Structures and activities of widely conserved small prokaryotic aminopeptidases-P clarify classification of M24B...

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• M24B peptidases include prolidases (cleave Xaa-Pro in dipeptides) and aminopeptidases-P (cleave Xaa-Pro in longer peptides).
• Bacterial small aminopeptidases-P (36-39 kDa) differ from the canonical Escherichia coli enzyme (50 kDa).
• Structure-function data for small bacterial aminopeptidases-P are limited.

Purpose of the Study:

• To elucidate the structure and function of small bacterial aminopeptidases-P.
• To investigate the substrate specificity and evolutionary relationships of these enzymes.
• To differentiate small aminopeptidases-P from other M24B peptidases.

Main Methods:

• X-ray crystallography of small aminopeptidases-P from E. coli and Deinococcus radiodurans.
• Enzymatic assays to determine substrate specificities.
• Mutagenesis studies focusing on the DXRY motif.

Main Results:

• Crystal structures revealed these enzymes are aminopeptidases-P, lacking a dipeptide-selectivity loop found in prolidases.
• Enzymatic assays confirmed aminopeptidase-P activity for bacterial enzymes and reclassified an archaeal enzyme.
• The DXRY motif is crucial for bacterial small aminopeptidases-P function, suggesting a shared evolutionary origin with human XPNPEP1/XPNPEP2.

Conclusions:

• Small bacterial aminopeptidases-P are distinct from prolidases, characterized by structural features and specific substrate cleavage.
• The DXRY motif plays a vital role in the function of bacterial small aminopeptidases-P.
• These findings clarify M24B peptidase classification and highlight evolutionary connections between bacterial and human enzymes.