Evolution of enzymatic activities in the orotidine 5'-monophosphate decarboxylase suprafamily: enhancing the promiscuous D-arabino-hex-3-ulose 6-phosphate synthase reaction catalyzed by 3-keto-L-gulonate 6-phosphate decarboxylase.
Evolution of enzymatic activities in the orotidine 5'-monophosphate decarboxylase suprafamily: enhancing the promiscuous D-arabino-hex-3-ulose 6-phosphate synthase reaction catalyzed by 3-keto-L-gulonate 6-phosphate decarboxylase.
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DOI：
10.1021/bi047815v
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发表时间：
2005-02
期刊：
影响因子：
2.9
通讯作者：
W. Yew;J. Akana;Eric L. Wise;I. Rayment;J. Gerlt
中科院分区：
文献类型：
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作者：
W. Yew;J. Akana;Eric L. Wise;I. Rayment;J. Gerlt
3-酮-l-谷氨酸6-磷酸脱羧酶（KGPDC）和d-阿拉伯糖-己-3-乌糖6-磷酸合成酶（HPS）是orotidine 5'-单磷酸脱羧酶（OMPDC）超家族的成员[Wise， E., Yew， W. S., Babbitt， P. C., Gerlt， J. A.，和Rayment， I.（2002）生物化学41,3861-3869]，一组同源酶共享（β / α）(8)桶折叠。李晓东，李晓东，李晓东，等。KGPDC在大肠杆菌K-12利用l-抗坏血酸的分解代谢途径中催化Mg（2+）依赖的脱羧反应[j]。184年,302 - 306年);HPS催化甲醛与d- 5-磷酸核酮糖之间的Mg（2+）依赖性醛缩[Kato， N., Ohashi， H., Hori， T., Tani， Y., and Ogata， K.（1977）农业。医学杂志。化学学报，41(1):1 - 4。我们之前对大肠杆菌中KGPDC的研究证实了一种稳定的顺式烯二酸酯中间体的存在[Yew， W. S., Wise， E., Rayment， I., and Gerlt， J. a . (2004) Biochemistry 43, 6427-6437；李晓明，李晓明，李晓明，等。（2004）生物化学学报（自然科学版），32(4):444 - 444。虽然hps催化反应的机制尚未被研究，但它也有望涉及一个Mg（2+）稳定的顺式烯二醇中间体。我们现在已经发现来自大肠杆菌的KGPDC和来自氨基甲基单胞菌的HPS在同源物催化的反应中都是天然混杂的。在同源KGPDC和HPS序列比对的基础上，将大肠杆菌KGPDC中的4个保守活性位点突变为HPS中的保守活性位点（E112D/R139V/T169A/R192A）：混交HPS活性的k（cat）值增加了170倍（E112D/R139V/T169A/R192A突变体），k(cat)/ k(m)值增加了260倍（E112D/R139V/T169A突变体）；在这两种情况下，KGPDC天然活性的动力学常数值都降低了。再加上随附稿件中报道的突变体结构[Wise， E. L., Yew， W. S., Akana， J., Gerlt， J. A.，和Rayment， I.，随附稿件]，这些研究表明，催化效率的巨大变化可以通过活性位点结构的适度变化来实现。因此，OMPDC超家族成员共有的（β / α）(8)桶状褶皱似乎非常适合新功能的进化。
3-Keto-l-gulonate 6-phosphate decarboxylase (KGPDC) and d-arabino-hex-3-ulose 6-phosphate synthase (HPS) are members of the orotidine 5'-monophosphate decarboxylase (OMPDC) suprafamily [Wise, E., Yew, W. S., Babbitt, P. C., Gerlt, J. A., and Rayment, I. (2002) Biochemistry 41, 3861-3869], a group of homologous enzymes that share the (beta/alpha)(8)-barrel fold. KGPDC catalyzes a Mg(2+)-dependent decarboxylation reaction in the catabolic pathway of l-ascorbate utilization by Escherichia coli K-12 [Yew, W. S., and Gerlt, J. A. (2002) J.Bacteriol. 184, 302-306]; HPS catalyzes a Mg(2+)-dependent aldol condensation between formaldehyde and d-ribulose 5-phosphate in formaldehyde-fixing methylotrophic bacteria [Kato, N., Ohashi, H., Hori, T., Tani, Y., and Ogata, K. (1977) Agric. Biol. Chem. 41, 1133-1140]. Our previous studies of the KGPDC from E. coli established the occurrence of a stabilized cis-enediolate intermediate [Yew, W. S., Wise, E., Rayment, I., and Gerlt, J. A. (2004) Biochemistry 43, 6427-6437; Wise, E., Yew, W. S., Gerlt, J. A., and Rayment, I. (2004) Biochemistry 43, 6438-6446]. Although the mechanism of the HPS-catalyzed reaction has not yet been investigated, it also is expected to involve a Mg(2+)-stabilized cis-enediolate intermediate. We now have discovered that the KGPDC from E. coli and the HPS from Methylomonas aminofaciens are both naturally promiscuous for the reaction catalyzed by the homologue. On the basis of the alignment of the sequences of orthologous KGPDC's and HPS's, four conserved active site residues in the KGPDC from E. coli were mutated to those conserved in HPS's (E112D/R139V/T169A/R192A): the value of the k(cat) for the promiscuous HPS activity was increased as much as 170-fold (for the E112D/R139V/T169A/R192A mutant), and the value of k(cat)/K(m) was increased as much as 260-fold (for the E112D/R139V/T169A mutant); in both cases, the values of the kinetic constants for the natural KGPDC activity were decreased. Together with the structures of mutants reported in the accompanying manuscript [Wise, E. L., Yew, W. S., Akana, J., Gerlt, J. A., and Rayment, I., accompanying manuscript], these studies illustrate that large changes in catalytic efficiency can be accomplished with only modest changes in active site structure. Thus, the (beta/alpha)(8)-barrel fold shared by members of the OMPDC suprafamily appears well-suited for the evolution of new functions.