Summary for peptidase M03.004: oligopeptidase A

Summary Alignment Tree Sequences Sequence features Distribution Literature Substrates Inhibitors

 

Names
MEROPS Nameoligopeptidase A
Other namesOpdA g.p. (Escherichia coli), prlC g.p. (Escherichia coli), proteinase In
Name and HistoryOligopeptidase A (OpdA) was firstly described by its capability to hydrolyze the peptide AcAla4 (Vimr et al., 1983) in extracts of Salmonella typhimurium dcp (peptidyl-dipeptidase Dcp) mutant. The opdA gene (previously called optA), homologous to prlC gene from Escherichia coli (Conlin et al., 1992), was mapped on the S. typhimurium chromosome on the same region as its homologue (Conlin & Miller, 1993).
Domain architecture
MEROPS Classification
Classification Clan MA >> Subclan MA(E) >> Family M3 >> Subfamily A >> M03.004
Holotypeoligopeptidase A (Salmonella typhimurium), Uniprot accession P27237 (peptidase unit: 1-680), MERNUM MER0001161
History Identifier created: Handbook of Proteolytic Enzymes (1998) Academic Press, London.
Activity
Catalytic typeMetallo
PeplistIncluded in the Peplist with identifier PL00139
NC-IUBMBSubclass 3.4 (Peptidases) >> Sub-subclass 3.4.24 (Metalloendopeptidases) >> Peptidase 3.4.24.70
EnzymologyBRENDA database
Proteolytic eventsCutDB database (1 cleavage)
PreparationConlin and Miller (Conlin & Miller, 1992) have purified OpdA from extracts of S. typhimurium strains transformed with the pBR328 plasmid carrying the opdA gene. Recombinant Escherichia coli OpdA has also been expressed with a C-terminal His-tag and purified in a Nickel-column with high efficiency (Paschoalin et al., 2005).
SpecificityOpdA is a zinc-dependent enzyme of the M3A subfamily and possess a conserved active site sequence motif (HEXXH). The enzyme was firstly characterized by its ability to cleave N-blocked peptides containing at least four amino acids, but not unblocked peptides with fewer than five amino acids (Vimr et al., 1983). It was determined that Gly or Ala on either side of the scissile bond would be permissive for hydrolysis (Novak & Dev, 1988). OpdA is able to cleave two important bioactive peptides, bradykinin (Phe-Ser bond) and neurotensin (Arg-Arg and Pro-Tyr bonds) (Paschoalin et al., 2005). Recently, a screening study using a series of fluorogenic peptides based on the bradykinin sequence determined the OpdA hydrolysis preference in the P1 position (Lorenzon et al., 2010). The general structure of the FRET peptides used in this study was Abz-GXSPFRQEDDnp (where X=different amino acids, Abz=o-aminobenzoyl and EDDnp=ethylenediamine 2,4-dinitrophenyl). OpdA cleaved all the peptides from this series only at the X–S bond. Recombinant OpdA exhibits high affinity for peptides containing the basic residue Arg in P1 position, whereas Lys and His were less susceptible to hydrolysis than Arg. The hydrophobic aromatic residues Phe and Tyr were also well accepted by the OpdA S1 subsite. In contrast, peptides containing acid residues such as Asp and Glu in the P1 position were poorly hydrolyzed by OpdA, while neutral, polar residues such as Asn and Ser, and nonpolar residues, such as Ala and Leu, did not show any important interaction with the S1 subsite.
pH optimumThe optimum pH is around 7 for the cleavage of the lead peptide Abz-GFSPFRQ-EDDnp by OpdA. The enzyme activity is not significantly modified by the increase in salt concentration (Lorenzon et al., 2010).
Substrate commentsOpdA can cleave the bioactive peptides bradykinin at the Phe-Ser bond and neurotensin at both Arg-Arg and Pro-Tyr bonds (Paschoalin et al., 2005).
Inhibitor commentsBesides the classic metallopeptidase inhibitors, OpdA is completely inhibited by the specific inhibitor of thimet oligopeptidase, JA-2, and partially by Pro-Ile, a selective neurolysin inhibitor (Paschoalin et al., 2005).
StructureOpdA is a zinc metallopeptidase from MA(E) subclan with 680 amino acid residues and a molecular mass of 77.1 kDa, forming an active monomer. The enzymes of the MA(E) subclan possess a highly conserved motif HEXXH that coordinates a single catalytic zinc ion and exhibit similar substrate specificities (Barrett et al., 1995, Rawlings & Barrett, 1995, Chu & Orlowski, 1985). The glutamate residue (Glu499) serves as the third zinc ligand. A comparative modeling study indicates that OpdA exhibits a similar number and sequential order of secondary structural elements compared to TOP, neurolysin and Dcp (Lorenzon et al., 2010). OpdA resembles a closed clam, where both adjacent shells enclose an elongated and deep cavity. The active site residues of OpdA are localized to the base of this cavity midway along its length, that provides access only to short peptides. In its open conformation, OpdA presents a bi-lobed structure. Two tyrosine residues present in the flexible loop move towards the active site when OpdA assumes a globular shape in its closed conformation. It was demonstrated by site-directed mutagenesis that Tyr607, present in the flexible loop, is crucial for substrate catalysis, once the substitution of this residue in OpdA was followed by a decrease in the kcat/Km value of about 100-fold with the Tyr607Phe mutant and 1000-fold with the Tyr607Ala mutant. This residue could be also relevant to stabilize the oxyanion intermediate formed after nucleophilic attack by water (Lorenzon et al., 2010).
PhysiologyDegradation of lipoprotein signal peptides, and other Intracellular oligopeptides. Role in maturation of bacteriophage P22 gp7 precursor.
Biological aspectsThe opdA gene is cotranscribed with a downstream open reading frame (yhiQ). opdA and yhiQ form an operon, whose transcription is induced by an increase in temperature and the induction is dependent on the heat shock sigma factor encoded by the rpoH(htpR) gene (Conlin & Miller, 2000). It was demonstrated that OpdA from E. coli is able to degrade fragments of signal peptide generated by protease IV from prolipoprotein (Novak & Dev, 1988) and it functions as a specific processing enzyme essential for the normal phage P22 development in S. typhimurium (Conlin et al., 1992). It was suggested that OpdA may play a role in the protein localization process, since certain alleles suppress the secretion defect of some signal sequence mutations (Emr et al., 1981, Trun & Silhavy, 1987, Trun & Silhavy, 1989). Due to its ability to hydrolyze small cytosolic peptides generated from breakdown of proteins by ATP-dependent proteases (Lon, HslUV and ClpAP), it has been suggested that OpdA could participate in multiple catabolic pathways in E. coli (Jain & Chan, 2007). Hydrolysis by OpdA of bradykinin and neurotensin, two biologically active peptides, has also been demonstrated (Paschoalin et al., 2005).
KnockoutNull mutations in the gene are not lethal and do not confer temperature sensitivity.
Distinguishing featuresSearching the HEFGH motif in the E. coli translated genome, OpdA and Dcp are the only oligopeptidases belonging to the M3A subfamily. Both enzymes have almost exactly the same molecular mass, are inhibited by metal chelators and zinc and both are stimulated by cobalt. However, the specifity is completely different, Dcp releases dipeptides sequentially from substrates with a free C-terminus and is inhibited by captopril. OpdA is completely inhibited by JA-2, a specific inhibitor of TOP, and partially by Pro-Ile, a selective neurolysin inhibitor. The FRET peptide Abz-GFSPFRQ-EDDnp has been used to distinguish between the bacterial and mammalian enzymes. OpdA cleaves this substrate at Phe-Ser bond, while TOP and neurolysin hydrolyse it at the Pro-Phe bond (Paschoalin et al., 2005).
Contributing authorsThaysa Paschoalin, Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, Rua Botucatu, 862, 8° andar, 04023-062 São Paulo, SP, Brazil.
Cleavage site specificity Explanations of how to interpret the following cleavage site sequence logo and specificity matrix can be found here.
Cleavage pattern-/a/P/-Scissile bond-/-/-/- (based on 10 cleavages)
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Specificity matrix
 
Amino acid P4 P3 P2 P1 P1' P2' P3' P4'
Gly 0 1 0 1 2 1 1 0
Pro 0 0 6 0 0 2 0 0
Ala 3 3 0 3 0 0 2 0
Val 0 2 0 0 0 2 0 0
Leu 0 0 1 1 2 0 0 0
Ile 0 0 0 0 0 0 0 0
Met 0 0 0 0 0 0 0 0
Phe 0 0 0 1 0 0 0 0
Tyr 0 1 0 0 0 0 1 0
Trp 0 0 0 0 0 0 0 0
Ser 1 0 0 0 0 0 0 0
Thr 0 0 0 0 0 0 0 0
Cys 0 0 0 0 0 0 0 0
Asn 0 0 0 0 0 0 0 0
Gln 0 0 0 0 0 0 0 0
Asp 0 0 0 0 0 0 0 0
Glu 0 0 0 1 0 0 0 0
Lys 0 0 0 2 1 0 0 1
Arg 0 0 0 1 0 0 0 0
His 0 0 0 0 0 0 0 0