. Comparison of DAP decarboxylase with protein sequences present in data bases revealed that bacterial DAP decarboxylases are homologous to mouse (Mus musculus) ornithine decarboxylase (E.C.4.1.1.17), the key enzyme in polyamine biosynthesis in mammals. On the other hand, no similarity was detected between DAP decarboxylases and other bacterial amino acid decarboxylases.      

Gene cloning and characterization of the very large NAD-dependent l-glutamate dehydrogenase from the psychrophile Janthinobacterium lividum, isolated from cold soil

NAD-dependent l-glutamate dehydrogenase (NAD-GDH) activity was detected in cell extract from the psychrophile Janthinobacterium lividum UTB1302, which was isolated from cold soil and purified to homogeneity. The native enzyme (1,065 kDa, determined by gel filtration) is a homohexamer composed of 170-kDa subunits (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Consistent with these findings, gene cloning and sequencing enabled deduction of the amino acid sequence of the subunit, which proved to be comprised of 1,575 amino acids with a combined molecular mass of 169,360 Da. The enzyme from this psychrophile thus appears to belong to the GDH family characterized by very large subunits, like those expressed by Streptomyces clavuligerus and Pseudomonas aeruginosa (about 180 kDa). The entire amino acid sequence of the J. lividum enzyme showed about 40% identity with the sequences from S. clavuligerus and P. aeruginosa enzymes, but the central domains showed higher homology (about 65%). Within the central domain, the residues related to substrate and NAD binding were highly conserved, suggesting that this is the enzyme's catalytic domain. In the presence of NAD, but not in the presence of NADP, this GDH exclusively catalyzed the oxidative deamination of l-glutamate. The stereospecificity of the hydride transfer to NAD was pro-S, which is the same as that of the other known GDHs. Surprisingly, NAD-GDH activity was markedly enhanced by the addition of various amino acids, such as l-aspartate (1,735%) and l-arginine (936%), which strongly suggests that the N- and/or C-terminal domains play regulatory roles and are involved in the activation of the enzyme by these amino acids.     

Primary and quaternary structure of the catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa. Extensive sequence homology with the anabolic ornithine carbamoyltransferases of Escherichia coli

We have determined the complete nucleotide sequence of the arcB gene from Pseudomonas aeruginosa strain PAO and we have purified the arcB product, the catabolic ornithine carbamoyltransferase (EC 2.1.3.3), to apparent homogeneity from the same strain. The N-terminal amino acid sequence, the total amino acid composition and the subunit size of the purified enzyme were in agreement with nucleotide sequencing results, which predict a polypeptide of 336 amino acids (Mr 38,108). Crosslinking experiments suggest that the native enzyme (apparent Mr approx. 420,000) basically consists of a trimer aggregating to form nonamers or dodecamers. The arcB gene of P. aeruginosa had strong homology with the argF and argI genes which code for the anabolic ornithine carbamoyltransferase isoenzymes in Escherichia coli; 63% of the nucleotides and 57% of the amino acids were absolutely conserved in arcB and argF. This indicates a close evolutionary relationship between these genes although their products have different physiological functions in the cell. Under conditions of induction (energy depletion) the catabolic ornithine carbamoyltransferase represented greater than or equal to 10% of the total cellular protein. Like other highly expressed Pseudomonas genes, the arcB gene was found not to use seven codons which correspond to minor or weakly interacting tRNA species in E. coli.     

Evaluation of inhibition of the carbenicillin-hydrolyzing beta-lactamase PSE-4 by the clinically used mechanism-based inhibitors

Characterization of the biochemical steps in the inactivation chemistry of clavulanic acid, sulbactam and tazobactam with the carbenicillin-hydrolyzing beta-lactamase PSE-4 from Pseudomonas aeruginosa is described. Although tazobactam showed the highest affinity to the enzyme, all three inactivators were excellent inhibitors for this enzyme. Transient inhibition was observed for the three inactivators before the onset of irreversible inactivation of the enzyme. Partition ratios (k(cat)/k(inact)) of 11, 41 and 131 were obtained with clavulanic acid, tazobactam and sulbactam, respectively. Furthermore, these values were found to be 14-fold, 3-fold and 80-fold lower, respectively, than the values obtained for the clinically important TEM-1 beta-lactamase. The kinetic findings were put in perspective by determining the computational models for the pre-acylation complexes and the immediate acyl-enzyme intermediates for all three inactivators. A discussion of the pertinent structural factors is presented, with PSE-4 showing subtle differences in interactions with the three inhibitors compared to the TEM-1 enzyme.     

Sequence analysis of a gene product synthesized by Xanthomonas sp. during growth on natural rubber latex

Xanthomonas sp. secretes an extracellular protein (Mr approximately 70+/-5 kDa) during growth on purified natural rubber [poly(1,4-cis-isoprene)] but not during growth on water-soluble carbon sources such as glucose or gluconate. A 1.3 kbp DNA fragment coding for an internal part of the structural gene of the 70 kDa protein was amplified by nested polymerase chain reaction (PCR) using amino acid sequence information obtained after Edman degradation of selected trypsin-generated peptides of the purified 70 kDa protein. The PCR product was used as a DNA probe to clone the complete structural gene from genomic DNA of Xanthomonas sp. The sequenced DNA contained a 2037 bp open reading frame which coded for a polypeptide of 678 amino acids (Mr 74.6 kDa) and which included the features of the N-terminal signal peptidase cleavage site (Mr approximately 72.9 kDa for the mature protein). Analysis of the amino acid sequence revealed the presence of two heme binding motifs (CXXCH) and a approximately 20 amino acids long sequence that is conserved in the Paracoccus denitrificans and Pseudomonas aeruginosa diheme cytochrome c peroxidases (CCPs). This region includes a histidine residue (H519 in Xanthomonas sp. and H265 and H271 in the Pseudomonas strains, respectively) that is essential for activity in CCPs and that is also conserved in other bacterial oxidases. Blast analysis confirmed the relatedness of the 70 kDa protein to heme-containing oxidases and suggested that it is a member of a new family of relatively large (approximately 500 to approximately 1000 amino acids) extracellular proteins with so far unknown function being only far related in amino acid sequence to P. denitrificans and P. aeruginosa CCPs.     

A novel R-stereoselective amidase from Pseudomonas sp. MCI3434 acting on piperazine-2-tert-butylcarboxamide.

A novel amidase acting on (R,S)-piperazine-2-tert-butylcarboxamide was purified from Pseudomonas sp. MCI3434 and characterized. The enzyme acted R-stereoselectively on (R,S)-piperazine-2-tert-butylcarboxamide to yield (R)-piperazine-2-carboxylic acid, and was tentatively named R-amidase. The N-terminal amino acid sequence of the enzyme showed high sequence identity with that deduced from a gene named PA3598 encoding a hypothetical hydrolase in Pseudomonas aeruginosa PAO1. The gene encoding R-amidase was cloned from the genomic DNA of Pseudomonas sp. MCI3434 and sequenced. Analysis of 1332 bp of the genomic DNA revealed the presence of one open reading frame (ramA) which encodes the R-amidase. This enzyme, RamA, is composed of 274 amino acid residues (molecular mass, 30 128 Da), and the deduced amino acid sequence exhibits homology to a carbon-nitrogen hydrolase protein (PP3846) from Pseudomonas putida strain KT2440 (72.6% identity) and PA3598 protein from P. aeruginosa strain PAO1 (65.6% identity) and may be classified into a new subfamily in the carbon-nitrogen hydrolase family consisting of aliphatic amidase, beta-ureidopropionase, carbamylase, nitrilase, and so on. The amount of R-amidase in the supernatant of the sonicated cell-free extract of an Escherichia coli transformant overexpressing the ramA gene was about 30 000 times higher than that of Pseudomonas sp. MCI3434. The intact cells of the E. coli transformant could be used for the R-stereoselective hydrolysis of racemic piperazine-2-tert-butylcarboxamide. The recombinant enzyme was purified to electrophoretic homogeneity from cell-free extract of the E. coli transformant overexpressing the ramA gene. On gel-filtration chromatography, the enzyme appeared to be a monomer. It had maximal activity at 45 degrees C and pH 8.0, and was completely inactivated in the presence of p-chloromercuribenzoate, N-ethylmaleimide, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Ag+, Cd2+, Hg2+, or Pb2+. RamA had hydrolyzing activity toward the carboxamide compounds, in which amino or imino group is connected to beta- or gamma-carbon, such as beta-alaninamide, (R)-piperazine-2-carboxamide (R)-piperidine-3-carboxamide, D-glutaminamide and (R)-piperazine-2-tert-butylcarboxamide. The enzyme, however, did not act on the other amide substrates for the aliphatic amidase despite its sequence similarity to RamA.     

Cloning of L-amino acid deaminase gene from Proteus vulgaris

The L-amino acid degrading enzyme gene from Proteus vulgaris was cloned and the nucleotide sequence of the enzyme gene was clarified. An open reading frame of 1,413 bp starting at an ATG methionine codon was found, which encodes a protein of 471 amino acid residues, the calculated molecular weight of which is 51,518. The amino acid sequence of P. vulgaris was 58.6% identical with the L-amino acid deaminase of P. mirabilis. A significantly conserved sequence was found around the FAD-binding sequence of flavo-proteins. The partially purified wild and recombinant enzymes had the same substrate specificity for L-amino acids to form the respective keto-acids, however not for D-amino acids.     

Genetics of xanthan production in Xanthomonas campestris: the xanA and xanB genes are involved in UDP-glucose and GDP-mannose biosynthesis.

The nucleotide sequence of a 3.4-kb EcoRI-PstI DNA fragment of Xanthomonas campestris pv. campestris revealed two open reading frames, which were designated xanA and xanB. The genes xanA and xanB encode proteins of 448 amino acids (molecular weight of 48,919) and 466 amino acids (molecular weight of 50,873), respectively. These genes were identified by analyzing insertion mutants which were known to be involved in xanthan production. Specific tests for the activities of enzymes involved in the biosynthesis of UDP-glucose and GDP-mannose indicated that the xanA gene product was involved in the biosynthesis of both glucose 1-phosphate and mannose 1-phosphate. The deduced amino acid sequence of xanB showed a significant degree of homology (59%) to the phosphomannose isomerase of Pseudomonas aeruginosa, a key enzyme in the biosynthesis of alginate. Moreover, biochemical analysis and complementation experiments with the Escherichia coli manA fragment revealed that xanB encoded a bifunctional enzyme, phosphomannose isomerase-GDP-mannose pyrophosphorylase.     

Proteus mirabilis amino acid deaminase: cloning, nucleotide sequence, and characterization of aad.

Proteus, Providencia, and Morganella species produce deaminases that generate alpha-keto acids from amino acids. The alpha-keto acid products are detected by the formation of colored iron complexes, raising the possibility that the enzyme functions to secure iron for these species, which do not produce traditional siderophores. A gene encoding an amino acid deaminase of uropathogenic Proteus mirabilis was identified by screening a genomic library hosted in Escherichia coli DH5 alpha for amino acid deaminase activity. The deaminase gene, localized on a cosmid clone by subcloning and Tn5::751 mutagenesis, was subjected to nucleotide sequencing. A single open reading frame, designated aad (amino acid deaminase), which appears to be both necessary and sufficient for deaminase activity, predicts a 473-amino-acid polypeptide (51,151 Da) encoded within an area mapped by transposon mutagenesis. The predicted amino acid sequence of Aad did not share significant amino acid sequence similarity with any other polypeptide in the PIR or SwissProt database. Amino acid deaminase activity in both P. mirabilis and E. coli transformed with aad-encoding plasmids was not affected by medium iron concentration or expression of genes in multicopy in fur, cya, or crp E. coli backgrounds. Enzyme expression was negatively affected by growth with glucose or glycerol as the sole carbon source but was not consistent with catabolite repression.     

Cloning and sequencing of a Deleya marina gene encoding for alginate lyase

A bacterial strain N-1 was isolated as a decomposer of alginate and identified as Deleya marina. The alyA encoding for alginate lyase was cloned into Escherichia coli. The structural gene, located on a 1.9-kb SalI fragment, revealed 1,122 bp encoding a mature protein of 348 amino acids and a signal peptide of 26 amino acids. The deduced amino acid sequence of the D. marina alginate lyase showed high homology to AlgL of Pseudomonas aeruginosa with 63% identity and belonging to class 1 by hydrophobic cluster analysis.     

Isolation of the braZ gene encoding the carrier for a novel branched-chain amino acid transport system in Pseudomonas aeruginosa PAO.

The braZ gene for a novel branched-chain amino acid transport system in Pseudomonas aeruginosa PAO was isolated and characterized. Determination of the nucleotide sequence showed that the braZ gene comprises 1,311 nucleotides specifying a protein of 437 amino acids. Hydropathy analysis suggested that the product is an integral membrane protein with 12 membrane-spanning segments. The amino acid sequence showed extensive homology to those of the braB and brnQ gene products, branched-chain amino acid carriers of P. aeruginosa and Salmonella typhimurium, respectively. By using the T7 RNA polymerase-promoter system, the braZ gene product was identified as a protein of an apparent Mr of 34,000 on a sodium dodecyl sulfate-polyacrylamide gel. Properties of the transport system encoded by braZ were studied by using P. aeruginosa PAO3537, defective in both the high- and low-affinity branched-chain amino acid transport systems (LIV-I and LIV-II, respectively). The transport system encoded by braZ was found to be another effective branched-chain amino acid transport system in P. aeruginosa PAO and was thus designated as LIV-III. This system is specific for isoleucine and valine, giving the same Km value of 12 microM for these amino acids. The system was found, however, to have a very low affinity for leucine, with a Km value of 150 microM, which contrasts with the substrate specificities of LIV-I and LIV-II.     

Characterization of a new (R)-hydroxynitrile lyase from the Japanese apricot Prunus mume and cDNA cloning and secretory expression of one of the isozymes in Pichia pastoris

PmHNL, a hydroxynitrile lyase from Japanese apricot ume (Prunus mume) seed was purified to homogeneity by ammonium sulfate fractionation and chromatographic steps. The purified enzyme was a monomer with molecular mass of 58 kDa. It was a flavoprotein similar to other hydroxynitrile lyases of the Rosaceae family. It was active over a broad temperature, and pH range. The N-terminal amino acid sequence (20 amino acids) was identical with that of the enzyme from almond (Prunus dulcis). Based on the N-terminal sequence of the purified enzyme and the conserved amino acid sequences of the enzymes from Pr. dulcis, inverse PCR method was used for cloning of a putative PmHNL (PmHNL2) gene from a Pr. mume seedling. Then the cDNA for the enzyme was cloned. The deduced amino acid sequence was found to be highly similar (95%) to that of an enzyme from Pr. serotina, isozyme 2. The recombinant Pichia pastoris transformed with the PmHNL2 gene secreted an active enzyme in glycosylated form.