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. HullKenneth G. HullBaylor Synthesis and Drug-Lead Discovery Laboratory, Department of Chemistry and Biochemistry, Baylor University, One Bear Place, Waco, Texas 76798-7348, United StatesMore by Kenneth G. Hull
- ,
- Daniel RomoDaniel RomoBaylor Synthesis and Drug-Lead Discovery Laboratory, Department of Chemistry and Biochemistry, Baylor University, One Bear Place, Waco, Texas 76798-7348, United StatesMore by Daniel Romo
- ,
- Frank M. RaushelFrank M. RaushelDepartment of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, Texas 77843-2128, United StatesDepartment of Chemistry, Texas A&M University, 3255 TAMU, College Station, Texas 77843-3255, United StatesMore by Frank M. Raushel
- ,
- James C. SacchettiniJames C. SacchettiniDepartment of Chemistry, Texas A&M University, 3255 TAMU, College Station, Texas 77843-3255, United StatesMore by James C. Sacchettini
- , and
- Margaret E. Glasner*Margaret E. Glasner*Email: [email protected]. Phone: (979) 458-0123.Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, Texas 77843-2128, United StatesMore by Margaret E. Glasner
Abstract
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.
Cited By
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