Cluster Analysis of Hydration Waters Around the Active Sites of Bacterial Alanine Racemase Using a 2-ns MD Simulation Hung-Chung Huang, 1 Daniel Jupiter, 1 Meikang Qiu, 2 James M. Briggs, 3 Vincent VanBuren 1 1 Department of Systems Biology and Translational Medicine, College of Medicine, TX A&M Health Science Center, Temple, TX 76504 2 Department of Electrical Engineering, University of New Orleans, New Orleans, LA 70148 3 Department of Biology and Biochemistry, University of Houston, Houston, TX 77204-5001 Received 11 September 2007; revised 6 November 2007; accepted 7 November 2007 Published online 19 November 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20893 This article was originally published online as an accepted preprint. The ‘‘Published Online’’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com INTRODUCTION A lanine racemase (AlaR), a pyridoxal 5 0 -phosphate- dependent (PLP-dependent) enzyme, is a bacterial enzyme that contributes to the formation of pepti- doglycan precursors via racemization of L-Alanine (L-Ala) to D-Alanine (D-Ala). This is the first step in the biosynthesis of the peptidoglycan. Because of its crucial role in bacterial cell wall biosynthesis and its nearly unique expression in bacteria, AlaR is considered to be an appropriate target for the design of inhibitors that will act as antibiotics. The AlaR enzyme from Geobacillus stearothermophilus is a homodimer, with subunits 1 and 2 interacting with each other noncovalently, head to tail (Figure 1). The AlaR mono- mer consists of an N-terminal a/b barrel domain and a C- terminal b barrel domain. A coenzyme PLP ring is covalently attached to the catalytic residue Lysine 39 (Lys39) in each subunit. The active site is formed by the catalytic residues from the N-terminal domain of one monomer and from the Cluster Analysis of Hydration Waters Around the Active Sites of Bacterial Alanine Racemase Using a 2-ns MD Simulation This article contains supplementary material available via the Internet at http:// www.interscience.wiley.com/jpages/0006-3525/suppmat. Correspondence to: H.-C. Huan; e-mail: hc.jhuang@tamu.edu or V. VanBuren; e-mail: vanburen@tamu.edu ABSTRACT: Structural data produced by a 2-ns molecular dynamics (MD) simulation on Geobacillus alanine racemase (AlaR; PDB: 1SFT) was used to study hydration around the two AlaR active sites. AlaR is a crucial enzyme for bacterial cell wall biosynthesis. It has been shown previously that the potency of an inhibitor can be increased by incorporating a functional group or atom that displaces hydration sites close to the substrate binding pocket of its target enzyme. The complete linkage algorithm was used for cluster analysis of the active site water positions from 126 solvent configurations sampled at regular intervals from the 2-ns MD simulation. Crystal waters in the 1SFT X-ray structure occupy most of the tightly bound water sites that were discovered. We show here that tightly bound water sites can be identified by cluster analysis of MD-generated coordinates starting with data supplied by a single X-ray structure, and we predict a highly conserved hydration site close to the carboxyl oxygen of L-Ala substrate. This approach holds promise for accelerating the drug design process. We also discuss an analysis of the well-known notion of residence time and introduce a new measure called retention time. # 2007 Wiley Periodicals, Inc. Biopolymers 89: 210–219, 2008. Keywords: alanine racemase; molecular dynamics; cluster analysis; complete linkage; water hydration sites Contract grant sponsor: National Institutes of Health Contract grant number: AI46340 V V C 2007 Wiley Periodicals, Inc. 210 Biopolymers Volume 89 / Number 3