Expression, Purification, and Bioinformatic Prediction of Mycobacterium tuberculosis Rv0439c as a Potential NADP+-Retinol Dehydrogenase

Tang, Wanggang; Gui, Chuanyue; Zhang, Tingting

doi:10.1007/s12033-023-00956-z

Cited by 1 publication

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“…Therefore, the substrate binding and catalytic residues cannot be identified from the available structural information. Fortunately, computational biology approaches, such as structural modeling, molecular docking, and molecular dynamic simulation, provide powerful tools for predicting substrate or coenzyme binding sites [2,8,9,29,30]. In this work, the three-dimensional structure of NgBDH was first modeled with AlphaFold2, which has an overall structure very similar to those of other members of the MDR family.…”

Section: Discussionmentioning

confidence: 99%

The critical role of residues Phe120 and Val161 of (2 R,3 R)‑2,3‑butanediol dehydrogenase from Neisseria gonorrhoeae as probed by molecular docking and site‐directed mutagenesis

Dong,

Zhang,

Gui

et al. 2024

J Basic Microbiol

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NAD+‐dependent (2 R,3 R)‑2,3‑butanediol dehydrogenase (BDH) from Neisseria gonorrhoeae (NgBDH) is a representative member of the medium‐chain dehydrogenase/reductase (MDR) superfamily. To date, little information is available on the substrate binding sites and catalytic residues of BDHs from this superfamily. In this work, according to molecular docking studies, we found that conserved residues Phe120 and Val161 form strong hydrophobic interactions with both (2 R,3 R)‑2,3‑butanediol (RR‐BD) and meso‐2,3‑butanediol (meso‐BD) and that mutations of these residues to alanine or threonine impair substrate binding. To further evaluate the roles of these two residues, Phe120 and Val161 were mutated to alanine or threonine. Kinetic analysis revealed that, relative to those of wild type, the apparent KM values of the Phe120Ala mutant for RR‐BD and meso‐BD increased 36‐ and 369‐fold, respectively; the catalytic efficiencies of this mutant with RR‐BD and meso‐BD decreased approximately 586‐ and 3528‐fold, respectively; and the apparent KM values of the Val161Ala mutant for RR‐BD and meso‐BD increased 4‐ and 37‐fold, respectively, the catalytic efficiencies of this mutant with RR‐BD and meso‐BD decreased approximately 3‐ and 28‐fold, respectively. Additionally, the Val161Thr mutant slightly decreased catalytic efficiencies (twofold with RR‐BD; 7.3‐fold with meso‐BD) due to an increase in KM (sixfold for RR‐BD; 24‐fold for meso‐BD) and a slight increase (2.8‐fold with RR‐BD; 3.3‐fold with meso‐BD) in kcat. These findings validate the critical roles of Phe120 and Val161 of NgBDH in substrate binding and catalysis. Overall, the current study provides a better understanding of the substrate binding and catalysis of BDHs within the MDR superfamily.

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Section: Discussionmentioning

confidence: 99%