From the State Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People's Republic of China.
J Biol Chem. 2014 Feb 28;289(9):6080-90. doi: 10.1074/jbc.M113.525535. Epub 2014 Jan 15.
Glycerol dehydrogenase (GDH) is an important polyol dehydrogenase for glycerol metabolism in diverse microorganisms and for value-added utilization of glycerol in the industry. Two GDHs from Klebsiella pneumoniae, DhaD and GldA, were expressed in Escherichia coli, purified and characterized for substrate specificity and kinetic parameters. Both DhaD and GldA could catalyze the interconversion of (3R)-acetoin/(2R,3R)-2,3-butanediol or (3S)-acetoin/meso-2,3-butanediol, in addition to glycerol oxidation. Although purified GldA appeared more active than DhaD, in vivo inactivation and quantitation of their respective mRNAs indicate that dhaD is highly induced by glycerol and plays a dual role in glycerol metabolism and 2,3-butanediol formation. Complementation in K. pneumoniae further confirmed the dual role of DhaD. Promiscuity of DhaD may have vital physiological consequences for K. pneumoniae growing on glycerol, which include balancing the intracellular NADH/NAD(+) ratio, preventing acidification, and storing carbon and energy. According to the kinetic response of DhaD to modified NADH concentrations, DhaD appears to show positive homotropic interaction with NADH, suggesting that the physiological role could be regulated by intracellular NADH levels. The co-existence of two functional GDH enzymes might be due to a gene duplication event. We propose that whereas DhaD is specialized for glycerol utilization, GldA plays a role in backup compensation and can turn into a more proficient catalyst to promote a survival advantage to the organism. Revelation of the dual role of DhaD could further the understanding of mechanisms responsible for enzyme evolution through promiscuity, and guide metabolic engineering methods of glycerol metabolism.
甘油脱氢酶（GDH）是多种微生物中甘油代谢的重要多元醇脱氢酶，也是工业中甘油增值利用的重要酶。本研究从肺炎克雷伯氏菌中表达并纯化了两种 GDH，即 DhaD 和 GldA，研究了它们的底物特异性和动力学参数。结果表明，DhaD 和 GldA 不仅可以催化甘油的氧化，还可以催化（3R）-乙酰基-（2R，3R）-2,3-丁二醇或（3S）-乙酰基-（meso）-2,3-丁二醇的相互转化。虽然纯化的 GldA 似乎比 DhaD 更活跃，但体内失活和各自 mRNA 的定量表明，dhaD 被甘油高度诱导，并在甘油代谢和 2,3-丁二醇形成中发挥双重作用。在肺炎克雷伯氏菌中的互补进一步证实了 DhaD 的双重作用。DhaD 的混杂性可能对以甘油为碳源生长的肺炎克雷伯氏菌具有重要的生理意义，包括平衡细胞内 NADH/NAD+的比值、防止酸化以及储存碳和能量。根据 DhaD 对修饰的 NADH 浓度的动力学响应，DhaD 似乎与 NADH 呈正同变相互作用，表明其生理作用可能受细胞内 NADH 水平的调节。两种功能性 GDH 酶的共存可能是由于基因复制事件。我们提出，虽然 DhaD 专门用于甘油的利用，但 GldA 起着后备补偿的作用，可以转变为更有效的催化剂，为生物体提供生存优势。DhaD 双重作用的揭示可以进一步了解通过混杂性导致的酶进化的机制，并为甘油代谢的代谢工程方法提供指导。
