Purification, properties, and oxygen reactivity of p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa

The monooxygenase, p-hydroxybenzoate hydroxylase (4-hydroxybenzoate, NADPH:oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) has been isolated and purified from Pseudomonas aeruginosa. The reaction catalysed is linked to the pathways for degradation of aromatic compounds by microorganisms. The enzyme has been quantitatively characterized in this paper for use in the mechanistic analysis of the protein by site-directed mutagenesis. This can be achieved when the results presented are used in combination with the information on the sequence and structure of the gene for this protein and the high-resolution crystallographic data for the protein from P. fluorescens. The protein is a dimer of identical sub-units in solution, and has one FAD per polypeptide with a monomeric molecular weight of 45,000. A full steady-state kinetic analysis was carried out at the optimum pH (8.0). A Vmax of 3750 min-1 at 25 degrees C was calculated, and the enzyme has a concerted-substitution mechanism, involving the substrates, NADPH, oxygen, and p-hydroxybenzoate. Extensive analyses of the reactions of reduced enzyme with oxygen were carried out. The quality of the data obtained confirmed the mechanisms of these reactions as proposed earlier by the authors for the enzyme from P. fluorescens. It was found that the amino acid residue differences between enzyme from P. fluorescence and aeruginosa do marginally change some observed transient state kinetic parameters, even though the structure of the enzyme shows they have no direct role in catalysis. Thus, transient state kinetic analysis is an excellent tool to examine the role of amino acid residues in catalysis.     

Substitution of Arg214 at the substrate-binding site of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.

The gene encoding p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was cloned in Escherichia coli to provide DNA for mutagenesis studies on the protein product. A plasmid containing a 1.65-kbp insert of P. fluorescens chromosomal DNA was obtained and its nucleotide sequence determined. The DNA-derived amino acid sequence agrees completely with the chemically determined amino acid sequence of the isolated protein. The enzyme is strongly expressed under influence of the vector-encoded lac promotor and is purified to homogeneity in a simple three-step procedure. The relation between substrate binding, the effector role of substrate and hydroxylation efficiency was studied by use of site-directed mutagenesis. Arg214, in ion-pair interaction with the carboxy moiety of p-hydroxybenzoate, was replaced with Lys, Gln and Ala, respectively. The affinity of the free enzymes for NADPH is unchanged, whereas the affinity for the aromatic substrate is strongly decreased. For enzymes Arg214-->Ala and Arg214-->Gln, the effector role of substrate is lost. For enzyme Arg214-->Lys, binding of p-hydroxybenzoate highly stimulates the rate of flavin reduction. In the presence of substrate or substrate analogues, the reduced enzyme Arg214-->Lys fails to stabilize the 4 alpha-hydroperoxyflavin intermediate, essential for efficient hydroxylation. Like the wild-type, enzyme Arg214-->Lys is susceptible to substrate inhibition. From spectral and kinetic results it is suggested that secondary binding of the substrate occurs at the re side of the flavin, where the nicotinamide moiety of NADPH is supposed to bind.     

Molecular cloning and sequence determination of the lpd gene encoding lipoamide dehydrogenase from Pseudomonas fluorescens

The lpd gene encoding lipoamide dehydrogenase (dihydrolipoamide dehydrogenase; EC 1.8.1.4) was isolated from a library of Pseudomonas fluorescens DNA cloned in Escherichia coli TG2 by use of serum raised against lipoamide dehydrogenase from Azotobacter vinelandii. Large amounts (up to 15% of total cellular protein) of the P. fluorescens lipoamide dehydrogenase were produced by the E. coli clone harbouring plasmid pCJB94 with the lipoamide dehydrogenase gene. The enzyme was purified to homogeneity by a three-step procedure. The gene was subcloned from plasmid pCJB94 and the complete nucleotide sequence of the subcloned fragment (3610 bp) was determined. The derived amino acid sequence of P. fluorescens lipoamide dehydrogenase showed 84% and 42% homology when compared to the amino acid sequences of lipoamide dehydrogenase from A. vinelandii and E. coli, respectively. The lpd gene of P. fluorescens is clustered in the genome with genes for the other components of the 2-oxoglutarate dehydrogenase complex.     

Cloning, sequencing and expression of the L-2-hydroxyisocaproate dehydrogenase-encoding gene of Lactobacillus confusus in Escherichia coli

The gene (L-HicDH) encoding L-2-hydroxyisocaproate dehydrogenase (L-HicDH) from Lactobacillus confusus was cloned in Escherichia coli. A 69-mer oligodeoxyribonucleotide probe, derived to be complementary to the N-terminal amino acid (aa) coding sequence, was used for screening. The complete nucleotide (nt) sequence of the L-HicDH gene was determined. The 5'-end of the mRNA was mapped by primer extension and the promoter identified. Downstream from the L-HicDH gene is a typical Rho-independent terminator. The aa sequence of L-HicDH, deduced from the nt sequence, has an overall similarity of 30% to the aa sequence of L-lactate dehydrogenase (L-LDH) from Lactobacillus casei. The aa residues involved in binding of coenzyme and substrate are highly conserved in L-HicDH with respect to prokaryotic and eukaryotic L-LDHs. The L-HicDH gene could be expressed under control of phage lambda 'Leftward' and 'rightward' promoters in E. coli up to 35% of total cell protein. The enzyme produced under these conditions exhibits full specific activity and is found exclusively in soluble form.     

Sequence and relatedness in other bacteria of the Pseudomonas aeruginosa oprP gene coding for the phosphate-specific porin P

The oprP gene encoding the Pseudomonas aeruginosa phosphate-specific outer membrane porin protein OprP was sequenced. Comparison of the derived amino acid sequence with the known sequences of other bacterial porins demonstrated that OprP could be no better aligned to these porin sequences than it could to the periplasmic phosphate-binding protein PhoS of Escherichia coli. Southern hybridization and restriction mapping of the oprP gene in 37 clinical isolates and the 17 serotype strains of P. aeruginosa revealed that restriction sites in the vicinity of the oprP gene were highly conserved. Several species from the Pseudomonas fluorescens rRNA homology group contained DNA that hybridized to an oprP gene probe.     

Cloning and nucleotide sequence of the aspartase gene of Pseudomonas fluorescens

The aspartase gene (aspA) of Pseudomonas fluorescens was cloned and the nucleotide sequence of the 2,066-base-pair DNA fragment containing the aspA gene was determined. The amino acid sequence of the protein deduced from the nucleotide sequence was confirmed by N- and C-terminal sequence analysis of the purified enzyme protein. The deduced amino acid composition also fitted the previous amino acid analysis results well (Takagi et al. (1984) J. Biochem. 96, 545-552). These results indicate that aspartase of P. fluorescens consists of four identical subunits with a molecular weight of 50,859, composed of 472 amino acid residues. The coding sequence of the gene was preceded by a potential Shine-Dalgarno sequence and by a few promoter-like structures. Following the stop codon there was a structure which is reminiscent of the Escherichia coli rho-independent terminator. The G + C content of the coding sequence was found to be 62.3%. Inspection of the codon usage for the aspA gene revealed as high as 80.0% preference for G or C at the third codon position. The deduced amino acid sequence was 56.3% homologous with that of the enzyme of E. coli W (Takagi et al. (1985) Nucl. Acids Res. 13, 2063-2074). Cys-140 and Cys-430 of the E. coli enzyme, which had been assigned as functionally essential (Ida & Tokushige (1985) J. Biochem. 98, 793-797), were substituted by Ala-140 and Ala-431, respectively, in the P. fluorescens enzyme.     

Molecular characterization of the extracellular poly(3-hydroxyoctanoic acid) [P(3HO)] depolymerase gene of Pseudomonas fluorescens GK13 and of its gene product.

phaZPfi, the gene encoding the extracellular poly(3-hydroxyoctanoic acid) depolymerase of Pseudomonas fluorescens GK13, was cloned, sequenced, and characterized. It comprises 837 bp and is transcribed as a monocistronic message of about 950 bp from a putative sigma 70-like promoter 32 bp upstream of the ATG start codon. The deduced protein of 278 amino acids reveals a typical leader peptide at its N terminus. When expressed in Escherichia coli, the mature depolymerase started with Ala-23, whereas the mature enzyme purified from P. fluorescens GK13 started with both Leu-34 and Arg-35 determining proteins of 26,687 and 26,573 Da, respectively. The depolymerase is a strongly hydrophobic protein and includes the lipase consensus sequence Gly-X-Ser-X-Gly, which is known for serine hydrolases. Replacement of the central residue, Ser-172, in the corresponding sequence (Gly-Ile-Ser-Ser-Gly) of PhaZPfl with alanine resulted in complete loss of enzyme activity, indicating that the poly(3-hydroxyoctanoic acid) depolymerase belongs to the family of serine hydrolases.     

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.     

解淀粉芽孢杆菌BamHI甲基转移酶基因克隆、功能鉴定与序列分析

从解淀粉芽孢杆菌Baillus amyloliquefaciens CICIM B2125中克隆了BamHI甲基转移酶基因(bamHIM),并在大肠杆菌JM109中得到了成功表达.该基因含有1 271 bp的开放阅读框(ORF),编码423个氨基酸,成熟蛋白分子量为49 kD.该基因在自身启动子引导下,表达了具有活性的BamHI甲基转移酶(M.BamHI).该酶可以将BamHI位点的碱基甲基化.氨基酸序列分析表明该酶存在有NADB_Rossmann结构域.     

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.      

Acinetobacter cyclohexanone monooxygenase: gene cloning and sequence determination.

The gene coding for cyclohexanone monooxygenase from Acinetobacter sp. strain NCIB 9871 was isolated by immunological screening methods. We located and determined the nucleotide sequence of the gene. The structural gene is 1,626 nucleotides long and codes for a polypeptide of 542 amino acids; 389 nucleotides 5' and 108 nucleotides 3' of the coding region are also reported. The complete amino acid sequence of the enzyme was derived by translation of the nucleotide sequence. From a comparison of the amino acid sequence with consensus sequences of nucleotide-binding folds, we identified a potential flavin-binding site at the NH2 terminus of the enzyme (residues 6 to 18) and a potential nicotinamide-binding site extending from residue 176 to residue 208 of the protein. An overproduction system for the gene to facilitate genetic manipulations was also constructed by using the tac promoter vector pKK223-3 in Escherichia coli.     

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.     

Protein engineering with synthetic Escherichia coli amber suppressor genes

We have constructed synthetic genes encoding different Escherichia coli suppressor tRNAs for use in amino acid substitution studies and protein engineering. We used oligonucleotides to assemble the genes for different tRNAs with the anticodon 5' CTA 3'. The suppressor genes are expressed from a synthetic promoter derived from the promoter sequence of the E. coli lipoprotein gene. The genes have been used to suppress an amber mutation in a protein coding sequence, and the resulting altered protein has been subjected to sequence analysis to determine the nature of the amino acid inserted at the amber site. Twelve amino acids can now be added in response to the amber codon. We have employed these suppressors to study amino acid substitutions in the lac repressor.