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J. Biol. Chem., Vol. 283, Issue 14, 9318-9327, April 4, 2008
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Molecular Basis of Prodrug Activation by Human Valacyclovirase, an 
-Amino Acid Ester Hydrolase*
13
From the
Department of Pharmaceutical Sciences and Center for Molecular Drug Targeting, University of Michigan, Ann Arbor, Michigan 48109-1065 and ¶Department of Biological Chemistry and ||Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109-2216
Chemical modification to improve biopharmaceutical properties, especially oral absorption and bioavailability, is a common strategy employed by pharmaceutical chemists. The approach often employs a simple structural modification and utilizes ubiquitous endogenous esterases as activation enzymes, although such enzymes are often unidentified. This report describes the crystal structure and specificity of a novel activating enzyme for valacyclovir and valganciclovir. Our structural insights show that human valacyclovirase has a unique binding mode and specificity for amino acid esters. Biochemical data demonstrate that the enzyme hydrolyzes esters of 
-amino acids exclusively and displays a broad specificity spectrum for the aminoacyl moiety similar to tricorn-interacting aminopeptidase F1. Crystal structures of the enzyme, two mechanistic mutants, and a complex with a product analogue, when combined with biochemical analysis, reveal the key determinants for substrate recognition; that is, a flexible and mostly hydrophobic acyl pocket, a localized negative electrostatic potential, a large open leaving group-accommodating groove, and a pivotal acidic residue, Asp-123, after the nucleophile Ser-122. This is the first time that a residue immediately after the nucleophile has been found to have its side chain directed into the substrate binding pocket and play an essential role in substrate discrimination in serine hydrolases. These results as well as a phylogenetic analysis establish that the enzyme functions as a specific -amino acid ester hydrolase. Valacyclovirase is a valuable target for amino acid ester prodrug-based oral drug delivery enhancement strategies.
Received for publication, November 20, 2007 , and in revised form, January 7, 2008.
The atomic coordinates and structure factors (codes 2OCG, 2OCI, 2OCK, and 2OCL) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
* This work was supported in part by National Institutes of Health Grant GM 37188 (to G. L. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1-S3 and Figs. S1-S4.
1 Present address: Dept. of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Ave., New Haven, CT 06520-8114.
2 To whom correspondence may be addressed: Dept. of Biological Chemistry, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI 48109-2216. Tel.: 734-615-2077; Fax: 734-763-6492; E-mail: zhaohui{at}umich.edu.
3 To whom correspondence may be addressed: Dept. of Pharmaceutical Sciences, University of Michigan, 428 Church St., Ann Arbor, MI 48109-1065. Tel.: 734-764-2226; Fax: 734-764-6282; E-mail: glamidon{at}umich.edu.
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