Departments of Biochemistry and Chemistry and Institute for Genomic Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Biochemistry. 2014 Apr 29;53(16):2722-31. doi: 10.1021/bi500264p. Epub 2014 Apr 15.
The continued increase in the size of the protein sequence databases as a result of advances in genome sequencing technology is overwhelming the ability to perform experimental characterization of function. Consequently, functions are assigned to the vast majority of proteins via automated, homology-based methods, with the result that as many as 50% are incorrectly annotated or unannotated ( Schnoes et al. PLoS Comput. Biol. 2009 , 5 ( 12 ), e1000605 ). This manuscript describes a study of the D-mannonate dehydratase (ManD) subgroup of the enolase superfamily (ENS) to investigate how function diverges as sequence diverges. Previously, one member of the subgroup had been experimentally characterized as ManD [dehydration of D-mannonate to 2-keto-3-deoxy-D-mannonate (equivalently, 2-keto-3-deoxy-D-gluconate)]. In this study, 42 additional members were characterized to sample sequence-function space in the ManD subgroup. These were found to differ in both catalytic efficiency and substrate specificity: (1) high efficiency (kcat/KM = 10(3) to 10(4) M(-1) s(-1)) for dehydration of D-mannonate, (2) low efficiency (kcat/KM = 10(1) to 10(2) M(-1) s(-1)) for dehydration of d-mannonate and/or D-gluconate, and 3) no-activity with either D-mannonate or D-gluconate (or any other acid sugar tested). Thus, the ManD subgroup is not isofunctional and includes D-gluconate dehydratases (GlcDs) that are divergent from the GlcDs that have been characterized in the mandelate racemase subgroup of the ENS (Lamble et al. FEBS Lett. 2004 , 576 , 133 - 136 ) (Ahmed et al. Biochem. J. 2005 , 390 , 529 - 540 ). These observations signal caution for functional assignment based on sequence homology and lay the foundation for the studies of the physiological functions of the GlcDs and the promiscuous ManDs/GlcDs.
由于基因组测序技术的进步，蛋白质序列数据库的规模不断扩大，以至于人们已经无法对其功能进行全面的实验性描述。因此，绝大多数蛋白质的功能都是通过自动化的同源性方法来确定的，其结果是多达 50%的蛋白质的注释是不正确的或没有被注释（Schnoes 等人，PLoS Comput Biol. 2009, 5 (12), e1000605）。本文描述了一项关于烯醇酶超家族（ENS）中 D-甘露糖酸脱水酶（ManD）亚组的研究，旨在探讨随着序列的变化，功能是如何发生分歧的。此前，该亚组的一个成员已被实验证实为 ManD[D-甘露糖酸脱水生成 2-酮-3-脱氧-D-甘露糖酸（相当于 2-酮-3-脱氧-D-葡萄糖酸）]。在这项研究中，还对 42 个额外的成员进行了特征描述，以在 ManD 亚组中对序列-功能空间进行采样。结果发现它们在催化效率和底物特异性上存在差异：（1）D-甘露糖酸脱水的高效率（kcat/KM=10(3)到 10(4)M(-1)s(-1)），（2）D-甘露糖酸和/或 D-葡萄糖酸脱水的低效率（kcat/KM=10(1)到 10(2)M(-1)s(-1)），以及（3）对 D-甘露糖酸或 D-葡萄糖酸（或任何其他测试的酸性糖）无活性。因此，ManD 亚组并非同工酶，其中包括与在 ENS 中的扁桃酸消旋酶亚组中已被描述的 D-葡萄糖酸脱水酶（GlcD）不同的 D-葡萄糖酸脱水酶（GlcD）（Lamble 等人，FEBS Lett. 2004, 576, 133-136）（Ahmed 等人，Biochem J. 2005, 390, 529-540）。这些观察结果表明，基于序列同源性进行功能分配需要谨慎，并为研究 GlcD 和混杂的 ManD/GlcD 的生理功能奠定了基础。
