Assessment of Substrate Inhibition of Bacterial Oligopeptidase B

Abstract

Oligopeptidase B (OPB; EC 3.4.21.83) from 2 Gram-negative bacteria, Stenotrophomonas maltophilia (Stm) and Serratia marcescens (Sem), and the Gram-positive bacterium Rhodococcus erythropolis (Re) were cloned and characterized to clarify their activities and substrate specificities using peptidyl-MCA substrates containing Arg or Lys. The cloned enzymes, Stm, Sem and ReOPBs, in addition to Escherichia coli OPB (EcOPB) were expressed using a pET expression system. Although the Stm and SemOPBs share 45% sequence identity to each other and up to 60% identity with respect to their catalytic domains, their activities towards MCA substrates were quite different. StmOPB is approximately 100-500 times more active than SemOPB and 3-30 times more active than EcOPB. The activity of ReOPB is comparable to that of StmOPB and it shares 40% and 36% identity to StmOPB and SemOPB, respectively. Some features of Stm, Re and EcOPBs are similar to those of previously cloned OPBs, which were also strongly inhibited by substrates, but SemOPB differs from all other OPBs in that it is not inhibited by substrates; even substrates containing double arginine at 35 µM did not inhibit SemOPB. On the other hand, the same substrates at only 5 µM inhibited the activity of the Stm, Re, and EcOPB. This phenomenon was not observed with substrates containing single or double lysine.Key words oligopeptidase B; prolyl oligopeptidase family; substrate inhibition; opportunistic bacteria; substrate specificityThe oligopeptidase B (OPB, EC 3.4.21.83) subfamily represents one of two branches of the prolyl oligopeptidase family of serine peptidases (clan SC, family S9).1) The substrate specificities of the subfamily enzymes are different, with prolyl oligopeptidase exclusively hydrolyzing peptide bonds at the C-terminal to proline residues in peptides, 2,3) while OPB demonstrates a trypsin-like substrate specificity, hydrolyzing peptide bonds on the C-terminal side of basic amino acid residues. Arginine-or lysine-containing peptides of no more than about 30 residues can be hydrolyzed, [4][5][6] since the N-terminal β-propeller domain of the enzymes blocks access of large globular proteins to the catalytic machinery.3) However, it has been demonstrated that OPB can cleave, in addition to low-molecular-mass peptides, several basic proteins in a restricted fashion, including human histones H1, H2A, H3, and H4.7) Peptides with Arg residues in both P1 and P2 are hydrolyzed at a much faster rate by OPB than peptides with only one Arg residue at the P1 site. 7,8) OPB is found in bacteria, plants and trypanosomatid pathogens, where it has been identified as a virulence factor and a potential drug target, since the genes coding for this enzyme are absent from mammals. The exact physiological function(s) and the physiological substrates of the OPB are unknown. Although the OPB was first cloned and characterized from Escherichia coli 4,9) and Moraxella lacunata, 6) bacterial OPBs have received much less attention than their homologues from protozoa. Thus their 3D stru...

Reversible Cyclic Thermal Inactivation of Oligopeptidase B from Serratia proteamaculans

“…( Leishmania major and L. amazonensis [ 4 ]). OpdB or the genes encoding this enzyme have been detected in prokaryotes, such as Escherichia coli [ 5 ], Moraxella lacunata [ 6 ], Salmonella enterica serovar typhimurium [ 7 ], Yersinia pestis [ 7 ], Serratia marcescens , Stenotrophomonas maltophilia and Rhodococcus erythropolis [ 8 ], in mycobacteria Mycobacterium tuberculosis and M. leprae [ 7 ], and in spirochetes Treponema denticola [ 9 ]. Members of the oligopeptidase B group are also found in some higher plants (e.g., Ambrosia artemisiifolia [ 10 ]).…”

Development of a fluorogenic small substrate for dipeptidyl peptidase-4

“…Stenotrophomonas dipeptidyl aminopeptidase IV was prepared from the E. coli DH5 containing pUC19-SDP4 as described previously [ 22 ]. The enzyme activity was assayed using H-Gly-Pro- 1 as a substrate following the procedure described previously [ 23 ]. The reaction mixture (total 100 μL) contained 0.1 M Tris–HCl (pH 8.0), 10–50 μM substrate and 10–400 ng of the enzyme.…”