The exquisite structure and reaction mechanism of bacterial Pz-peptidase A toward collagenous peptides: X-ray crystallographic structure analysis of PZ-peptidase a reveals differences from mammalian thimet oligopeptidase.
Pz-peptidase A, from the thermophilic bacterium Geobacillus collagenovorans MO-1, hydrolyzes a synthetic peptide substrate, 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz-PLGPR), which contains a collagen-specific tripeptide sequence, -Gly-Pro-X-, but does not act on collagen proteins themselves. The mammalian enzyme, thimet oligopeptidase (TOP), which has comparable functions with bacterial Pz-peptidases but limited identity at the primary sequence level, has recently been subjected to x-ray crystallographic analysis; however, no crystal structure has yet been reported for complexes of TOP with substrate analogues. Here, we report crystallization of recombinant Pz-peptidase A in complex with two phosphinic peptide inhibitors (PPIs) that also function as inhibitors of TOP and determination of the crystal structure of these complexes at 1.80-2.00 Å resolution. The most striking difference between Pz-peptidase A and TOP is that there is no channel running the length of bacterial protein. Whereas the structure of TOP resembles an open bivalve, that of Pz-peptidase A is closed and globular. This suggests that collagenous peptide substrates enter the tunnel at the top gateway of the closed Pz-peptidase A molecule, and reactant peptides are released from the bottom gateway after cleavage at the active site located in the center of the tunnel. One of the two PPIs, PPI-2, which contains the collagen-specific sequence, helped to clarify the exquisite structure and reaction mechanism of Pz-peptidase A toward collagenous peptides. This study describes the mode of substrate binding and its implication for the mammalian enzymes.
Kawasaki
Akio
A
From the Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Shimogamo, Sakyo, Kyoto 606-8522, Japan.
Nakano
Hiroaki
H
Hosokawa
Allin
A
Nakatsu
Toru
T
Kato
Hiroaki
H
Watanabe
Kunihiko
K
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D056506:Geobacillus / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics; D011480:Protease Inhibitors / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase
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