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J. Bacteriol. doi:10.1128/JB.01029-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Structure-Activity Relationship of Gelatinase Biosynthesis-Activating Pheromone of Enterococcus faecalis

Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 812-8581, Japan; Department of Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan; Department of Functional Metabolic Design, Bio-architecture Center, Kyushu University, Fukuoka 812-8581, Japan

^* To whom correspondence should be addressed. Email: nakayama{at}agr.kyushu-u.ac.jp.

	Abstract

The expression of pathogenicity-related extracellular proteases, namely, gelatinase and serine protease, in Enterococcus faecalis is positively regulated by a quorum sensing system mediated by an autoinducing peptide called gelatinase biosynthesis-activating pheromone (GBAP). GBAP is an 11-amino-acid-residue cyclic peptide containing a lactone linkage. To study the structure-activity relationship of GBAP, we synthesized a series of GBAP analogues and evaluated their activities by a gelatinase-inducing assay and newly developed receptor-binding assays, in which fluorescence-labeled peptides bound onto FsrC-overexpressing Lactococcus lactis cell surface were observed by fluorescent microscopy and quantified by using fluorophotometr. Alanine scanning analysis of GBAP showed that the entire ring region was involved in the GBAP agonist activity while side chains of the tail region were not strictly recognized. The alanine substitution of Phe⁷ or Trp¹⁰ almost abolished their receptor-binding ability and GBAP agonist activity, suggesting that these 2 aromatic side-chains are strongly involved in receptor interaction and activation. Furthermore, Trp¹⁰ substitution with natural and unnatural aromatic amino acids, except pentafluorophenylalanine, caused no loss of the agonist activity. This suggested the importance of a negative electrostatic potential created by an -electron cloud on the aromatic ring surface. Structural analysis of GBAP with nuclear magnetic resonance (NMR) spectroscopy revealed that the ring region adopted a hairpin-like fold and was tightly packed into a compact form. The side-chain of Trp¹⁰ was partially buried in the core structure, contributing to the stabilization of the compact form, while that of Phe⁷ was extended from the core structure into the solvent and was probably directly involved in receptor binding.