Second-Shell Amino Acid R266 Helps Determine N-Succinylamino Acid Racemase Reaction Specificity in Promiscuous N-Succinylamino Acid Racemase/o-Succinylbenzoate Synthase Enzymes
- Dat P. TruongDat P. TruongDepartment of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, Texas 77843-2128, United StatesMore by Dat P. Truong
- Simon RousseauSimon RousseauDepartment of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, Texas 77843-2128, United StatesMore by Simon Rousseau
- Benjamin W. MachalaBenjamin W. MachalaDepartment of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, Texas 77843-2128, United StatesMore by Benjamin W. Machala
- Jamison P. HuddlestonJamison P. HuddlestonDepartment of Chemistry, Texas A&M University, 3255 TAMU, College Station, Texas 77843-3255, United StatesMore by Jamison P. Huddleston
- Mingzhao ZhuMingzhao ZhuBaylor Synthesis and Drug-Lead Discovery Laboratory, Department of Chemistry and Biochemistry, Baylor University, One Bear Place, Waco, Texas 76798-7348, United StatesMore by Mingzhao Zhu
- Kenneth G. Hull
- Daniel Romo
- Frank M. RaushelFrank M. RaushelDepartment of Chemistry, Texas A&M University, 3255 TAMU, College Station, Texas 77843-3255, United StatesMore by Frank M. Raushel
- James C. Sacchettini
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- Margaret E. Glasner*
Catalytic promiscuity is the coincidental ability to catalyze nonbiological reactions in the same active site as the native biological reaction. Several lines of evidence show that catalytic promiscuity plays a role in the evolution of new enzyme functions. Thus, studying catalytic promiscuity can help identify structural features that predispose an enzyme to evolve new functions. This study identifies a potentially preadaptive residue in a promiscuous N-succinylamino acid racemase/o-succinylbenzoate synthase (NSAR/OSBS) enzyme from Amycolatopsis sp. T-1-60. This enzyme belongs to a branch of the OSBS family which includes many catalytically promiscuous NSAR/OSBS enzymes. R266 is conserved in all members of the NSAR/OSBS subfamily. However, the homologous position is usually hydrophobic in other OSBS subfamilies, whose enzymes lack NSAR activity. The second-shell amino acid R266 is close to the catalytic acid/base K263, but it does not contact the substrate, suggesting that R266 could affect the catalytic mechanism. Mutating R266 to glutamine in Amycolatopsis NSAR/OSBS profoundly reduces NSAR activity but moderately reduces OSBS activity. This is due to a 1000-fold decrease in the rate of proton exchange between the substrate and the general acid/base catalyst K263. This mutation is less deleterious for the OSBS reaction because K263 forms a cation−π interaction with the OSBS substrate and/or the intermediate, rather than acting as a general acid/base catalyst. Together, the data explain how R266 contributes to NSAR reaction specificity and was likely an essential preadaptation for the evolution of NSAR activity.
This article is cited by 1 publications.
- Jie Gu, Yan Xu, Yao Nie. Role of distal sites in enzyme engineering. Biotechnology Advances 2023, 63 , 108094. https://doi.org/10.1016/j.biotechadv.2023.108094