Crystal structure of a novel prolidase from Deinococcus radiodurans identifies new subfamily of bacterial prolidases

Abstract

Xaa-Pro peptidases (XPP) are dinuclear peptidases of MEROPS M24B family that hydrolyze Xaa-Pro iminopeptide bond with a trans-proline at the second position of the peptide substrate. XPPs specific towards dipeptides are called prolidases while those that prefer longer oligopeptides are called aminopeptidases P. Though XPPs are strictly conserved in bacterial and archaeal species, the structural and sequence features that distinguish between prolidases and aminopeptidases P are not always clear. Here, we report 1.4 Å resolution crystal structure of a novel XPP from Deinococcus radiodurans (XPPdr). XPPdr forms a novel dimeric structure via unique dimer stabilization loops of N-terminal domains such that their C-terminal domains are placed far apart from each other. This novel dimerization is also the consequence of a different orientation of N-terminal domain in XPPdr monomer than those in other known prolidases. The enzymatic assays show that it is a prolidase with broad substrate specificity. Our structural, mutational, and molecular dynamics simulation analyses show that the conserved Arg46 of N-terminal domain is important for the dipeptide selectivity. Our BLAST search found XPPdr orthologs with conserved sequence motifs which correspond to unique structural features of XPPdr, thus identify a new subfamily of bacterial prolidases.

Biochemical Characterization of a Novel Alkaline-Tolerant Xaa-Pro Dipeptidase from Aspergillus phoenicis

“…The recombinant ApXPD characterized in this study, however, shows a preference for positively charged Lys-Pro (Tables 1 and 2). Additionally, most prolidases do not hydrolyze Pro-Pro and Gly-Pro except MtXPD [4], XcXPD [4], AnPEPP [16], PlOPAA [39], DrXPP [43], and HsPEPDI [46]. As indicated by the kinetic study, ApXPD may also show allosteric behavior (Table 2).…”

“…In vitro studies have shown that most prolidases ever reported utilize Mn 2+ ion as cofactors (Table S1) [4,28,[37][38][39][40], whereas PfPEPQ, PhPROL, and ScXPD require Co 2+ in their active sites under aerobic assay conditions [25,34,41], as is the case of XPD from A. phoenicis ATCC 14332 (Figure 7). Furthermore, the enzymatic activities of LdPEPQ, LlXPDII, and TsPROL are dependent on Zn 2+ [26,42,43]. In contrast, the activities of AsOPAA, AuOPAA, LcXPD, PfpepQ, PhPROL, and XmXPD were strongly or even completely inactivated by high concentrations of Zn 2+ (Table S1), which is due to the fact that Zn binding decreases the volume of active site pocket for accumulating the substrates [30].…”

“…There are also some differences in dipeptide substrate specificities among the prolidases that have been biochemically characterized. Their main activity is Xaa-Pro dipeptides hydrolysis, although several prolidases can cleave dipeptides with proline at the N-terminus or dipeptides that do not contain a proline residue [40,43,45]. Most prolidases show broad substrate specificities and prefer Xaa-Pro dipeptides containing N-terminal hydrophobic amino acids, whereas PfPEPQ [25], PhPROL [34], and EcPEPQ [37] show affinity for negatively charged and/or hydrophilic amino acids at the N-terminus.…”

“…XPDs in the other three subfamilies, however, prolidases from the unassigned subfamily use a novel arginine-dependent mechanism for dipeptide selectivity. According to the results of structural, mutational, and/or molecular dynamics simulation analyses, the corresponding arginine residues employed by XcXPD [4], DrXPP [43], and LlXPDII [48] have been found to be Arg72, Arg46, and Arg40, respectively, while the equivalent residue in ApXPD is Arg141 (Figure 2). Further investigations are therefore needed to understand the precise mechanism of prolidases from the unassigned subfamily used for dipeptide selectivity.…”

Enzymatic Hydrolysis of Tertiary Amide Bonds by anti Nucleophilic Attack and Protonation

“…The scarce family of Xaa-Pro peptidases capable of hydrolyzing trans proline-based tertiary amide bonds , includes aminopeptidase P acting on polyamides (also referred to as X-prolyl aminopeptidase) and prolidases acting on dipeptide substrates. ,, Although Xaa-Pro peptidases can display different domain architectures, they share a dinuclear active site embedded in a catalytic domain composed of a curved β-sheet referred to as the “pita bread” fold. − The bimetallic catalytic center, typically composed of two manganese atoms in oxidation state +2, displays overall conserved amino acid ligands within this enzyme superfamily. The metals interact with a nucleophilic μ-hydroxide activated by a catalytic glutamate, both of which reside on the same face of the peptide bond. ,, Interestingly, initial visual inspection of available structures of Escherichia coli aminopeptidase P complexed with inhibitors (PDB 1A16 and 1N51 ) revealed a histidine (H243) in spatial arrangement that would fulfill the geometrical criterion for the general acid shown in Figure d.…”