Evolutionary relationships among bacterial carbamoyltransferases

An immunological approach was used for the study of ornithine carbamoyltransferase (OTCase) evolution in bacteria. Antisera were prepared against the anabolic and catabolic OTCases of Pseudomonas aeruginosa and Aeromonas formicans as well as against OTCase and putrescine carbamoyltransferases from Streptococcus faecalis; these antisera were then tested against the unpurified OTCases, either anabolic or catabolic, of 34 bacterial strains. Extensive cross-reactions were observed between the antisera to catabolic OTCases from P. aeruginosa, A. formicans and S. faecalis and the catabolic enzymes from other species or genera. These antisera cross-reacted also with the anabolic OTCases of strains of the Enterobacteriaceae but not with the anabolic OTCases of the same species or of other species or genera. The cross-reaction measured between the antisera against P. aeruginosa anabolic OTCase and the anabolic OTCases of other Pseudomonas were largely in agreement with the phylogenic subdivision of Pseudomonas proposed by N. J. Palleroni. The correlation was also significantly higher with the anabolic enzyme of an archaeobacterium, Methanobacterium thermoaceticum, than with the catabolic or anabolic OTCases from other genera in the eubacterial line. The antiserum raised against A. formicans anabolic OTCase was quite specific for its antigen and appeared to be raised against the heaviest of the various oligomeric structures of the enzyme.     

Purification and characterization of Arabidopsis ornithine transcarbamoylase (OTCase), a member of a distinct and evolutionarily-conserved group of plant OTCases

We affinity-purified an ornithine transcarbamoylase (carbamoyl phosphate:L-ornithine carbamoyltransferase; EC 2.1.3.3) 676-fold to near homogeneity from leaf tissues of Arabidopsis thaliana L. cv. Columbia. The purified OTCase protein exhibited a molecular mass of 37 kDa on SDS-PAGE gels and exhibited a pI = 6.8. A 41-kDa polypeptide was immunoprecipitated from Arabidopsis leaf poly(A)⁺ RNA in vitro translation products by pea OTCase antiserum. This precursor OTCase (pOTCase) is the predicted size (41 170 Da) for a polypeptide encoded by an Arabidopsis OTCase cDNA. Characteristics of N-terminal residues of the deduced amino acid sequence of this pOTCase suggest that it is a chloroplast-targeted protein. The sequences of plant OTCases suggest that they represent a distinct and evolutionarily-conserved group of OTCases. No evidence was found for OTCase isoenzymes in Arabidopsis leaf tissues. The Arabidopsis pOTCase was poorly-expressed in Escherichia coli strain TB-2, an OTCase-deficient mutant, and did not complement the mutant on arginine-minus selection medium.     

Converting catabolic ornithine carbamoyltransferase to an anabolic enzyme

Pseudomonas aeruginosa has an anabolic and a catabolic ornithine carbamoyltransferase (OTCase). In vitro, these homologous enzymes catalyze the same reaction (ornithine + carbamoyl phosphate (CP) in equilibrium citrulline + Pi), yet in vivo they function unidirectionally owing to specific kinetic properties. The catabolic OTC-ase cannot promote the anabolic reaction (citrulline formation) in vivo because of a sigmoidal CP saturation curve and a high CP concentration for half-maximal velocity. The structural basis for this kinetic specialization was examined. The catabolic OTCase lost most of its homotropic cooperativity and gained anabolic activity when an amino acid residue near the CP binding site, Glu-106, was replaced by alanine or glycine. In the anabolic OTCase of Escherichia coli the glutamine residue corresponding to Glu-106 was exchanged for glutamate; however, in this case no CP cooperativity was acquired. Thus, in catabolic OTCase, sequence features in addition to Glu-106 are important for sigmoidal CP saturation, and such a sequence was identified in the C-terminal part. By an in vivo gene fusion technique the 9 C-terminal amino acids of catabolic OTCase were replaced by the homologous 8 amino acids from anabolic OTCase of E. coli; the hybrid enzyme had a markedly reduced homotropic cooperativity. This gene fusion method should be generally useful for directed enzyme evolution.     

Catabolic ornithine transcarbamylase of Halobacterium halobium (salinarium): purification, characterization, sequence determination, and evolution.

Halobacterium halobium (salinarium) is able to grow fermentatively via the arginine deiminase pathway, which is mediated by three enzymes and one membrane-bound arginine-ornithine antiporter. One of the enzymes, catabolic ornithine transcarbamylase (cOTCase), was purified from fermentatively grown cultures by gel filtration and ammonium sulfate-mediated hydrophobic chromatography. It consists of a single type of subunit with an apparent molecular mass of 41 kDa. As is common for proteins of halophilic Archaea, the cOTCase is unstable below 1 M salt. In contrast to the cOTCase from Pseudomonas aeruginosa, the halophilic enzyme exhibits Michaelis-Menten kinetics with both carbamylphosphate and ornithine as substrates with Km values of 0.4 and 8 mM, respectively. The N-terminal sequences of the protein and four peptides were determined, comprising about 30% of the polypeptide. The sequence information was used to clone and sequence the corresponding gene, argB. It codes for a polypeptide of 295 amino acids with a calculated molecular mass of 32 kDa and an amino acid composition which is typical of halophilic proteins. The native molecular mass was determined to be 200 kDa, and therefore the cOTCase is a hexamer of identical subunits. The deduced protein sequence was compared to the cOTCase of P. aeruginosa and 14 anabolic OTCases, and a phylogenetic tree was constructed. The halobacterial cOTCase is more distantly related to the cOTCase than to the anabolic OTCase of P. aeruginosa. It is found in a group with the anabolic OTCases of Bacillus subtilis, P. aeruginosa, and Mycobacterium bovis.     

Cloning, Sequence, and Expression of the L-(+) Lactate Dehydrogenase of Streptococcus bovis

The ldh gene encoding the fructose-1,6-diphosphate-dependent L-(+) lactate dehydrogenase from the ruminal bacterium Streptococcus bovis was cloned and sequenced. A genomic library of S. bovis JB1 DNA was constructed in lambda ZAP II and screened by use of a heterologous probe derived from the cloned Streptococcus mutans ldh gene. Several clones were isolated that contained a common 2.9-kb fragment as determined by restriction analysis. Nucleotide sequence analysis revealed a 987-bp open reading frame with extensive homology to Streptococcus thermophilus and S. mutans ldh nucleic acid and amino acid sequences. Expression of the cloned S. bovis ldh gene in Escherichia coli was confirmed by the ability to complement the ldh mutation of E. coli FMJ39, by using an in-gel activity screen and by enzymatic assay. Increased LDH activity was observed in S. bovis JB1 containing the cloned ldh genes on a multicopy plasmid. Received: 15 October 1996 / Accepted: 3 December 1996     

Immunogenic protein MPB57 from Mycobacterium bovis BCG: Molecular cloning, nucleotide sequence and expression

Using a single-probe method, we have cloned the gene for an immunogenic MPB57 protein of Mycobacterium bovis BCG. The nucleotide sequence includes an ORF of 300 base pairs encoding a protein of 99 amino acids with an M_r of 10 818. This cloned gene was expressed in an Escherichia coli expression vector to give a mature protein which reacted with a polyclonal antibody raised against MPB57.     

Identification of intracellular amylase activity in Streptococcus bovis and Streptococcus salivarius

The ruminal bacterium Streptococcus bovis has been demonstrated to produce an extracellular amylase activity. We previously reported on the cloning of a gene from S. bovis encoding for what was initially believed to be the extracellular amylase. DNA sequence analyses indicated that the amylase produced by the cloned gene did not match the N-terminus amino acid sequence of the purified extracellular amylase and contained no apparent leader sequence for secretion. Analyses of crude extracts demonstrated the presence of an intracellular amylase in S. bovis JB1 that differed in molecular weight (56,000) from that of the extracellular amylase (70,000). The 56,000 molecular weight amylase was identical to the amylase produced by Escherichia coli containing the cloned amylase gene. Low levels of intracellular amylase activity were also detected in other strains of S. bovis and also Streptococcus salivarius. Introduction of the plasmid pVA838 containing the cloned amylase gene into S. bovis and S. sanguis resulted in enhanced intracellular amylase production by both organisms. The amylase gene has been sequenced, and analysis of the deduced amino acid sequence for the amylase indicates a high degree of similarity with secreted amylases from Bacillus species.Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Molecular cloning of the phospholipase D gene from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. YU100 and its expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The gene for phospholipase D (PLD) of Streptomyces sp. YU100 was cloned from λ phage library and hetero-logously expressed in Escherichia coli. Using an amplified gene fragment based on the consensus sequences of streptomycetes PLDs, λ phage library of Streptomyces sp. YU100 chromosomal DNA was screened. The sequencing result of BamHI-digested 3.8 kb fragment in a positive phage clone revealed the presence of an open reading frame of a full sequence of PLD gene encoding a 540-amino acid protein including 33-amino acid signal peptide. The deduced amino acid sequence showed a high homology with other Streptomyces PLDs, having the highly conserved ‘HKD’ motifs. The PLD gene excluding signal peptide sequence was amplified and subcloned into a pET-32b(+) expression vector in E. coli BL21(DE3). The recombinant PLD was purified by nickel affinity chromatography and compared the enzyme activity with wild-type PLD. The results imply that the recombinant PLD produced by E. coli had the nearly same enzyme activity as PLD from Streptomyces sp. YU100.     

Cloning and Sequence Analysis of cDNA encoding Rhizopus niveus Lipase

Complementary DNA encoding Rhizopus niveus lipase (RNL) was isolated from the R. niveus IF04759 cDNA library using a synthetic oligonucleotide corresponding to the amino acid sequence of the enzyme. A clone, which had an insert of 1.0 kilobase pairs, was found to contain the coding region of the enzyme. The lipase gene was expressed in Escherichia coli as a lacZ fusion protein. The mature RNL consisted of 297 amino acid residues with a molecular mass of 32 kDa. The RNL sequence showed significant overall homology to Rhizomucor miehei lipase and the putative active site residues were strictly conserved.     

Association of the Rv0679c protein with lipids and carbohydrates in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>/<Emphasis Type="Italic">Mycobacterium bovis</Emphasis> BCG

The Rv0679c gene in Mycobacterium tuberculosis H37Rv encodes a protein with a predicted molecular mass of 16,586 Da consisting of 165 amino acids which contains a putative N-terminal signal sequence and a consensus lipoprotein-processing motif. Globomycin treatment, Triton X-114 separation and mass spectrometry analyses clarified a property of the Rv0679c protein as a lipoprotein. In addition, trifluoromethanesulphonic acid treatment of the lysate revealed an association of the recombinant Rv0679c protein with carbohydrates. The Rv0679c protein homolog of Mycobacterium bovis BCG was also expressed as the protein associated with lipids and carbohydrates. In Western blot analysis, each of the protein homolog and Lipoarabinomannan (LAM) was detected as a similar pattern by anti-Rv0679c and anti-LAM antibodies, respectively. Interestingly, the Rv0679c protein was detected in commercially available LAM purified from M. tuberculosis. Inhibition assay of LAM synthesis in M. bovis BCG by ethambutol showed an altered migration pattern of the Rv0679c protein to low molecular mass similar to that of LAM. The results suggest that the Rv0679c protein exists as a tight complex with LAM in M. tuberculosis/M. bovis BCG.     

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.     

Cloning of a superoxide dismutase gene from Listeria ivanovii by functional complementation in Escherichia coli and characterization of the gene product.

Summary A gene encoding superoxide dismutase (EC 1.15.1.1., SOD) was isolated from a plasmid library of chromosomal DNA from Listeria ivanovii by functional complementation of an SOD-negative Escherichia coli host. The nucleotide sequence of the cloned gene was determined and contained an open reading frame which codes for a protein of 202 amino acid residues (calculated molecular weight 22 755 Da including the amino-terminal methionine residue). Comparison of the deduced amino acid sequence of L. ivanovii SOD with previously reported SOD amino acid sequences revealed considerable homologies with Fe- and Mn-dependent SODs. Enzymatic analyses using cell lysates and the purified recombinant enzyme indicated that this SOD is manganese-dependent. The recombinant SOD accounted for up to 30% of the total soluble protein in recombinant E. coli and protected sodA sodB mutants against the toxic effects of paraquat. Subunits of the recombinant Listeria SOD and of both E. coli SODS formed enzymatically active hybrids in vivo.Some of our preliminary observations have been published as a conference report of SOD V (Jerusalem, 1989) in Free Rad Res Commun (1991) 12–13:371     

Cloning and Characterization of the Gene Encoding Pyrroloquinoline Quinone-dependent Poly (vinyl alcohol) Dehydrogenase of Pseudomonas sp. Strain VM15C

A gene library of poly (vinyl alcohol) (PVA)-degrading Pseudomonas sp. strain VM15C was constructed in Escherichia coli with the vector pUC18. Screening of this library with a chromogenic PVA dehydrogenase assay resulted in the isolation of a clone that carries the gene (pdh) for the PVA dehydrogenase, and the entire nucleotide sequence of its structural gene was determined. The gene encodes a protein of 639 amino acid residues (68,045 Da) and in the deduced amino acid sequence, some putative functional sites, a signal sequence, a heme c-binding site, and a PQQ-binding site, were detected. The amino acid sequence showed low similarity to other types of quinoprotein dehydrogenases. PVA dehydrogenase expressed in E. coli clones required PQQ. Ca²⁺, and Mg²⁺ stimulated the activity. PVA-dependent heme c reduction occurred with exogenous PQQ in cell extracts of the E. coli clone. The PVA dehydrogenase in the E. coli clone was localized in the cytoplasm.     

Metabolic enzymes from psychrophilic bacteria: challenge of adaptation to low temperatures in ornithine carbamoyltransferase from Moritella abyssi

The enzyme ornithine carbamoyltransferase (OTCase) of Moritella abyssi (OTCase(Mab)), a new, strictly psychrophilic and piezophilic bacterial species, was purified. OTCase(Mab) displays maximal activity at rather low temperatures (23 to 25 degrees C) compared to other cold-active enzymes and is much less thermoresistant than its homologues from Escherichia coli or thermophilic procaryotes. In vitro the enzyme is in equilibrium between a trimeric state and a dodecameric, more stable state. The melting point and denaturation enthalpy changes for the two forms are considerably lower than the corresponding values for the dodecameric Pyrococcus furiosus OTCase and for a thermolabile trimeric mutant thereof. OTCase(Mab) displays higher K(m) values for ornithine and carbamoyl phosphate than mesophilic and thermophilic OTCases and is only weakly inhibited by the bisubstrate analogue delta-N-phosphonoacetyl-L-ornithine (PALO). OTCase(Mab) differs from other, nonpsychrophilic OTCases by substitutions in the most conserved motifs, which probably contribute to the comparatively high K(m) values and the lower sensitivity to PALO. The K(m) for ornithine, however, is substantially lower at low temperatures. A survey of the catalytic efficiencies (k(cat)/K(m)) of OTCases adapted to different temperatures showed that OTCase(Mab) activity remains suboptimal at low temperature despite the 4.5-fold decrease in the K(m) value for ornithine observed when the temperature is brought from 20 to 5 degrees C. OTCase(Mab) adaptation to cold indicates a trade-off between affinity and catalytic velocity, suggesting that optimization of key metabolic enzymes at low temperatures may be constrained by natural limits.     

Production of a recombinant chitin oligosaccharide deacetylase from Vibrio parahaemolyticus in the culture medium of Escherichia coli cells

An open reading frame (ORF) encoding chitin oligosaccharide deacetylase (Pa-COD) gene and its signal sequence was cloned from the Vibrio parahaemolyticus KN1699 genome and its sequence was analyzed. The ORF encoded a 427 amino acid protein, including the 22 amino acid signal sequence. The deduced amino acid sequence was highly similar to several bacterial chitin oligosaccharide deacetylases in carbohydrate esterase family 4. An expression plasmid containing the gene was constructed and inserted into Escherichia coli cells and the recombinant enzyme was secreted into the culture medium with the aid of the signal peptide. The concentration of the recombinant enzyme in the E. coli culture medium was 150 times larger than that of wild-type enzyme produced in the culture medium by V. parahaemolyticus KN1699. The recombinant enzyme was purified to homogeneity from culture supernatant in an overall yield of 16%. Substrate specificities of the wild-type and the recombinant enzymes were comparable.     

Cloning, sequencing and heterologous expression of the monoamine oxidase gene from Aspergillus niger

The gene encoding the flavin-containing monoamine oxidase (MAO-N) of the filamentous fungus Aspergillus niger was cloned. MAO-N is the first nonvertebrate monoamine oxidase described to date. Three partial cDNA clones, isolated from an expression library, were used to identify and clone the structural gene (maoN) from an A. niger genomic DNA library. The maoN gene was sequenced, and analysis revealed an open reading frame that codes for a protein of 495 amino acids with a calculated molecular mass of 55.6 kDa. Sequencing of an internal proteolytic fragment of the purified enzyme confirmed the derived amino acid sequence. Analysis of the deduced amino acid sequence indicates that MAO-N is structurally related to the human monoamine oxidases MAO-A and MAO-B. In particular, the regions known to be involved in the binding of the FAD cofactor show a high degree of homology; however, the conserved cysteine residue to which the flavin cofactor is covalently bound in the mammalian forms is absent in the fungal enzyme. MAO-N has the C-terminal tripeptide Ala-Arg-Leu, which corresponds to the consensus targeting sequence found in many peroxisomal enzymes. The full-length cDNA for MAO-N was expressed in Escherichia coli from the T7 promoter of the expression vector pET3a, yielding a soluble and fully active enzyme form.     

A Novel Glycerol Kinase from Flavobacterium meningosepticum: Characterization,Gene Cloning and Primary Structure

A thermostable glycerol kinase (FGK) was purified 34-fold to homogeneity from Flavobacterium meningosepticum. The molecular masses of the enzyme were 200 kDa by gel filtration and 50 kDa by SDS-PAGE. The Km for glycerol and ATP were 0.088 and 0.030 mM, respectively. The enzyme was stable at 65°C for 10 min and at 37°C for two weeks. The enzyme gene was cloned into Escherichia coli and its complete DNA was sequenced. The FGK gene consists of an open reading frame of 1494-bp encoding a protein of 498 amino acids. The deduced amino acid sequence of the gene had 40-60% similarity to those of glycerol kinases from other origins and the amino acid sequence of the putative active site residue reported for E. coli GK is identical to the corresponding sequence of FGK except for one amino acid residue.     

Molecular Cloning and Expression of Proteinaceous α-Amylase Inhibitor Gene from Streptomyces nitrosporeus in Escherichia coli

The proteinaceous α-amylase inhibitor, T-76, gene was cloned by screening a Streptomyces nitrosporeus genomic library using a deoxyinosine-containing probe corresponding to the amino acid sequence of the inhibitor. The nucleotide sequence of the insert of a positive clone had an open reading frame of 330 bp that encoded a polypeptide of 110 amino acid residues with a calculated molecular mass of 11,306 daltons. The polypeptide begins with proximal basic amino acids and a region rich in hydrophobic amino acids that possibly act as a signal peptide for secretion, which is followed by a sequence consistent with the amino-terminal amino acid sequence of the T-76 inhibitor. Escherichia coli cells harboring the plasmid derivatives for expression produced the inhibitor in their periplasmic space. The amino-terminal sequence of the inhibitor produced by an E. coli transformant was identical to that of the T-76 inhibitor secreted by S. nitrosporeus. The amino acid sequence of the inhibitor deduced from nucleotide sequence showed significant homology to other proteinaceous α-amylase inhibitors.