Molecular dynamics complemented by site-directed mutagenesis reveals significant difference between the interdomain salt bridge networks stabilizing oligopeptidases B from bacteria and protozoa in their active conformations

First Crystal Structure of Bacterial Oligopeptidase B in an Intermediate State: The Roles of the Hinge Region Modification and Spermine

“…Thus, it is possible to assume that the disruption of SB Arg151-Asp617 is rather favorable for catalysis. Neither alanine nor glutamate substitution of Arg151 caused significant PSP inhibition [29], which confirms that SB Arg151-Asp617 is not a functional analog of the TbOpB SB1, and the mechanism of catalytic activation proposed for protozoan OpB is not compatible with both the amino acid sequence of PSP and structural data presented here. Determination of the mechanism of catalytic activation of bacterial OpB require further experimental and/or computational studies, but prior their conduction we had to confirm that the intermediate state was not an artefact of crystallization and clarify its relation with both the hinge modification and spermine presence.…”

“…The partial restoration of the catalytic activity of PSPmod was also achieved by the alanine substitution of Glu125 acidic residue from the β-propeller domain: PSPmodE125A possesses hydrolysis efficiency toward the substrates, from 19 to 26%, of PSP (Figure 1D), demonstrating increases in hydrolysis efficiency (k cat /K m ) over PSPmod by about 6-to 9-fold (Figure 1F). According to our previous in silico modelling, Glu125 also participated in the putative interdomain SB network and its alanine substitution increased the activities of PSP toward BAPNA and dibasic substrates by about 8-and about 2-fold, respectively [28,29].…”

“…Kinetic parameters of substrate hydrolysis by wild-type and modified PSP variants were determined as described in [28,29]. Briefly, hydrolysis of Nα-benzoyl-D,L-arginine-pnitroanilide (BAPNA) (Sigma-Aldrich, St. Louis, MI, USA) and two other p-nitroanilide (pNA) substrates, Z-RR-pNA and Z-KR-pNA (Z = benzyloxycarbonyl) (Bachem AG, Budendorf, Switzerland), was monitored as an increase in the absorption at 405 nm (25 • C) due to the formation of free p-nitroaniline (∆ε405 = 10.400 M −1 × cm −1 ).…”

“…At the same time, such characteristics of substrate specificity of PSP as enhanced activity towards dibasic substrates or preference to the Arg residue over Lys in the P2 position have not been changed (Figure 1E). The hinge region modification included the substitution of the functionally important residue Glu75, which, according to our previous homology modeling and molecular dynamics studies, participated in the putative interdomain SB network stabilizing PSP in the closed conformation [28,29]. The alanine substitution of Glu75 caused a loss in activity of wild-type PSP toward three p-Na substrates, from 82 to 92% [28].…”

“…A common mechanism of catalytic activation for all three branches of POP was suggested, which highlighted the importance of the interdomain interface and particularly of one of the interdomain salt bridges (SB1 in TbOpB) in the transition of the enzymes between two states [26]. It is intriguing that the residues forming this SB1 were not conserved in γ-proteobacterial OpB [28,29], including the well-studied enzymes from E. coli [30], Salmonella enterica [31] and Serratia proteomaculans [32]. This difference strongly suggests there is no direct transfer of the activation mechanism proposed for protozoan OpB to the bacterial enzymes and requires applications of the structural data obtained for OpB from bacteria to elucidate the mechanisms underlying their catalytic activation.…”