Institute of Molecular and Cellular Biology and Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan.
PLoS One. 2012;7(10):e48301. doi: 10.1371/journal.pone.0048301. Epub 2012 Oct 31.
Lysine racemase, a pyridoxal 5'-phosphate (PLP)-dependent amino acid racemase that catalyzes the interconversion of lysine enantiomers, is valuable to serve as a novel non-antibiotic selectable marker in the generation of transgenic plants. Here, we have determined the first crystal structure of a lysine racemase (Lyr) from Proteus mirabilis BCRC10725, which shows the highest activity toward lysine and weaker activity towards arginine. In addition, we establish the first broad-specificity amino acid racemase (Bar) structure from Pseudomonas putida DSM84, which presents not only the highest activity toward lysine but also remarkably broad substrate specificity. A complex structure of Bar-lysine is also established here. These structures demonstrate the similar fold of alanine racemase, which is a head-to-tail homodimer with each protomer containing an N-terminal (α/β)(8) barrel and a C-terminal β-stranded domain. The active-site residues are located at the protomer interface that is a funnel-like cavity with two catalytic bases, one from each protomer, and the PLP binding site is at the bottom of this cavity. Structural comparisons, site-directed mutagenesis, kinetic, and modeling studies identify a conserved arginine and an adjacent conserved asparagine that fix the orientation of the PLP O3 atom in both structures and assist in the enzyme activity. Furthermore, side chains of two residues in α-helix 10 have been discovered to point toward the cavity and define the substrate specificity. Our results provide a structural foundation for the design of racemases with pre-determined substrate specificity and for the development of the non-antibiotic selection system in transgenic plants.
赖氨酸消旋酶是一种依赖于吡哆醛 5′-磷酸（PLP）的氨基酸消旋酶，可催化赖氨酸对映异构体的相互转化，可作为一种新型非抗生素选择标记物，用于转基因植物的产生。在这里，我们确定了来自变形杆菌 BCRC10725 的赖氨酸消旋酶（Lyr）的第一个晶体结构，该结构对赖氨酸表现出最高的活性，对精氨酸的活性较弱。此外，我们建立了来自假单胞菌 DSM84 的第一个广泛特异性氨基酸消旋酶（Bar）结构，该结构不仅对赖氨酸表现出最高的活性，而且具有显著广泛的底物特异性。还建立了 Bar-赖氨酸的复合物结构。这些结构展示了丙氨酸消旋酶的相似折叠，丙氨酸消旋酶是一种头尾同源二聚体，每个单体包含一个 N 端（α/β）（8）桶和一个 C 端β-折叠结构域。活性位点残基位于单体界面处，是一个漏斗状腔，其中两个催化碱，一个来自每个单体，PLP 结合位点位于该腔的底部。结构比较、定点突变、动力学和建模研究确定了一个保守的精氨酸和一个相邻的保守天冬酰胺，它们在两个结构中固定了 PLP O3 原子的方向，并有助于酶活性。此外，已经发现α-螺旋 10 中的两个侧链指向腔并定义了底物特异性。我们的研究结果为设计具有预定底物特异性的消旋酶和开发转基因植物中的非抗生素选择系统提供了结构基础。
