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首页> 外文期刊>Biochemistry >Active site loop dynamics of a class IIa fructose 1,6-bisphosphate aldolase from Mycobacterium tuberculosis
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Active site loop dynamics of a class IIa fructose 1,6-bisphosphate aldolase from Mycobacterium tuberculosis

机译：IIA类果糖的有源部位环动态1,6-双磷酸醛糖酶免于结核分枝杆菌

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摘要

Class II fructose 1,6-bisphosphate aldolases (FBAs, EC 4.1.2.13) comprise one of two families of aldolases. Instead of forming a Schiff base intermediate using an ε-amino group of a lysine side chain, class II FBAs utilize Zn(II) to stabilize a proposed hydroxyenolate intermediate (HEI) in the reversible cleavage of fructose 1,6-bisphosphate, forming glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). As class II FBAs have been shown to be essential in pathogenic bacteria, focus has been placed on these enzymes as potential antibacterial targets. Although structural studies of class II FBAs from Mycobacterium tuberculosis (MtFBA), other bacteria, and protozoa have been reported, the structure of the active site loop responsible for catalyzing the protonation-deprotonation steps of the reaction for class II FBAs has not yet been observed. We therefore utilized the potent class II FBA inhibitor phosphoglycolohydroxamate (PGH) as a mimic of the HEI- and DHAP-bound form of the enzyme and determined the X-ray structure of the MtFBA-PGH complex to 1.58 ?. Remarkably, we are able to observe well-defined electron density for the previously elusive active site loop of MtFBA trapped in a catalytically competent orientation. Utilization of this structural information and site-directed mutagenesis and kinetic studies conducted on a series of residues within the active site loop revealed that E169 facilitates a water-mediated deprotonation-protonation step of the MtFBA reaction mechanism. Also, solvent isotope effects on MtFBA and catalytically relevant mutants were used to probe the effect of loop flexibility on catalytic efficiency. Additionally, we also reveal the structure of MtFBA in its holoenzyme form.
机译：II类果糖1,6-双磷酸醛糖酶（FBA，EC 4.1.2.13）包含两个醛糖酶的两个家族之一。使用赖氨酸侧链的ε-氨基的ε-氨基，II类FBA使用Zn（II）来稳定提出的羟基酚酸酯中间体（Hei），以在果糖1,6-双磷酸盐的可逆裂解中稳定一个羟基磷酸酯，形成甘油醛3-磷酸​​盐和二羟基丙酮磷酸盐（DHAP）。由于II类FBA已被证明在致病细菌中是必不可少的，则将重点放在这些酶中作为潜在的抗菌靶标。虽然已经报道了II类FBA的结构研究，但是已经报道了来自分枝杆菌（MTFBA），其他细菌和原生动物的结构研究，但尚未观察到负责催化II类FBA的反应的质子化 - 去质子化步骤的活性位点环的结构。因此，我们利用效率的II型FBA抑制剂磷酸亚羟肟酸酯（PGH）作为酶的HEI和DHAP键合的模拟，并确定MTFBA-PGH复合物的X射线结构至1.58？。值得注意的是，我们能够观察到以催化称职的定位捕获的MTFBA先前难以捉摸的活性位点的明确定义的电子密度。在活性部位环内的一系列残留物中使用该结构信息和定向诱变和动力学研究显示E169促进MTFBA反应机制的水介导的去质子化 - 质子化步骤。此外，对MTFBA和催化相关突变体的溶剂同位素作用用于探测环形柔韧性对催化效率的影响。另外，我们还以其全酶形式揭示MTFBA的结构。

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  • 来源
    《Biochemistry》 |2013年第5期|共14页
  • 作者

    Pegan S.D.; Rukseree K.; Capodagli G.C.; Baker E.A.; Krasnykh O.; Franzblau S.G.; Mesecar A.D.;

  • 作者单位

    Eleanor Roosevelt Institute Department of Chemistry and Biochemistry University of Denver 2190 E. Iliff Ave. Olin 202 Denver CO 80208 United States;

    National Center for Genetic Engineering and Biotechnology (BIOTEC) NSTDA Thailand Science Park Tha Khlong 12120 Thailand;

    Eleanor Roosevelt Institute Department of Chemistry and Biochemistry University of Denver 2190 E. Iliff Ave. Olin 202 Denver CO 80208 United States;

    Eleanor Roosevelt Institute Department of Chemistry and Biochemistry University of Denver 2190 E. Iliff Ave. Olin 202 Denver CO 80208 United States;

    Institute for Tuberculosis Research University of Illinois at Chicago Chicago IL 60612 United States;

    Institute for Tuberculosis Research University of Illinois at Chicago Chicago IL 60612 United States;

    Department of Biological Sciences and Chemistry Purdue Center for Cancer Research Purdue University West Lafayette IN 47907 United States;

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  • 正文语种 eng
  • 中图分类 生物化学;
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