进化orotidine酶活动的5 '一磷酸脱羧酶suprafamily:提高滥交D-arabino-hex-3-ulose 6-phosphate合酶所催化的反应3-keto-L-gulonate 6-phosphate脱羧酶。
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紫杉WS, Akana J、明智的EL Rayment我Gerlt农协
进化orotidine酶活动的5 '一磷酸脱羧酶suprafamily:提高滥交D-arabino-hex-3-ulose 6-phosphate合酶所催化的反应3-keto-L-gulonate 6-phosphate脱羧酶。
生物化学,2005年2月15日,44(6):1807 - 15所示。
- PubMed ID
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15697206 (在PubMed]
- 文摘
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3-Keto-l-gulonate 6-phosphate脱羧酶(KGPDC)和d-arabino-hex-3-ulose 6-phosphate合成酶(HPS)的成员orotidine 5 '一磷酸脱羧酶(OMPDC) suprafamily[智慧,E。紫杉,w·S。,巴比特,p . C。Gerlt, j。,Rayment i(2002)生物化学41岁,3861 - 3869年),一组同源酶分享(β/α)(8)桶。KGPDC催化镁(2 +)端依赖脱羧反应的分解途径l-ascorbate由大肠杆菌利用k - 12(紫杉,w·S。Gerlt, j . a (2002) J.Bacteriol。184年,302 - 306年);HPS催化镁(2 +)端依赖之间的醇醛缩合甲醛和d-ribulose 5-phosphate在formaldehyde-fixing methylotrophic细菌(加藤,N。大桥,H。Hori T。塔尼语,Y。理事长绪方,k(1977)阿格利司。医学杂志。化学41岁,1133 - 1140]。KGPDC从大肠杆菌的先前的研究建立一个稳定cis-enediolate中间的发生[紫杉,w·S。明智的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.