title: The phosphate of pyridoxal-5'-phosphate is an acid/base catalyst in the mechanism of Pseudomonas fluorescens kynureninase.
abstract: Kynureninase (L-kynurenine hydrolase, EC 3.7.1.3) catalyzes the hydrolytic cleavage of L-kynurenine to L-alanine and anthranilic acid. The proposed mechanism of the retro-Claisen reaction requires extensive acid/base catalysis. Previous crystal structures showed that Tyr226 in the Pseudomonas fluorescens enzyme (Tyr275 in the human enzyme) hydrogen bonds to the phosphate of the pyridoxal-5'-phosphate (PLP) cofactor. This Tyr residue is strictly conserved in all sequences of kynureninase. The human enzyme complexed with a competitive inhibitor, 3-hydroxyhippuric acid, showed that the ligand carbonyl O is located 3.7 Å from the phenol of Tyr275 (Lima, S., Kumar, S., Gawandi, V., Momany, C. & Phillips, R. S. (2009) J. Med. Chem. 52, 389-396). We prepared a Y226F mutant of P. fluorescens kynureninase to probe the role of this residue in catalysis. The Y226F mutant has approximately 3000-fold lower activity than wild-type, and does not show the pKa values of 6.8 on kcat and 6.5 and 8.8 on k(cat)/K(m) seen for the wild-type enzyme (Koushik, S. V., Moore, J. A. III, Sundararaju, B. & Phillips, R. S. (1998) Biochemistry 37, 1376-1382). Wild-type kynureninase shows a resonance at 4.5 ppm in (31)P-NMR, which is shifted to 5.0, 3.3 and 2.0 ppm when the potent inhibitor 5-bromodihydrokynurenine is added. However, Y226F kynureninase shows resonances at 3.6 and 2.5 ppm, and no change in the peak position is seen when 5-bromodihydrokynurenine is added. Taken together, these results suggest that Tyr226 mediates proton transfer between the substrate and the phosphate, which accelerates formation of external aldimine and gem-diol intermediates. Thus, the phosphate of PLP acts as an acid/base catalyst in the mechanism of kynureninase.
authors.affiliation: Department of Biochemistry and Molecular Biology, University Georgia, Athens, GA, USA; Department of Chemistry, University Georgia, Athens, GA, USA.
authors.forename: Robert S
authors.initials: RS
authors.lastname: Phillips
authors.forename: Israel
authors.initials: I
authors.lastname: Scott
authors.forename: Riya
authors.initials: R
authors.lastname: Paulose
authors.forename: Akshay
authors.initials: A
authors.lastname: Patel
authors.forename: Taylor Colt
authors.initials: TC
authors.lastname: Barron
mesh_terms: D002384:Catalysis; D006867:Hydrolases / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D010710:Phosphates / Q000737:chemistry*; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D011732:Pyridoxal Phosphate / Q000378:metabolism*
chemical_list: D010710:Phosphates; D011732:Pyridoxal Phosphate; D006867:Hydrolases; C021480:kynureninase
keywords: 31P-NMR; acid-base catalysis; enzyme mechanism; mutagenesis; pyridoxal-5′-phosphate