The roles of Ser-36, Asp-132 and Asp-201 in the reaction of Pseudomonas fluorescens Kynureninase

Publication date: Available online 14 May 2019Source: Biochimica et Biophysica Acta (BBA) - Proteins and ProteomicsAuthor(s): Robert S. Phillips, Mori Crocker, Richard Lin, O. Elijah Idowu, David K. McCannon, Santiago LimaAbstractKynureninase from Pseudomonas fluorescens (Pfkynase) catalyzes the pyridoxal-5′-phosphate (PLP) dependent hydrolytic cleavage of L-kynurenine to give anthranilate and L-alanine. Asp-132 and Asp-201 are located in the structure near the pyridine NH of the PLP, with Asp-201 forming a hydrogen bond. Mutation of Asp-132 to alanine and glutamate and Asp-201 to glutamate results in reduced catalytic activity with L-kynurenine and β-benzoyl-L-alanine, but not O-benzoyl-l-serine. D132A, D132E D201E and S36A mutant Pfkynases all can form quinonoid and vinylogous amide intermediates with β-benzoyl-L-alanine, similar to wild-type enzyme. D132A, D132E, and D201E Pfkynase react more slowly with β-benzoyl-L-alanine and benzaldehyde to form an aldol product absorbing at 490 nm than wild-type, with D132E reacting the slowest. The 1H NMR spectra of wild-type and D201E Pfkynase are very similar in the low field region from 10 to 18 ppm, but that of D132A Pfkynase is missing a resonance at 13.1 ppm. These results show that these residues modulate the reactivity of the PLP at different stages during the reaction cycle. Ser-36 is located near the expected location of the carbonyl oxygen of the substrate. Mutation of Ser-36 to alanine results in a 230-fold redu...
Source: Biochimica et Biophysica Acta (BBA) Proteins and Proteomics - Category: Biochemistry Source Type: research