4FP1

P. putida mandelate racemase co-crystallized with 3,3,3-trifluoro-2-hydroxy-2-(trifluoromethyl) propionic acid

Summary for 4FP1

• Entry DOI: 10.2210/pdb4fp1/pdb
• Related: 3UXK 3UXL 4FP0
• Descriptor: Mandelate racemase, MAGNESIUM ION, 3,3,3-trifluoro-2-hydroxy-2-(trifluoromethyl)propanoic acid, ... (4 entities in total)
• Functional Keywords: enolase superfamily enzyme, isomerase-isomerase inhibitor complex, isomerase/isomerase inhibitor
• Biological source: Pseudomonas putida
• Total number of polymer chains: 2
• Total formula weight: 83081.95

Authors

Lietzan, A.D.,St.Maurice, M. (deposition date: 2012-06-21, release date: 2013-06-26, Last modification date: 2023-09-13)

Primary citation

Nagar, M.,Lietzan, A.D.,St Maurice, M.,Bearne, S.L.
Potent inhibition of mandelate racemase by a fluorinated substrate-product analogue with a novel binding mode.
Biochemistry, 53:1169-1178, 2014

PubMed Abstract: Mandelate racemase (MR) from Pseudomonas putida catalyzes the Mg(2+)-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Because trifluorolactate is also a substrate of MR, we anticipated that replacing the phenyl rings of the competitive, substrate-product analogue inhibitor benzilate (Ki = 0.7 mM) with trifluoromethyl groups might furnish an inhibitor. Surprisingly, the substrate-product analogue 3,3,3-trifluoro-2-hydroxy-2-(trifluoromethyl)propanoate (TFHTP) was a potent competitive inhibitor [Ki = 27 ± 4 μM; cf. Km = 1.2 mM for both (R)-mandelate and (R)-trifluorolactate]. To understand the origins of this high binding affinity, we determined the X-ray crystal structure of the MR-TFHTP complex to 1.68 Å resolution. Rather than chelating the active site Mg(2+) with its glycolate moiety, like other ground state analogues, TFHTP exhibited a novel binding mode with the two trifluoromethyl groups closely packed against the 20s loop and the carboxylate bridging the two active site Brønsted acid-base catalysts Lys 166 and His 297. Recognizing that positioning a carboxylate between the Brønsted acid-base catalysts could yield an inhibitor, we showed that tartronate was a competitive inhibitor of MR (Ki = 1.8 ± 0.1 mM). The X-ray crystal structure of the MR-tartronate complex (1.80 Å resolution) revealed that the glycolate moiety of tartronate chelated the Mg(2+) and that the carboxylate bridged Lys 166 and His 297. Models of tartronate in monomers A and B of the crystal structure mimicked the binding orientations of (S)-mandelate and that anticipated for (R)-mandelate, respectively. For the latter monomer, the 20s loop appeared to be disordered, as it also did in the X-ray structure of the MR triple mutant (C92S/C264S/K166C) complexed with benzilate, which was determined to 1.89 Å resolution. These observations indicate that the 20s loop likely undergoes a significant conformational change upon binding (R)-mandelate. In general, our observations suggest that inhibitors of other enolase superfamily enzymes may be designed to capitalize on the recognition of the active site Brønsted acid-base catalysts as binding determinants.

PubMed: 24472022
DOI: 10.1021/bi401703h

Experimental method

X-RAY DIFFRACTION (1.68 Å)