7S8W

Amycolatopsis sp. T-1-60 N-succinylamino acid racemase/o-succinylbenzoate synthase R266Q mutant in complex with N-succinylphenylglycine

Summary for 7S8W

• Entry DOI: 10.2210/pdb7s8w/pdb
• Descriptor: N-succinylamino acid racemase/O-succinylbenzoate synthase, N-succinyl-L-phenylglycine, MAGNESIUM ION, ... (5 entities in total)
• Functional Keywords: complex, racemase, dehydratase, mutant, isomerase
• Biological source: Amycolatopsis sp.
• Total number of polymer chains: 4
• Total formula weight: 159017.25

Authors

Truong, D.P.,Rousseau, S.,Sacchettini, J.C.,Glasner, M.E. (deposition date: 2021-09-20, release date: 2021-10-06, Last modification date: 2023-10-18)

Primary citation

Truong, D.P.,Rousseau, S.,Machala, B.W.,Huddleston, J.P.,Zhu, M.,Hull, K.G.,Romo, D.,Raushel, F.M.,Sacchettini, J.C.,Glasner, M.E.
Second-Shell Amino Acid R266 Helps Determine N -Succinylamino Acid Racemase Reaction Specificity in Promiscuous N -Succinylamino Acid Racemase/ o -Succinylbenzoate Synthase Enzymes.
Biochemistry, 60:3829-3840, 2021

PubMed 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 -succinylamino acid racemase/-succinylbenzoate synthase (NSAR/OSBS) enzyme from 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 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.

PubMed: 34845903
DOI: 10.1021/acs.biochem.1c00627

Experimental method

X-RAY DIFFRACTION (2.9 Å)