NAD+-依赖(2R,来自淋病奈瑟菌(NgBDH)的3R)‑2,3‑丁二醇脱氢酶(BDH)是中链脱氢酶/还原酶(MDR)超家族的代表性成员。迄今为止,关于该超家族BDHs的底物结合位点和催化残基的信息很少。在这项工作中,根据分子对接研究,我们发现保守残基Phe120和Val161与两者形成强疏水相互作用(2R,3R)-2,3-丁二醇(RR-BD)和内消旋-2,3-丁二醇(内消旋-BD),并且这些残基向丙氨酸或苏氨酸的突变会损害底物结合。为了进一步评估这两个残基的作用,Phe120和Val161突变为丙氨酸或苏氨酸。动力学分析显示,相对于野生型,RR-BD和meso-BD的Phe120Ala突变体的表观KM值增加了36倍和369倍,该突变体与RR-BD和meso-BD的催化效率分别降低了约586和3528倍,分别;RR-BD和meso-BD的Val161Ala突变体的表观KM值增加了4倍和37倍,分别,该突变体具有RR-BD和meso-BD的催化效率降低了约3倍和28倍,分别。此外,由于KM的增加(RR-BD的六倍;meso-BD的24倍)和Kcat的轻微增加(RR-BD的2.8倍;meso-BD的3.3倍),Val161Thr突变体的催化效率略有降低(RR-BD的两倍;meso-BD的3.3倍)。这些发现验证了NgBDH的Phe120和Val161在底物结合和催化中的关键作用。总的来说,本研究为MDR超家族中BDHs的底物结合和催化提供了更好的理解。
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.