6VIM

P. putida mandelate racemase co-crystallized with phenylboronic acid

Summary for 6VIM

• Entry DOI: 10.2210/pdb6vim/pdb
• Descriptor: Mandelate racemase, MAGNESIUM ION, PHENYL BORONIC ACID, ... (5 entities in total)
• Functional Keywords: inhibitor, boronate, isomerase, isomerase-inhibitor complex, isomerase/inhibitor
• Biological source: Pseudomonas putida
• Total number of polymer chains: 8
• Total formula weight: 332537.87

Authors

Grandinetti, L.,Sharma, A.N.,Bearne, S.L.,St Maurice, M. (deposition date: 2020-01-13, release date: 2020-08-05, Last modification date: 2024-10-23)

Primary citation

Sharma, A.N.,Grandinetti, L.,Johnson, E.R.,St Maurice, M.,Bearne, S.L.
Potent Inhibition of Mandelate Racemase by Boronic Acids: Boron as a Mimic of a Carbon Acid Center.
Biochemistry, 59:3026-3037, 2020

PubMed Abstract: Boronic acids have been successfully employed as inhibitors of hydrolytic enzymes. Typically, an enzymatic nucleophile catalyzing hydrolysis adds to the electrophilic boron atom forming a tetrahedral species that mimics the intermediate(s)/transition state(s) for the hydrolysis reaction. We show that -substituted phenylboronic acids (PBAs) are potent competitive inhibitors of mandelate racemase (MR), an enzyme that catalyzes a 1,1-proton transfer rather than a hydrolysis reaction. The value for PBA was 1.8 ± 0.1 μM, and -Cl-PBA exhibited the most potent inhibition ( = 81 ± 4 nM), exceeding the binding affinity of the substrate by ∼4 orders of magnitude. Isothermal titration calorimetric studies with the wild-type, K166M, and H297N MR variants indicated that, of the two Brønsted acid-base catalysts Lys 166 and His 297, the former made the greater contribution to inhibitor binding. The X-ray crystal structure of the MR·PBA complex revealed the presence of multiple H-bonds between the boronic acid hydroxyl groups and the side chains of active site residues, as well as formation of a His 297 N-B dative bond. The dramatic upfield change in chemical shift of 27.2 ppm in the solution-phase B nuclear magnetic resonance spectrum accompanying binding of PBA by MR was consistent with an sp-hybridized boron, which was also supported by density-functional theory calculations. These unprecedented findings suggest that, beyond substituting boron at carbon centers participating in hydrolysis reactions, substitution of boron at the acidic carbon center of a substrate furnishes a new approach for generating inhibitors of enzymes catalyzing the deprotonation of carbon acid substrates.

PubMed: 32786399
DOI: 10.1021/acs.biochem.0c00478

Experimental method

X-RAY DIFFRACTION (2 Å)