Echt Stefanie, Bauer Stefanie, Steinbacher Stefan, Huber Robert, Bacher Adelbert, Fischer Markus
Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Garching, Germany.
J Mol Biol. 2004 Aug 20;341(4):1085-96. doi: 10.1016/j.jmb.2004.06.053.
A synthetic gene specifying a putative 3,4-dihydroxy-2-butanone 4-phosphate synthase of Candida albicans directed the synthesis of a 22.5 kDa peptide in a recombinant Escherichia coli strain. The recombinant protein was purified to apparent homogeneity by two chromatographic steps and was shown to catalyze the formation of L-3,4-dihydroxy-2-butanone 4-phosphate from ribulose 5-phosphate at a rate of 332 nmol mg(-1) min(-1). Hydrodynamic studies indicated a native molecular mass of 41 kDa in line with a homodimer structure. The protein was crystallized in its apoform. Soaking yielded crystals in complex with the substrate ribulose 5-phosphate. The structures were solved at resolutions of 1.6 and 1.7 angstroms, respectively, using 3,4-dihydroxy-2-butanone 4-phosphate synthase of E. coli for molecular replacement. Structural comparison with the orthologs of Magnaporthe grisea and Methanococcus jannaschii revealed a hitherto unknown conformation of the essential acidic active-site loop.
一个合成基因编码白色念珠菌假定的3,4-二羟基-2-丁酮4-磷酸合酶，该基因在重组大肠杆菌菌株中指导合成了一种22.5 kDa的肽。通过两步色谱法将重组蛋白纯化至表观均一，结果表明其能以332 nmol mg⁻¹ min⁻¹的速率催化5-磷酸核酮糖形成L-3,4-二羟基-2-丁酮4-磷酸。流体动力学研究表明其天然分子量为41 kDa，符合同二聚体结构。该蛋白以脱辅基形式结晶。浸泡后得到与底物5-磷酸核酮糖结合的晶体。分别使用大肠杆菌的3,4-二羟基-2-丁酮4-磷酸合酶进行分子置换，以1.6埃和1.7埃的分辨率解析了结构。与稻瘟病菌和詹氏甲烷球菌的直系同源物进行结构比较，发现了必需酸性活性位点环迄今未知的构象。
