Structural insights into the broad substrate specificity of carboxypeptidase T from Thermoactinomyces vulgaris

Abstract

The crystal structures of carboxypeptidase T (CpT) complexes with phenylalanine and arginine substrate analogs -benzylsuccinic acid and (2-guanidinoethylmercapto)succinic acid -were determined by the molecular replacement method at resolutions of 1.57 A and 1.62 A to clarify the broad substrate specificity profile of the enzyme. The conservative Leu211 and Leu254 residues (also present in both carboxypeptidase A and carboxypeptidase B) were shown to be structural determinants for recognition of hydrophobic substrates, whereas Asp263 was for recognition of positively charged substrates. Mutations of these determinants modify the substrate profile: the CpT variant Leu211Gln acquires carboxypeptidase B-like properties, and the CpT variant Asp263Asn the carboxypeptidase A-like selectivity. The Pro248-Asp258 loop interacting with Leu254 and Tyr255 was shown to be responsible for recognition of the substrate's C-terminal residue. Substrate binding at the S1 0 subsite leads to the ligand-dependent shift of this loop, and Leu254 side chain movement induces the conformation rearrangement of the Glu277 residue crucial for catalysis. This is a novel insight into the substrate selectivity of metallocarboxypeptidases that demonstrates the importance of interactions between the S1 0 subsite and the catalytic center.

New microbial resource: microbial diversity, function and dynamics in Chinese liquor starter

“…Meanwhile, the highly abundant no-rank Actinobacteria in JF liquor starter (J3 and J4) could produce volatile organic compounds; the secondary metabolic products of Actinomyces could also contribute to flavor development 38 . Thermoactinomyces can produce plenty of thermostable extracellular proteolytic enzymes, e.g., alkaline proteinase 39 , glutamyl endopeptidase 40 and carboxypeptidase 41 . This study further found that Bacillales and no-rank Actinobacteria were the dominant communities when the temperature increased to the highest point.…”

The nature of the ligand’s side chain interacting with the S1'-subsite of metallocarboxypeptidase T (from Thermoactinomyces vulgaris) determines the geometry of the tetrahedral transition complex

“…The kinetic parameters of ZAAL, ZAAE and ZAAR hydrolysis by CPT were determined as described earlier [16].…”

“…Other factors important for an enzyme’s catalytic activity and substrate specificity can be related to the non-productive binding of a substrate or necessity to reposition the substrate in the course of its catalytic conversion in the active site of the enzyme. In CPT catalysis it has been shown, for example, that the side chain of the positively charged substrate should undergo repositioning when moving from the enzyme-substrate complex to the transition state, whereas the side chain of the hydrophobic substrate does not need repositioning [16]. However, it is not clear how different the structural organization of the transition complexes is at conversion of different substrates by CPT and what factors influence its substrate specificity.…”

Structure of the complex of carboxypeptidase B and N-sulfamoyl-L-arginine

“…It is used in the insulin industry and in drug development as a model for blood carboxypeptidase B (Bunnage et al, 2007). We used carboxypeptidase B as a prototype to investigate the structural basis of the wide substrate selectivity of carboxypeptidase T from Thermoactinomyces vulgaris (Grishin et al, 2008;Akparov et al, 2015). Because these investigations were conducted for the rational design of metallocarboxypeptidase B with improved or changed selectivity, they required detailed information about the active site in order to provide a three-dimensional structure of the complex with the transition-state analogue of an arginine substrate.…”