Sphingomonas sp. KT-1 PahZ2 Structure Reveals a Role for Conformational Dynamics in Peptide Bond Hydrolysis

Abstract

Poly(aspartic acid) (PAA) is a common water-soluble polycarboxylate used in a broad range of applications. PAA biodegradation and environmental assimilation were first identified in river water bacterial strains, Sphingomonas sp. KT-1 and Pedobacter sp. KP-2. Within Sphingomonas sp. KT-1, PahZ1 KT-1 cleaves β-amide linkages to oligo(aspartic acid) and then is degraded by PahZ2 KT-1 . Recently, we reported the first structure for PahZ1 KT-1 . Here, we report novel structures for PahZ2 KT-1 bound to either Gd 3+ /Sm 3+ or Zn 2+ cations in a dimeric state consistent with M28 metallopeptidase family members. PahZ2 KT-1 monomers include a dimerization domain and a catalytic domain with dual Zn 2+ cations. MD methods predict the putative substrate binding site to span across the dimerization and catalytic domains, where NaCl promotes the transition from an open conformation to a closed conformation that positions the substrate adjacent to catalytic zinc ions. Structural knowledge of PahZ1 KT-1 and PahZ2 KT-1 will allow for protein engineering endeavors to develop novel biodegradation reagents.

Novel DNA-Binding Activity Exhibited by Poly(aspartic acid) Hydrolase-1 Inhibits Poly(aspartic acid) Hydrolase Activity

“…Assays were performed by preincubating a constant amount of enzyme with increasing amounts of plasmid DNA from 0.1 to 1000 ng for 30 min to prebind DNA to the enzyme, followed by subsequent addition of the tPAA substrate and incubation for 15 min to allow for biodegradation to occur. Gel permeation chromatography (GPC) was used to analyze the amount of tPAA degradation catalyzed by the wild-type enzymes alone, as described previously, , and when varied DNA amounts were present.…”