| Summary: | 博士 === 國立清華大學 === 分子與細胞生物研究所 === 100 === Lysine racemase, an enzyme that catalyzes the interconversion of lysine enantiomers, is valuable to serve as a non-antibiotic selectable marker in the generation of transgenic plants. Here, we have determined the crystal structures of a novel lysine racemase (Lyr) from Proteus mirabilis BCRC10725 and a broad-specificity amino acid racemase (Bar) from Pseudomonas putida DSM84. Lyr showed a level of high acitivity towards lysine and weaker activity towards arginine, whereas the lyr gene was found to share highest identity with the putative alanine racemases. In contrast, the Bar protein presented not only the highest activity towards lysine but also remarkably broad substrate specificity. The structural model of Lyr was established by molecular replacement method within the template: Bar structure, which was solved by using the multiwavelength anomalous dispersion (MAD) method of SeMet-Bar crystal. Both structures demonstrate the fold of alanine racemase, which consists of an N-terminal domain with eight-stranded α/β barrels containing the pyridoxal 5’-phosphate (PLP) cofactor and a C-terminal domain primarily composed of β-strands. Both active sites of Lyr and Bar show that the PLP cofactor forms an aldimine linkage to Lys74 and Lys75 in Lyr and Bar, respectively, as a protonated Schiff base. Two conserved residues are presented in both Lyr (Lys74, Tyr299’) and Bar (Lys75, Tyr301’) to serve as the catalytic bases for racemization. Structural comparison and analysis of substrate binding sites and active residues would assist to clarify the mechanism of variant substrate selectivity. Two key residues (Thr391, Ser394 in Lyr and Ala393, Tyr396 in Bar), which are both located at -helix 10 and point toward the active site, are discovered to play an important role in the substrate specificity. The racemization activity toward lysine of the Lyr double-mutant (T391Y-S394Y) reduces to only 6% compared with the wild-type protein. In addition, two important residues are also investigated in both Lyr and Bar (Arg173, Asn174 and Arg174, Asn175, respectively) and are all in charge of the enzyme activity. Moreover, molecular modeling using different substrates with Lyr or Bar structure assists to elucidate the difference of substrate specificity between Lyr and Bar. A Bar-lysine liganded crystal structure is also established here and helps to prove that the docking approach used in this study is dependable according to the structural similarity between docking model and crystal structure of Bar-lysine complex. Together, our results suggest a proposed mechanism for the reversible racemization reaction of both enzymes described here and point out the key residues responsible for the substrate specificity and enzyme activity. This work provides a structural foundation for the design of racemases with pre-determined substrate specificity and assists in improving the reliability of the non-antibiotic selection system for transgenic plants.
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