The phosphate of pyridoxal‐5′‐phosphate is an acid/base catalyst in the mechanism of Pseudomonas fluorescens kynureninase

Abstract

Kynureninase (l‐kynurenine hydrolase, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/7/1/3.html) 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 kcat/Km 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 31P‐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.

General base-general acid catalysis by terpenoid cyclases

“…45,46 The residual activity was hypothesized to be due to a water-mediated hydride shift in the absence of Y226. 44 These data support the proposed role of the PLP phosphate group as the general base that deprotonates Y226 in catalysis.…”

“…44 The mutation resulted in a reduction of the k cat by about 2800-fold for the substrate L-kynurenine, from 16 s −1 for the wild-type enzyme to 5.8 × 10 −3 s −1 for Y226F kynureninase. The k cat / K m saw a 375-fold reduction from 2.0×10 5 M −1 s −1 for the wild-type enzyme to 530 M −1 s −1 for Y226F kynureninase.…”

Chemical Inhibition of Kynureninase Reduces Pseudomonas aeruginosa Quorum Sensing and Virulence Factor Expression

“…The KynU enzyme is a dimer of identical subunits whose chemistry has been of great interest for nearly a century. ,, It is also highly conserved across multiple kingdoms and has been implicated in a number of diseases of the central nervous system . We showed that compound 7 has a K i value of 140.4 μM against P .…”

“…The KynU enzyme is a dimer of identical subunits whose chemistry has been of great interest for nearly a century. 22,58,59 It is also highly conserved across multiple kingdoms and has been implicated in a number of diseases of the central nervous system. 60 We showed that compound 7 has a K i value of 140.4 μM against P. aeruginosa rKynU, which is of the same order of magnitude as was reported for S-phenyl-L-cysteine (700 μM) and an order of magnitude higher than S-phenyl-L-cysteine S,Sdioxide (3.9 μM), both reported by Dua et al 23 These compounds are structurally related to compound 7, in that compound 7 is the sulfoxide form of S-phenyl-L-cysteine, and Sphenyl-L-cysteine S,S-dioxide is the sulfone form.…”

Current Advances on Structure-Function Relationships of Pyridoxal 5′-Phosphate-Dependent Enzymes

“…The phosphate group of PLP was proposed to interact with the substrate L-serine hydroxyl group and contributed to the critical intermediate formation and stereospecific orientation of formed quinonoid or carbanionic intermediates (Beattie et al, 2013). The phosphate group was also suggested functioning as an acid/base catalyst to promote proton transfer to aid in external aldimine formation and accelerating gem-diol intermediate formation in kynureninase-mediated hydrolytic cleavage reaction (Phillips et al, 2014).…”