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首页> 外文期刊>Biochemistry >Mechanistic and Bioinformatic Investigation of a Conserved Active Site Helix in α-Isopropylmalate Synthase from Mycobacterium tuberculosis, a Member of the DRE-TIM Metallolyase Superfamily
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Mechanistic and Bioinformatic Investigation of a Conserved Active Site Helix in α-Isopropylmalate Synthase from Mycobacterium tuberculosis, a Member of the DRE-TIM Metallolyase Superfamily

机译：DRE-TIM金属分解酶超家族成员结核分枝杆菌α-异丙基苹果酸合酶中保守的活性位点螺旋的机制和生物信息学研究

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The characterization of functionally diverse enzyme superfamilies provides the opportunity to identify evolutionarily conserved catalytic strategies, as well as amino acid substitutions responsible for the evolution of new functions or specificities. Isopropylmalate synthase (IPMS) belongs to the DRE-TIM metallolyase superfamily. Members of this superfamily share common active site elements, including a conserved active site helix and an HXH divalent metal binding motif, associated with stabilization of a common enolate anion intermediate. These common elements are overlaid by variations in active site architecture resulting in the evolution of a diverse set of reactions that include condensation, lyase/aldolase, and carboxyl transfer activities. Here, using IPMS, an integrated biochemical and bioinformatics approach has been utilized to investigate the catalytic role of residues on an active site helix that is conserved across the superfamily. The construction of a sequence similarity network for the DRE-TIM metallolyase superfamily allows for the biochemical results obtained with IPMS variants to be compared across superfamily members and within other condensation-catalyzing enzymes related to IPMS. A comparison of our results with previous biochemical data indicates an active site arginine residue (R80 in IPMS) is strictly required for activity across the superfamily, suggesting that it plays a key role in catalysis, most likely through enolate stabilization. In contrast, differential results obtained from substitution of the C-terminal residue of the helix (Q84 in IPMS) suggest that this residue plays a role in reaction specificity within the superfamily.
机译：功能多样的酶超家族的表征为鉴定进化上保守的催化策略以及负责新功能或特异性进化的氨基酸取代提供了机会。苹果酸异丙酯合酶（IPMS）属于DRE-TIM金属裂解酶超家族。该超家族成员共享共同的活性位点元素，包括保守的活性位点螺旋和HXH二价金属结合基序，与稳定烯醇酸阴离子中间体有关。这些共同的元素被活性位点结构的变化所覆盖，从而导致包括缩合，裂解酶/醛缩酶和羧基转移活性在内的多种反应的发展。在这里，使用IPMS，已经采用了一种综合的生物化学和生物信息学方法来研究残基在整个超家族中保守的活性位点螺旋上的催化作用。 DRE-TIM金属分解酶超家族的序列相似性网络的构建允许在整个超家族成员之间以及与IPMS相关的其他缩合催化酶中比较使用IPMS变体获得的生化结果。将我们的结果与以前的生化数据进行比较，结果表明，超家族中的活性完全需要一个活性位点的精氨酸残基（IPMS中的R80），这表明它在催化中起着关键作用，很可能是通过烯醇稳定。相反，从螺旋的C末端残基（IPMS中的Q84）取代获得的不同结果表明，该残基在超家族中的反应特异性中起作用。

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