The Adenylation Domain of Tyrocidine Synthetase 1

Abstract

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, 287-2981. 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 Arg416, 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 Lys186 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 Lys186 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.

NMR Identification of an Acyl-adenylate Intermediate in the Aryl-aldehyde Oxidoreductase Catalyzed Reaction

“…Whether benzoyl-AMP is actually released from the enzyme active site during catalysis is not known. Benzoyl-AMP, has a significantly lower (67-fold) apparent K m (9.66 Ϯ 0.71 M), a higher (1.3-fold) V max (7.50 Ϯ 0.18 mol min Ϫ1 mg Ϫ1 ) than those for benzoic acid (7). These results indicate that steady-state levels of benzoyl-AMP are low during the course of the reduction of benzoic acid to benzaldehyde.…”

“…Nocardia Aryl-aldehyde Oxidoreductase-The enzyme was produced and purified from cells of Nocardia sp. NRRL 5646 and characterized by our methods (7). The enzyme preparation used in this work had a specific activity of 6.5 units/mg of protein.…”

Mutational analysis of 4‐coumarate:CoA ligase identifies functionally important amino acids and verifies its close relationship to other adenylate‐forming enzymes

“…The presence of several conserved peptide motifs in all available 4CL amino acid sequences has been repeatedly observed by computer assisted sequence alignments [2,8,10–12]. The Box I motif, SSGTTGLPKGV, is not only almost absolutely conserved in 4CLs, but highly similar motifs are also found in luciferases, acetyl‐CoA synthetases, long‐chain fatty acyl‐CoA synthetases and peptide synthetases [13,14]. The presence of this putative nucleotide‐binding motif has even been used as one important criterion to establish a superfamily of adenylate‐forming enzymes [13].…”

Crystal Structure of DltA

“…Comparison of all available structures reveals that conserved charged residues known to be crucial for catalysis (31,32,(47)(48)(49)(50) exchange their partners in going from one conformational state to another. This intriguing facet of A-domains, overlooked in previous studies, suggests that only small energy differences separate the individual states.…”