Research paper Site-directed mutagenesis of the conserved Ala348 and Gly350 residues at the putative active site of Bacillus kaustophilus leucine aminopeptidase Meng-Chun Chi a,d , Jai-Shin Liu b , Wen-Ching Wang b , Long-Liu Lin c, * , Hsien-Bin Huang d, ** a Department of Chemistry and Biochemistry, National Chung Cheng University, Chiayi, Taiwan b Institute of Molecular and Cellular Biology, and Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan c Department of Applied Chemistry, National Chiayi University, 300 University Road, Chiayi 600, Taiwan d Department of Life Sciences and Institute of Molecular Biology, National Chung Cheng University, Chiayi, Taiwan Received 18 July 2007; accepted 30 November 2007 Available online 26 December 2007 Abstract Leucine aminopeptidase (LAP) is an exopeptidase that catalyzes the hydrolysis of amino acid residues from the amino terminus of proteins and peptides. Sequence alignment shows that the conserved Ala348 and Gly350 residues of Bacillus kaustophilus LAP (BkLAP) are located right next to a coordinated ligand. We further investigated the roles of these two residues by performing computer modeling and site-directed mutagenesis. Based on the modeling, the carbonyl group of Ala348 interacts with Asn345 and Asn435, and that of Gly350 with Ile353 and Leu354, where these interactions might maintain the zinc-coordinated residues at their correct positions. Replacement of Ala348 with Arg resulted in a dramatic reduction in LAP activity. A complete loss of the activity was also observed in A348E, A348V, and the Gly350 variants. Measurement of intrinsic tryptophan fluorescence revealed alteration of the microenvironment of aromatic amino acid residues, while circular dichroism spectra were nearly identical for wild-type and all mutant enzymes. Protein modeling and site-directed mutagenesis suggest that residues Ala348 and Gly350 are essential for BkLAP in maintaining a stable active-site environment for the catalytic reaction. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Leucine aminopeptidase; Bacillus kaustophilus; Protein modeling; Site-directed mutagenesis; Tryptophan emission fluorescence; Circular dichroism 1. Introduction Dinuclear metallohydrolases are a large class of enzymes that catalyze the degradation of peptide, b-lactam, and ester bonds, making them important components in cellular protein turnover, antibiotic resistance, and homeostasis [1,2]. These enzymes have been implicated in angiogenesis, processing of angiogenic peptides, blood pressure regulation, HIV infec- tion, and resistance of bacteria to certain antibiotics [3e7]. One of the first discovered and most widely studied dinuclear metallohydrolases with respect to sequence, composition, structure and mechanism of action is the M17 leucine aminopeptidase (LAP) (EC 3.4.11.1). This enzyme is of biological and medical significance due to its altered activity observed in some diseases, such as eye lens aging and cataract [8]. For practical applications, LAPs have been shown to im- prove the bitter off-taste of food hydrolytes [9] and to convert L-homophenylalanyl amide into L-homophenylalanine, the ver- satile intermediate for a class of angiotensin I-converting enzyme inhibitors [10]. The structure of LAP, its mechanism of action, and diver- sity in structure and function were recently reviewed [11,12]. The enzyme from bovine eye lens (BlLAP) is the most extensively studied in its native form of three-dimen- sional structure [13,14] as well as those complexed with the inhibitors, amastatin [15], bestatin [16,17], leucinal [14] and phosphonic analogue of leucine [18]. BlLAP is built by six identical protomers to form a 10-A ˚ high solvent cavity in the center of the hexamer [19]. Each monomer consists of * Corresponding author. Tel.: þ886 5 271 7969; fax: þ886 5 271 7901. ** Corresponding author. Fax: þ886 5 2720411. E-mail addresses: llin@mail.ncyu.edu.tw (L.-L. Lin), biohbh@ccu.edu.tw (H.-B. Huang). 0300-9084/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2007.11.011 Available online at www.sciencedirect.com Biochimie 90 (2008) 811e819 www.elsevier.com/locate/biochi