Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 May;290(9):2449-2462.
doi: 10.1111/febs.16640. Epub 2022 Oct 17.

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

Tanakan Paladkong  1 , Panu Pimviriyakul  2 , Jittima Phonbuppha  3 , Somchart Maenpuen  4 , Pimchai Chaiyen  1   3 , Ruchanok Tinikul  1
Affiliations
Free article

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

Tanakan Paladkong et al. FEBS J. 2023 May.
Free article

Abstract

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, ~ 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 (kcat ) decreased ~ 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 pKa 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 pKa 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.

Keywords: aldehyde dehydrogenase superfamily; nucleophilicity; reactive cysteine; succinic semialdehyde dehydrogenase.

PubMed Disclaimer

Similar articles

See all similar articles

Cited by

References

    1. Swenby NP, Picklo MJ. The conserved R166 residue of ALDH5A (succinic semialdehyde dehydrogenase) has multiple functional roles. Chem Biol Interact. 2009;178:70-4.
    1. Muñoz-Clares RA, González-Segura L, Riveros-Rosas H, Julián-Sánchez A. Amino acid residues that affect the basicity of the catalytic glutamate of the hydrolytic aldehyde dehydrogenases. Chem Biol Interact. 2015;234:45-58.
    1. de Carvalho LPS, Ling Y, Shen C, Warren JD, Rhee KY. On the chemical mechanism of succinic semialdehyde dehydrogenase (GabD1) from Mycobacterium tuberculosis. Arch Biochem Biophys. 2011;509:90-9.
    1. Cao J, Singh NK, Locy RD. Characterization of the recombinant succinic semi-aldehyde dehydrogenase from Saccharomyces cerevisiae. Yeast. 2014;31:411-20.
    1. Rothacker B, Ilg T. Functional characterization of a Drosophila melanogaster succinic semialdehyde dehydrogenase and a non-specific aldehyde dehydrogenase. Insect Biochem Mol Biol. 2008;38:354-66.

Publication types

LinkOut - more resources