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首页> 外文期刊>The FEBS journal. >Mechanistic roles of the neighbouring cysteine in enhancing nucleophilicity of catalytic residue in a two-cysteine succinic semialdehyde dehydrogenase
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Mechanistic roles of the neighbouring cysteine in enhancing nucleophilicity of catalytic residue in a two-cysteine succinic semialdehyde dehydrogenase

机译：Mechanistic roles of the neighbouring cysteine in enhancing nucleophilicity of catalytic residue in a two-cysteine succinic semialdehyde dehydrogenase

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摘要

Succinic semialdehyde dehydrogenase (SSADH) catalyses the conversion of succinic semialdehyde into succinic acid and two electrons are transferred to NAD(P)(+) to yield NAD(P)H. Our previous work has already reported the catalytic role of Cys289 of two-cysteine SSADH from Acinetobacter baumannii (AbSSADH). However, the mechanistic role of the neighbouring conserved Cys291 and Glu255 remains unexplored. In this study, the functional roles of Cys291 and Glu255 in AbSSADH catalysis have been characterized. Results demonstrated that the E255A activity was almost completely lost, similar to 7000-fold lower than the wild-type (WT), indicating that Glu255 is very crucial and directly involved in AbSSADH catalysis. However, the C291A and C291S variants activity and catalytic turnover (k(cat)) decreased similar to 2-fold and 9-fold respectively. To further characterize the functional roles of Cys291, we employed two pH-dependent methods; pre-steady-state burst amplitude and NADP-enzyme adduct formation. The results showed that the pK(a) values of catalytic Cys289 measured for the WT and C291A reactions were 7.8 and 8.7-8.8, respectively, suggesting that Cys291 can lower the pK(a) of Cys289 and consequently trigger the deprotonation of a Cys289 thiol. In addition, the Cys291 also plays a role in disulfide/sulfhydryl redox regulation for AbSSADH activity. Hence, we demonstrated for the first time the dual functions of Cys291 in enhancing the nucleophilicity of the catalytic Cys289 and regulating a disulfide/sulfhydryl redox switch for AbSSADH catalysis. The mechanistic insights into the nucleophilicity enhancement of the catalytic cysteine of AbSSADH might be applicable to understanding how the microenvironment increases cysteine reactivity in other enzymes in the aldehyde dehydrogenase superfamily.

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