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Journal of Bacteriology, January 2009, p. 641-650, Vol. 191, No. 2
0021-9193/09/$08.00+0     doi:10.1128/JB.01029-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Structure-Activity Relationship of Gelatinase Biosynthesis-Activating Pheromone of Enterococcus faecalis{triangledown}

Kenzo Nishiguchi,1 Koji Nagata,2 Masaru Tanokura,2 Kenji Sonomoto,1,3 and Jiro Nakayama1*

Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 812-8581, Japan,1 Department of Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan,2 Department of Functional Metabolic Design, Bio-Architecture Center, Kyushu University, Fukuoka 812-8581, Japan3

Received 25 July 2008/ Accepted 28 October 2008

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 the FsrC-overexpressing Lactococcus lactis cell surface were observed by fluorescent microscopy and quantified by using a fluorophotometer. 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 abilities and GBAP agonist activities, suggesting that these two aromatic side chains are strongly involved in receptor interaction and activation. Furthermore, the Trp10 substitution with natural and unnatural aromatic amino acids, except pentafluorophenylalanine, caused no loss of agonist activity. This suggested the importance of a negative electrostatic potential created by an {pi}-electron cloud on the aromatic ring surface. Structural analysis of GBAP with nuclear magnetic resonance 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.

* Corresponding author. Mailing address: Laboratory of Microbial Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Phone: 81-92-642-3020. Fax: 81-92-642-3021. E-mail: nakayama{at}agr.kyushu-u.ac.jp

{triangledown} Published ahead of print on 7 November 2008.

Journal of Bacteriology, January 2009, p. 641-650, Vol. 191, No. 2
0021-9193/09/$08.00+0     doi:10.1128/JB.01029-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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