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Mechanistic Studies of the Streptomyces bingchenggensis Aldolase-Dehydratase: Implications for Substrate and Reaction Specificity in the Acetoacetate Decarboxylase-like Superfamily.
Biochemistry ( IF 2.9 ) Pub Date : 2019-09-20 , DOI: 10.1021/acs.biochem.9b00652
Lisa S Mydy 1 , Robert W Hoppe 1 , Trevor M Hagemann 1 , Alan W Schwabacher 1 , Nicholas R Silvaggi 1
Biochemistry ( IF 2.9 ) Pub Date : 2019-09-20 , DOI: 10.1021/acs.biochem.9b00652
Lisa S Mydy 1 , Robert W Hoppe 1 , Trevor M Hagemann 1 , Alan W Schwabacher 1 , Nicholas R Silvaggi 1
Affiliation
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The acetoacetate decarboxylase-like superfamily (ADCSF) is a little-explored group of enzymes that may contain new biocatalysts. The low level of sequence identity (∼20%) between many ADCSF enzymes and the confirmed acetoacetate decarboxylases led us to investigate the degree of diversity in the reaction and substrate specificity of ADCSF enzymes. We have previously reported on Sbi00515, which belongs to Family V of the ADCSF and functions as an aldolase-dehydratase. Here, we more thoroughly characterize the substrate specificity of Sbi00515 and find that aromatic, unsaturated aldehydes yield lower KM and higher kcat values compared to those of other small electrophilic substrates in the condensation reaction. The roles of several active site residues were explored by site-directed mutagenesis and steady state kinetics. The lysine-glutamate catalytic dyad, conserved throughout the ADCSF, is required for catalysis. Tyrosine 252, which is unique to Sbi00515, is hypothesized to orient the incoming aldehyde in the condensation reaction. Transient state kinetics and an intermediate-bound crystal structure aid in completing a proposed mechanism for Sbi00515.
中文翻译：
bingchenggensis链霉菌醛缩酶脱水酶的机理研究：乙酰乙酸脱羧酶样超家族中底物和反应特异性的含义。
乙酰乙酸类脱羧酶样超家族（ADCSF）是一种酶，其探索性很强，可能包含新的生物催化剂。许多ADCSF酶与已确认的乙酰乙酸酯脱羧酶之间的序列同一性较低（约20％），这使我们研究了ADCSF酶反应的多样性程度和底物特异性。我们先前曾报道过Sbi00515，它属于ADCSF的V族，起醛缩酶脱水酶的作用。在这里，我们更彻底地表征了Sbi00515的底物特异性，发现芳香族不饱和醛的K M值较低，而k cat的值较高。缩合反应中与其他小亲电底物相比 通过定点诱变和稳态动力学探索了几个活性位点残基的作用。在整个ADCSF中保守的赖氨酸-谷氨酸催化二聚体是催化所必需的。Sbi00515特有的酪氨酸252被认为可以在缩合反应中定向进入的醛。瞬态动力学和中间结合的晶体结构有助于完成Sbi00515的拟议机理。
更新日期：2019-09-21
中文翻译：
bingchenggensis链霉菌醛缩酶脱水酶的机理研究：乙酰乙酸脱羧酶样超家族中底物和反应特异性的含义。
乙酰乙酸类脱羧酶样超家族（ADCSF）是一种酶，其探索性很强，可能包含新的生物催化剂。许多ADCSF酶与已确认的乙酰乙酸酯脱羧酶之间的序列同一性较低（约20％），这使我们研究了ADCSF酶反应的多样性程度和底物特异性。我们先前曾报道过Sbi00515，它属于ADCSF的V族，起醛缩酶脱水酶的作用。在这里，我们更彻底地表征了Sbi00515的底物特异性，发现芳香族不饱和醛的K M值较低，而k cat的值较高。缩合反应中与其他小亲电底物相比 通过定点诱变和稳态动力学探索了几个活性位点残基的作用。在整个ADCSF中保守的赖氨酸-谷氨酸催化二聚体是催化所必需的。Sbi00515特有的酪氨酸252被认为可以在缩合反应中定向进入的醛。瞬态动力学和中间结合的晶体结构有助于完成Sbi00515的拟议机理。
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