or in proper orientation for catalysis. However, there is no hydrogen bond between Gln360 and phosphate group in AlrTt. In addition, mutation of Gln360 to hydrophobic and 9 / 18 Sturcure and Substrate Selection of a Thermostable Alanine Racemase Fig 3. Relative MedChemExpress LY-411575 activity of the saturation mutants of Gln360 in L-Ala racemization, and the relative activity of wild-type and Q360Y mutant in racemization of ten L-amino acids. The racemase activities of the saturation mutants of Gln360 relative to wild-type AlrTt are represented by columns with means SD of quadruplicate experiments. The racemase activities of ten L-amino acids relative to L-Ala catalyzed by wild-type AlrTt are represented by columns with means SD of quadruplicate experiments. The relative amino acids specificity of Q360Y towards ten L-amino acids is shown in same profile as. doi:10.1371/journal.pone.0133516.g003 neutral amino acids that could not form hydrogen bonds surprisingly increased the racemase activity, indicating that forming hydrogen bond with PLP is not the primary role of residues at 
Gln360 position, instead its hydrophobic interaction with surrounding environment is more important in racemization. In AlrTt structure, we observed a hydrophobic patch localized underneath Gln360 and phosphate group of PLP. This hydrophobic patch is conserved both in sequence and conformation among bacterial alanine racemase. It is formed by residues from Pro225 to Arg237 at helix h2 and Pro359 in 11. The backbone amide group of Gly226-Ile227 in this patch forms hydrogen bond with OP1 of the phosphate group. In AlrBst, A228 and Asp343 form hydrogen bonds with Asn353, dragging 11 into the active site pocket, thus side chain of Tyr354 is pointing towards the phosphate group to form the hydrogen bond. 10 / 18 Sturcure and Substrate Selection of a Thermostable Alanine Racemase Fig 4. The hydrophobic patch and its interaction with active site pocket. The hydrophobic patch in AlrTt is colored in gray, the active site pocket is shown in orange, the substrate and key amino acids mediating the interactions are shown in sticks, the hydrogen bonding interactions are indicated. The hydrophobic patch and active site pocket of the other three bacterial alanine racemases are shown in same view: the PLP-D-Ala complex of AlrBst. PLP complex of AlrCd, PLP and DLY complex of DadXpao. doi:10.1371/journal.pone.0133516.g004 Identical interactions are also observed in AlrCd. In the catabolic alanine racemase DadXpao, the hydrophobic patch adopts similar conformation, but the hydrogen bonding are replaced by hydrophobic interactions. Although Ala334, Gly330 and Ala331 replaced PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19748594 the charged residues, 11 is still closely associated with the hydrophobic patch. When hydrophobic amino acids are introduced at Gln360, they first interact with Pro359 and residues like Ile227, Phe230 and Tyr234 in the hydrophobic patch to form a stable substrate binding pocket. Second, their side chains steric pack with the aromatic ring of Tyr44, which was given an optimal orientation for hydrogen bonding with the phosphate group. Thus, mutation of Gln360 to hydrophobic residues could enhance the hydrophobic interactions, provide a stronger steric hindrance to form a stable and strictly constrained substrate binding pocket for proper PLP immobilization, thus the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19748686 racemase activity of AlrTt was increased. Smaller amino acids did not affect the racemase activity, indicating the side chain of 11 / 18 Sturcure and Substrate Selection of a
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