Naslov
The Adenylation Domain of Tyrocidine Synthetase 1: Structural and Functional Role of the Interdomain Linker Region and the (S,T)GT(T,S)GxPKG Core Sequence

Autori
Dieckmann, Ralf ; Pavela-Vrančič, Maja ; Pfeifer, Eva ; von Doehren, Hans ; Kleinkauf, Horst

Izvornik
European journal of biochemistry (0014-2956) 247 (1997); 1074-1082

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
Peptide synthetases; secondary structure; adenylate domain; domain movements; loop regions; limited proteolysis; kinetic analysis.

Sažetak
Sequence analysis of peptide synthetases revealed extensive structure similarity with firefly luciferase, whose crystal structure has recently become available, providing evidence for the localization of the active site at the interface between two subdomains separated by a distorted linker region [Conti, E., Franks, N.P., & Brick, P. (1996) Structure 4, 287-298]. The functional importance of two flexible loops, corresponding to the linker region of firefly luciferase and the highly conserved (S,T)GT(T,S)GxPKG core sequence, has been studied in view of the proposed conformational changes by the use of mutant analysis, limited proteolysis and chemical modification of tyrocidine synthetase 1. Substitution of the highly conserved Arg-416, residing in the loop separating the subdomains of the adenylation domain, resulted in profound loss of activity. Limited proteolysis of the mutant suggested significant structural changes as manifested by lack of protection to degradation in the presence of substrates, revealing a probable disturbance of the induced-fit mechanism regulating the transformation from an open to a closed conformation. Mutants, obtained by replacement of the conserved Lys-186 from the (S,T)GT(T,S)GxPKG core sequence, displayed only minor differences in substrate binding affinity despite significant reduction of catalytic efficiency. Residue Lys-186, appears to play an important role in either stabilization of the bound substrate through charge-charge interactions, and/or fixing of the loop for maintainance of the active site conformation. This is the first direct evidence for conformational changes related to catalysis and substrate binding in peptide synthetases.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

POVEZANOST RADA