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/

Sequence analysis of peptide synthetases revealed extensive structure similarity with firefly luciferase, whose crystal structure has recent ly become available, providing evidence for the localization Of the ac tive site at the interface between two subdomains separated by distort ed linker region [Conti, E., Franks, N. P. & Brick, P. (1996) Structur e 4, 287 - 298]. The functional importance of two flexible loops, corr esponding to the linker region of firefly luciferase and the highly co nserved (S/T)GT(T/S)GXPKG core sequence, has been studied in view of t he proposed conformational changes by the use of mutant analysis, limi ted proteolysis and chemical modification of tyrocidine synthetase 1. Substitution of the highly conserved Arg416, residing in the loop sepa rating the subdomains of the adenylation domain, resulted in profound loss of activity. Limited proteolysis of the mutant suggested signific ant structural changes as manifested by lack of protection to degradat ion 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 cons erved Lys 186 from the (S/T)GT(T/S)GXPKG core sequence, displayed only minor differences in substrate-binding affinity despite significant r eduction of catalytic efficiency. Residue Lys186 appears to play an im portant role in either stabilization of the bound substrate through ch arge-charge-interactions, and/or fixing of the loop for maintainance o f the active-site conformation.