7MQX

P. putida mandelate racemase forms an oxobenzoxaborole adduct with 2-formylphenylboronic acid

Summary for 7MQX

• Entry DOI: 10.2210/pdb7mqx/pdb
• Descriptor: Mandelate racemase, MAGNESIUM ION, (3S)-2,1-benzoxaborole-1,3(3H)-diol, ... (5 entities in total)
• Functional Keywords: boronate, isomerase, isomerase-inhibitor complex, isomerase-isomerase inhibitor complex, isomerase/isomerase inhibitor
• Biological source: Pseudomonas putida
• Total number of polymer chains: 8
• Total formula weight: 333320.56

Authors

Grandinetti, L.,Bearne, S.L.,St.Maurice, M. (deposition date: 2021-05-06, release date: 2021-08-11, Last modification date: 2024-10-23)

Primary citation

Douglas, C.D.,Grandinetti, L.,Easton, N.M.,Kuehm, O.P.,Hayden, J.A.,Hamilton, M.C.,St Maurice, M.,Bearne, S.L.
Slow-Onset, Potent Inhibition of Mandelate Racemase by 2-Formylphenylboronic Acid. An Unexpected Adduct Clasps the Catalytic Machinery.
Biochemistry, 2021

PubMed Abstract: -Carbonyl arylboronic acids such as 2-formylphenylboronic acid (2-FPBA) are employed in biocompatible conjugation reactions with the resulting iminoboronate adduct stabilized by an intramolecular N-B interaction. However, few studies have utilized these reagents as active site-directed enzyme inhibitors. We show that 2-FPBA is a potent reversible, slow-onset inhibitor of mandelate racemase (MR), an enzyme that has served as a valuable paradigm for understanding enzyme-catalyzed abstraction of an α-proton from a carbon acid substrate with a high p. Kinetic analysis of the progress curves for the slow onset of inhibition of wild-type MR using a two-step kinetic mechanism gave and * values of 5.1 ± 1.8 and 0.26 ± 0.08 μM, respectively. Hence, wild-type MR binds 2-FPBA with an affinity that exceeds that for the substrate by ∼3000-fold. K164R MR was inhibited by 2-FPBA, while K166R MR was not inhibited, indicating that Lys 166 was essential for inhibition. Unexpectedly, mass spectrometric analysis of the NaCNBH-treated enzyme-inhibitor complex did not yield evidence of an iminoboronate adduct. B nuclear magnetic resonance spectroscopy of the MR·2-FPBA complex indicated that the boron atom was sp-hybridized (δ 6.0), consistent with dative bond formation. Surprisingly, X-ray crystallography revealed the formation of an N-B dative bond between Lys 166 and 2-FPBA with intramolecular cyclization to form a benzoxaborole, rather than the expected iminoboronate. Thus, when -carbonyl arylboronic acid reagents are employed to modify proteins, the structure of the resulting product depends on the protein architecture at the site of modification.

PubMed: 34339165
DOI: 10.1021/acs.biochem.1c00374

Experimental method

X-RAY DIFFRACTION (1.914 Å)