Determination of three amino acids causing alteration of proteolytic activities of staphylococcal glutamyl endopeptidases

Abstract

Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus warneri secrete glutamyl endopeptidases, designated GluV8, GluSE, and GluSW, respectively. The order of their protease activities is GluSE < GluSW << GluV8. In the present study, we investigated the mechanism that causes these differences. Expression of chimeric proteins between GluV8 and GluSE revealed that the difference is primarily attributed to amino acid residues 170-195, which define the intrinsic protease activity, and additionally to residues 119-169, which affect the proteolytic sensitivity. Among nine substitutions present in residues 170-195 of the three proteases, the substitutions at positions 185, 188, and 189 were responsible for the changes in their activities, and the combination of W185, V188, and P189, which naturally occurs in GluV8, exerts the highest protease activity. W185 and P189 were indispensable for full activity, but V188 could be replaced by hydrophobic amino acids. These three amino acid residues appear to create a substrate-binding pocket together with the catalytic triad and the N-terminal V1, and therefore define the K(m) values of the proteases. We also describe a method to produce a chimeric form of GluSE and GluV8 that is resistant to proteolysis, and therefore possesses 4-fold higher activity than the wild-type recombinant GluV8.

Secreted Proteases Control Autolysin-mediated Biofilm Growth of Staphylococcus aureus

“…Nemoto and co-workers (73, 74) characterized S. aureus glutamyl endopeptidase V8 and identified, in addition to catalytic Ser 237 (Esp Ser 235 ), the N-terminal Val 69 (Esp Val 67 ) residue as essential for substrate cleavage. S. aureus V8 protease with an N-terminal truncation to Ile 70 (Esp Ile 68 ) was inactive, and mutants with an altered N-terminal residue Val 69 (even with conserved substitutions) were also inactive, which is indicative of a strict requirement of the N-terminal Val residue for enzyme activity (75).…”

“…The N-terminal Val 67 in Esp is positioned with its α-amino group located adjacent to the conserved Thr 230 (V8 Thr 232 ) and Asn 259 (V8 Asn 261 ), pointed into S1 pocket, within hydrogen bonding distance. Similarly, the His 250 (V8 His 252 ) residue on loop 2, conserved among glutamyl endopeptidases (73), having hydrogen bonds with side chains of conserved Tyr 226 (V8 Tyr 228 ) and Thr 230 (V8 Thr 232 ), is also suitably positioned to interact with the substrate acidic P1 residue.…”

“…8 d ) that can be associated with differences in substrate specificity. Extensive mutational analysis of V8 and Esp by Nemoto et al (73) localized the difference in their specificities to Tyr 251 (V8 Trp 253 ) and Asp 255 (V8 Pro 257 ) residues on Esp loop 2. Substitutions at these positions affected mainly the K m with constant k cat values, suggesting that these residues affect only substrate binding affinities (73).…”

“…Extensive mutational analysis of V8 and Esp by Nemoto et al (73) localized the difference in their specificities to Tyr 251 (V8 Trp 253 ) and Asp 255 (V8 Pro 257 ) residues on Esp loop 2. Substitutions at these positions affected mainly the K m with constant k cat values, suggesting that these residues affect only substrate binding affinities (73). The K m value of native Esp harboring Tyr 251 -Val 254 -Asp 255 on loop 2 was larger than that of V8 with Trp 253 -Val 256 -Pro 257 , but with almost similar k cat values (76).…”

NVS and Staphylococci in the Oral Cavity – A Cause of Infective Endocarditis

“…Val 1 is required to exert proper maturation mediated by cleavage between the Xaa-Val bond and for proteolytic activity itself, with Trp 185 , Val 188 and Pro 189 also involved in proteolytic activity (Fig. 9) (Nemoto et al, 2009 (Table 5). These residues can be involved in substrate affinity, which implicates the mechanism of alteration in proteolytic activity among the members of this family.…”