Department of Chemistry, University of Manitoba, 144 Dysart Rd, Winnipeg, R3T 2N2, Manitoba, Canada.
FASEB J. 2010 Feb;24(2):404-14. doi: 10.1096/fj.09-138297. Epub 2009 Sep 28.
Gabaculine is a potent inhibitor of the vitamin B6-dependent key enzyme in chlorophyll biosynthesis, glutamate-1-semialdehyde aminomutase (GSAM). The inhibition effect is caused by an enzymatic deprotonation of the neurotoxin and requires the aldimine (PLP) form of the cofactor at the active site. In this study, we show that a single-point mutation confers resistance to gabaculine. A combined functional and structural analysis of wild-type GSAM in complex with gabaculine and the GSAM(M248I) form allowed us to decipher in atomic detail the molecular basis of this unique resistance. Interestingly, the gabaculine tolerance is caused by the absence of an essential water molecule that has a dual functional role. It serves as a nucleophilic shuttle for the hydroxyl anion along the reaction pathway and holds active-site Lys273 in a catalytically competent conformation. The single-point mutant is not able to fix this catalytic water between the beta-branched side chain of Ile248 and Lys273. As a consequence, the mutant enzyme is trapped in a gabaculine-insensitive but still enzymatically active amine (PMP) form.
加巴喷丁是一种强抑制剂，可抑制叶绿素生物合成过程中维生素 B6 依赖性关键酶——谷氨酸-1-半醛亚胺氨基转移酶（GSAM）。这种抑制作用是由于神经毒素的酶促去质子化引起的，需要在活性部位的辅助因子亚胺（PLP）形式。在这项研究中，我们表明单点突变赋予了对加巴喷丁的抗性。野生型 GSAM 与加巴喷丁和 GSAM（M248I）形式的复合物的综合功能和结构分析，使我们能够详细地解析这种独特抗性的分子基础。有趣的是，加巴喷丁耐受性是由缺乏必需水分子引起的，该水分子具有双重功能。它作为亲核试剂沿着反应途径沿羟基阴离子，并保持活性位点 Lys273 处于催化能力构象。单点突变体不能在 Ile248 和 Lys273 的β支链侧链之间固定这种催化水。结果，突变酶被捕获在加巴喷丁不敏感但仍具有酶活性的胺（PMP）形式中。
