Fischer Markus, Romisch Werner, Schiffmann Susanne, Kelly Mark, Oschkinat Hartmut, Steinbacher Stefan, Huber Robert, Eisenreich Wolfgang, Richter Gerald, Bacher Adelbert
Institut für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany.
J Biol Chem. 2002 Nov 1;277(44):41410-6. doi: 10.1074/jbc.M206863200. Epub 2002 Aug 27.
The hypothetical protein predicted by the open reading frame MJ0055 of Methanococcus jannaschii was expressed in a recombinant Escherichia coli strain under the control of a synthetic gene optimized for translation in an eubacterial host. The recombinant protein catalyzes the formation of the riboflavin precursor 3,4-dihydroxy-2-butanone 4-phosphate from ribulose 5-phosphate at a rate of 174 nmol mg(-1) min(-1) at 37 degrees C. The homodimeric 51.6-kDa protein requires divalent metal ions, preferentially magnesium, for activity. The reaction involves an intramolecular skeletal rearrangement as shown by (13)C NMR spectroscopy using [U-(13)C(5)]ribulose 5-phosphate as substrate. A cluster of charged amino acid residues comprising arginine 25, glutamates 26 and 28, and aspartates 21 and 30 is essential for catalytic activity. Histidine 164 and glutamate 185 were also shown to be essential for catalytic activity.
詹氏甲烷球菌（Methanococcus jannaschii）开放阅读框MJ0055预测的假定蛋白，在为真细菌宿主翻译优化的合成基因控制下，于重组大肠杆菌菌株中表达。该重组蛋白在37℃时，以174 nmol mg⁻¹ min⁻¹的速率催化从5-磷酸核糖生成核黄素前体3,4-二羟基-2-丁酮4-磷酸。这种同二聚体的51.6 kDa蛋白的活性需要二价金属离子，优先是镁离子。如以[U-(¹³)C₅]5-磷酸核糖为底物通过¹³C NMR光谱所示，该反应涉及分子内骨架重排。由精氨酸25、谷氨酸26和28以及天冬氨酸21和30组成的一组带电荷氨基酸残基对催化活性至关重要。组氨酸164和谷氨酸185也显示对催化活性至关重要。
