d the racemase 
activity of DadXTt. These observations draw our attention to the role of Gln360 in alanine racemization. In order to understand the biochemical properties of AlrTt, especially the role of the nonconserved residue Gln360 in alanine racemization, we determined the crystal structure of AlrTt in complex with L-Ala at 2.7 resolution, and investigated its racemase activity by saturation mutagenesis of Gln360. Overall architecture of AlrTt is similar to typical bacterial alanine racemase. However, presence of Gln360 and conformational changes of the active site residues destabilized PLP immobilization, resulted in the low racemase activity of AlrTt. Introduction of 2 / 18 Sturcure and Substrate Selection of a Thermostable Alanine Racemase hydrophobic amino acids at Gln360 increased the overall catalytic efficiency of the enzyme. Through interactions with a hydrophobic patch near the active site pocket, these hydrophobic amino acids, especially Tyr residue steric block the entry and turnover of larger amino acids, enhance the substrate specificity of AlrTt. This work for the first time revealed a preference of hydrophobic amino acids at Gln360 position for substrate selection, further explored the racemization mechanism of bacterial alanine racemase, it will contribute useful information for antibiotics development. Materials and Methods Construction of the expression plasmids for wild-type and mutant AlrTt The alanine racemase gene alrTt was amplified by PCR using the genomic DNA of T. tengcongensis MB4 as template and a pair of primers. PCR products were purified using gel extraction kit, and then inserted into TA cloning vector pMD19-T to construct plasmid pMD-AlrTt. The plasmid was verified by DNA sequencing and digested with restriction enzyme NheI and XhoI, the gene fragment encoding full-length AlrTt was then ligated into a pET-28a vector to obtain the expression plasmid pET-28a-AlrTt. The QuikChange Site-directed Mutagenesis kit was used for mutation of residues Ser173 to Asp173, Gln360 to Tyr360 and Gln360 to other residues in the substrate entryway of AlrTt. PCR products were obtained with primer pairs S173D-F’ and S173D-R’, Q360Y-F’ and MedChemExpress ONX-0914 Q360Y-R’, S-Q360-F’ and S-Q360-R’ summarized in S1 Enzyme expression and purification Full length AlrTt was cloned into pET28a vector with both N- and C-terminal 6His tag and overexpressed in E. coli BL21 cells. The cells were lysed by sonication in buffer containing 50 mM NaH2PO4 pH8.0, 300 mM NaCl and 20 mM imidazole. The cell PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19747578 lysates were centrifuged at 26,664 g for 10 min. The soluble fractions were applied to Ni-NTA column, and the bound AlrTt was eluted by 250 mM imidazole. After buffer exchange into 25 mM Tris-HCl pH8.0, 200 mM NaCl, 10 M PLP, AlrTt was further purified by size exclusion chromatography using a Superdex 200 10/300 GL column. The purified AlrTt was collected and dialyzed against 10 mM Tris-Cl pH8.0, 10 M PLP, and concentrated with an Amicon Ultra-15 Centrifugal Filter Device for crystallization. Enzyme expression and purification of AlrTt mutants were same as wild-type. Crystallization Crystallization was performed using hanging-drop vapor diffusion method. Purified AlrTt was incubated with L-Ala and PLP at 1:1.5:1.5 molar ratio at 277 K for 2 h before crystallization. Diffracted crystals were PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19748686 obtained at 289 K by mixing 1 L L-Ala and PLP incubated AlrTt with 1 L reservoir solution and equilibrating the mixture against 300 L reservoir solution. 3 / 18 Sturcure and