School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia.
FEBS J. 2010 Sep;277(18):3844-60. doi: 10.1111/j.1742-4658.2010.07788.x. Epub 2010 Aug 16.
We report the first characterization of an L-arginine:glycine amidinotransferase from a prokaryote. The enzyme, CyrA, is involved in the pathway for biosynthesis of the polyketide-derived hepatotoxin cylindrospermopsin from Cylindrospermopsis raciborskii AWT205. CyrA is phylogenetically distinct from other amidinotransferases, and structural alignment shows differences between the active site residues of CyrA and the well-characterized human L-arginine:glycine amidinotransferase (AGAT). Overexpression of recombinant CyrA in Escherichia coli enabled biochemical characterization of the enzyme, and we confirmed the predicted function of CyrA as an L-arginine:glycine amidinotransferase by (1) H NMR. As compared with AGAT, CyrA showed narrow substrate specificity when presented with substrate analogs, and deviated from regular Michaelis-Menten kinetics in the presence of the non-natural substrate hydroxylamine. Studies of initial reaction velocities and product inhibition, and identification of intermediate reaction products, were used to probe the kinetic mechanism of CyrA, which is best described as a hybrid of ping-pong and sequential mechanisms. Differences in the active site residues of CyrA and AGAT are discussed in relation to the different properties of both enzymes. The enzyme had maximum activity and maximum stability at pH 8.5 and 6.5, respectively, and an optimum temperature of 32 °C. Investigations into the stability of the enzyme revealed that an inactivated form of this enzyme retained an appreciable amount of secondary structure elements even on heating to 94 °C, but lost its tertiary structure at low temperature (T(max) of 44.5 °C), resulting in a state reminiscent of a molten globule. CyrA represents a novel group of prokaryotic amidinotransferases that utilize arginine and glycine as substrates with a complex kinetic mechanism and substrate specificity that differs from that of the eukaryotic L-arginine:glycine amidinotransferases.
我们首次对一种来自原核生物的 L-精氨酸：甘氨酸氨基转移酶进行了表征。该酶 CyrA 参与从 Cylindrospermopsis raciborskii AWT205 生物合成聚酮衍生的肝毒素环胞嘧啶的途径。CyrA 在系统发育上与其他氨基转移酶不同，结构比对显示 CyrA 的活性位点残基与经过充分表征的人类 L-精氨酸：甘氨酸氨基转移酶（AGAT）之间存在差异。在大肠杆菌中过表达重组 CyrA 使该酶的生化特性得以阐明，我们通过 1 H NMR 证实了 CyrA 作为 L-精氨酸：甘氨酸氨基转移酶的预测功能。与 AGAT 相比，当使用底物类似物时，CyrA 的底物特异性较窄，并且在非天然底物羟胺存在下偏离常规米氏动力学。通过研究初始反应速度和产物抑制，并鉴定中间反应产物，探究了 CyrA 的动力学机制，该机制最好描述为乒乓和顺序机制的混合。CyrA 和 AGAT 的活性位点残基的差异与两种酶的不同性质有关。该酶在 pH 8.5 时具有最大活性，在 pH 6.5 时具有最大稳定性，最适温度为 32°C。对该酶稳定性的研究表明，即使在加热至 94°C 时，该酶的失活形式仍保留相当数量的二级结构元件，但在低温下（T(max)为 44.5°C）失去其三级结构，导致类似于熔融球蛋白的状态。CyrA 代表了一类新型的原核氨基转移酶，它们利用精氨酸和甘氨酸作为底物，具有复杂的动力学机制和不同于真核 L-精氨酸：甘氨酸氨基转移酶的底物特异性。
