Cloning and nucleotide sequence of opdA, the gene encoding oligopeptidase A in Salmonella typhimurium

Conlin, C A; Miller, Charles G.

doi:10.1128/jb.174.5.1631-1640.1992

Cited by 37 publications

(28 citation statements)

References 61 publications

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“…Other thiolutin resistance mutations have been mapped to 11 and 85 map units in E. coli (36). opdA maps at 76 map units on the S. typhimurium chromosome (9), and priC, the E. coli homolog of opdA (10), maps at 71 map units on the E. coli chromosome (39). Although the relationship between the thiolutin resistance mutations and opd4 is unknown, the map positions of the E. coli mutations suggest that they affect different genes.…”

Section: Methodsmentioning

confidence: 99%

“…Substrate specificity studies indicate that OpdA is an endopeptidase with a preference for Ala and Gly residues (27,28,41). Its inhibition by metal chelators (9,28) suggests that it is a metallopeptidase.…”

mentioning

confidence: 99%

“…The predicted amino acid sequence of the 77-kDa OpdA protein contains a thermolysin-type metalloprotease Zn2+-binding site (9,22). The amino acid sequence shows strong similarity to another bacterial peptidase (dipeptidyl carboxypeptidase, encoded by dcp) and to a mammalian metalloprotease (rat metallopeptidase EP 24.15) that is involved in the processing of peptide hormones (9).…”

mentioning

confidence: 99%

“…The gene encoding OpdA, opdA, has been cloned and sequenced (9). The predicted amino acid sequence of the 77-kDa OpdA protein contains a thermolysin-type metalloprotease Zn2+-binding site (9,22).…”

mentioning

confidence: 99%

“…OpdA has been purified from both Escherichia coli (28) and S. typhimurium (9). Novak et al (28) and Novak and Dev (27) have shown that OpdA is a signal peptide peptidase.…”

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confidence: 99%

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Oligopeptidase A is required for normal phage P22 development

1992

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The opdA gene of SalmoneUla typhimurium encodes an endoprotease, oligopeptidase A (OpdA). Strains carying opd4 mutations were deficient as hosts for phage P22. P22 and the closely related phages L and A3 formed tiny plaques on an opdA host. SalmoneUla phages 9NA, KB1, and ES18.hl were not affected by opdA mutations. Although opd4 strains displayed normal doubling times and were infected by P22 as efficiently as opdA+ strains, the burst size of infectious particles from an opd4 host was less than 1/10 of that from an opdA + host. This decrease resulted from a reduced efficiency of plating of particles from an opdAl infection. In the absence of a functional opdA4 gene, most ofthe P22 particles are defective. To identify the target of OpdA action, P22 mutants which formed plaques larger than wild-type plaques on an opdA mutant lawn were isolated.Marker rescue experiments using cloned fragments of P22 DNA localized these mutations to a 1-kb fragment.The nucleotide sequence of this fragment and a contiguous region (including all of both P22 gene 7 and gene 14) was determined. The mutations leading to opdA independence affected the region of gene 7 coding for the amino terminus of gp7, a protein required for DNA injection by the phage. Comparison of the nucleotide sequence with the N-terminal amino acid sequence of gp7 suggested that a 20-amino-acid peptide is removed from gp7 during phage development. Further experiments showed that this processing was opd4 dependent and rapid (half-life, <2 min) and occurred in the absence of other phage proteins. The opd4-independent mutations lead to mutant forms of gp7 which function without processing.Oligopeptidase A (OpdA) was identified by Vimr and Miller (42) as one of two enzymes in extracts of Salmonella typhimurium which hydrolyzed N-acetyl-L-Ala4. OpdA has been purified from both Escherichia coli (28) and S. typhimurium (9). Novak et al. (28) and Novak and Dev (27) have shown that OpdA is a signal peptide peptidase. In vitro, OpdA is the major soluble enzyme able to hydrolyze the E. coli lipoprotein signal peptide. Substrate specificity studies indicate that OpdA is an endopeptidase with a preference for Ala and Gly residues (27,28,41). Its inhibition by metal chelators (9, 28) suggests that it is a metallopeptidase.The gene encoding OpdA, opdA, has been cloned and sequenced (9). The predicted amino acid sequence of the 77-kDa OpdA protein contains a thermolysin-type metalloprotease Zn2+-binding site (9,22). The amino acid sequence shows strong similarity to another bacterial peptidase (dipeptidyl carboxypeptidase, encoded by dcp) and to a mammalian metalloprotease (rat metallopeptidase EP 24.15) that is involved in the processing of peptide hormones (9). Evidence presented in the accompanying article shows that opdA4 is the Salmonella homolog of the E. coliprlC gene (10).Mutant alleles of prlC suppress the localization defect of certain signal peptide mutations (39).Mutations in opdA were originally isolated by screening for mutants unable to use AcAla4 as a sole nitro...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

“…OpdA has been purified from both Escherichia coli (28) and S. typhimurium (9). Novak et al (28) and Novak and Dev (27) have shown that OpdA is a signal peptide peptidase.…”

mentioning

confidence: 99%

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Oligopeptidase A is required for normal phage P22 development

1992

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Oligopeptidase A

Schomburg

Stephan

1998

Enzyme Handbook 16

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Heterologous Expression, Purification and Characterization of an Oligopeptidase A from the Pathogen Leptospira interrogans

et al. 2018

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Oligopeptidases are enzymes involved in the degradation of short peptides (generally less than 30 amino acids in size) which help pathogens evade the host defence mechanisms. Leptospira is a zoonotic pathogen and causes leptospirosis in mammals. Proteome analysis of Leptospira revealed the presence of oligopeptidase A (OpdA) among other membrane proteins. To study the role of oligopeptidase in leptospirosis, the OpdA of L. interrogans was cloned and expressed in Escherichia coli with a histidine tag (His-tag). The protein showed maximum expression at 37 °C with 0.5 mM of IPTG after 2 h of induction. Recombinant OpdA protein was purified to homogeneity using Ni-affinity chromatography. The purified OpdA showed more than 80% inhibition with a serine protease inhibitor but the activity was reduced to 30% with the cysteine protease inhibitor. The peptidase activity was increased significantly in the presence of Zn at a neutral pH. Inhibitor assay indicate the presence of more than one active sites for peptidase activity as reported with the OpdA of E. coli and Salmonella. Over-expression of OpdA in E. coli BL21 (DE3) did not cause any negative effects on normal cell growth and viability. The role of OpdA as virulence factor in Leptospira and its potential as a therapeutic and diagnostic target in leptospirosis is yet to be identified.

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Cloning and nucleotide sequence of opdA, the gene encoding oligopeptidase A in Salmonella typhimurium

Cited by 37 publications

References 61 publications

Oligopeptidase A is required for normal phage P22 development

Oligopeptidase A is required for normal phage P22 development

Oligopeptidase A

Heterologous Expression, Purification and Characterization of an Oligopeptidase A from the Pathogen Leptospira interrogans

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