Structures and activities of widely conserved small prokaryotic aminopeptidases‐P clarify classification of M24B peptidases

Abstract

M24B peptidases cleaving Xaa‐Pro bond in dipeptides are prolidases whereas those cleaving this bond in longer peptides are aminopeptidases‐P. Bacteria have small aminopeptidases‐P (36‐39 kDa), which are diverged from canonical aminopeptidase‐P of Escherichia coli (50 kDa). Structure‐function studies of small aminopeptidases‐P are lacking. We report crystal structures of small aminopeptidases‐P from E. coli and Deinococcus radiodurans, and report substrate‐specificities of these proteins and their ortholog from Mycobacterium tuberculosis. These are aminopeptidases‐P, structurally close to small prolidases except for absence of dipeptide‐selectivity loop. We noticed absence of this loop and conserved arginine in canonical archaeal prolidase (Maher et al., Biochemistry. 43, 2004, 2771‐2783) and questioned its classification. Our enzymatic assays show that this enzyme is an aminopeptidase‐P. Further, our mutagenesis studies illuminate importance of DXRY sequence motif in bacterial small aminopeptidases‐P and suggest common evolutionary origin with human XPNPEP1/XPNPEP2. Our analyses reveal sequence/structural features distinguishing small aminopeptidases‐P from other M24B peptidases.

Structure-Function and Industrial Relevance of Bacterial Aminopeptidase P

“…Zn A is penta-coordinated and exhibits distorted trigonal-bipyramidal geometry; Zn B is tetracoordinated and shows tetrahedral geometry. Based on their structure, small APPro was almost identical to small type prolidase except for the absence of the loop containing residue of XER motif in small APPro (Figure 2) [19]. This loop has been identified in small prolidase of Xanthomonas campestris, which houses Arg residue that is responsible for dipeptide selectivity.…”

“…This could potentially classify small APPro from the closely related M24B peptidases [56]. Mutagenesis of the residues of DXRY of DrsmAPPro revealed that the Interestingly, small prokaryotic APPros have been reported from E. coli, L. lactis, D. radiodurans, and M. tuberculosis [19,47,[56][57][58]. The size of the polypeptide in the small APPro is around 36-39 kDa, which is smaller compared with the two-domain APPro (48-50 kDa) and three-domain APPro (70-88 kDa).…”

“…Mutagenesis of the residues of DXRY of DrsmAPPro revealed that the sequence motif is in fact crucial for enzymatic activity. Crystal structures of EcsmAPPro (PDB ID: 5GIQ) and DrsmAPPro (PDB ID: 5CNX) were solved to 2.6 Å and 1.8 Å, respectively [19]. Both proteins adopt a two-domain structure typical of the M24B subfamily and form dimers in solution.…”

“…A proline-specific aminopeptidase, aminopeptidase P (APPro, or Xaa-Pro aminopeptidase; E.C 3.4.11.9, MEROPS M24.004) is a metalloprotease that cleaves N-terminal amino acid residue from polypeptide chains where the second residue is proline [12,[15][16][17][18][19]. The unique and special structure of proline stimulates conformational constraints on the peptide bond, shielding it from the common degradation process [20].…”

Structural features and functional implications of proteins enabling the robustness of Deinococcus radiodurans

“…Mn and Zn metal ions, which come from either protein copurification or the reservoir solution for crystallization, are two divalent metal ions frequently observed in the currently solved structures of Deinococcus proteins. Mn ions are found in the structures of RecJ [15] , RNase J [72] , MazG [73] , MntH [69] , and SodA [59] , and the structures of PprI [57] , RecO [74] , [75] , RecR [76] , [77] , RecQ [40] , DinB [78] , PolX [79] , Dps1 [66] , LuxS [80] , and several peptidases [81] , [82] contain Zn ions.…”