Second-Shell Amino Acid R266 Helps Determine N‑Succinylamino Acid Racemase Reaction Specificity in Promiscuous N‑Succinylamino Acid Racemase/o‑Succinylbenzoate Synthase Enzymes
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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.
author list (cited authors)
Truong, D. P., Rousseau, S., Machala, B. W., Huddleston, J. P., Zhu, M., Hull, K. G., ... Glasner, M. E.
complete list of authors
Truong, Dat P||Rousseau, Simon||Machala, Benjamin W||Huddleston, Jamison P||Zhu, Mingzhao||Hull, Kenneth G||Romo, Daniel||Raushel, Frank M||Sacchettini, James C||Glasner, Margaret E