Three-Dimensional Structure of Kynureninase from Pseudomonas fluorescens^,

Abstract

Kynureninase [E.C. 3.7.1.3] is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the hydrolytic cleavage of l-kynurenine to anthranilic acid and l-alanine. Sequence alignment with other PLP-dependent enzymes indicated that kynureninase is in subgroup IVa of the aminotransferases, along with nifS, CsdB, and serine-pyruvate aminotransferase, which suggests that kynureninase has an aminotransferase fold. Crystals of Pseudomonas fluorescens kynureninase were obtained, and the structure was solved by molecular replacement using the CsdB coordinates combined with multiple isomorphous heavy atom replacement. The coordinates were deposited in the PDB (ID code 1QZ9). The structure, refined to an R factor of 15.5% to 1.85 A resolution, is dimeric and has the aminotransferase fold. The structure also confirms the prediction from sequence alignment that Lys-227 is the PLP-binding residue in P. fluorescens kynureninase. The conserved Asp-201, expected for an aminotransferase fold, is located near the PLP nitrogen, but Asp-132 is also strictly conserved and at a similar distance from the pyridinium nitrogen. Mutagenesis of both conserved aspartic acids shows that both contribute equally to PLP binding, but Asp-201 has a greater role in catalysis. The structure shows that Tyr-226 donates a hydrogen bond to the phosphate of PLP. Unusual among PLP-dependent enzymes, Trp-256, which is also strictly conserved in kynureninases from bacteria to humans, donates a hydrogen bond to the phosphate through the indole N1-hydrogen.

Reaction Mechanism and Molecular Basis for Selenium/Sulfur Discrimination of Selenocysteine Lyase

“…The highest Z scores were calculated to be 62.4 for human SCL (an as yet unpublished structure, PDB 2HDY), 49 (42). This result shows that the structure of SCL is quite similar to that of human SCL, and more similar to those of group I cysteine desulfurases (IscS and NifS-like protein) than those of group II cysteine desulfurases (SufS and CsdB) (35,43) (Fig.…”

Hydrolytic Polyketide Shortening by Ayg1p, a Novel Enzyme Involved in Fungal Melanin Biosynthesis

“…3.7.1. X), which includes fumarylacetoacetate hydrolase (18), kynureninase (19), and ␤-diketone hydrolase (20). Fumarylacetoacetate hydrolase is a typical ␤-keto acid hydrolase that converts fumarylacetoacetate to fumarate and acetoacetate.…”

Initial characterization of a recombinant kynureninase from Trypanosoma cruzi identified from an EST database

“…Kynureninase activity was measured spectrophotometrically at 37°C by following the decrease in absorbance at 370 nm as 3-hydroxy-DL-kynurenine was hydrolyzed to 3-hydroxyanthranilate and L-alanine or at 360 nm when L-kynurenine was hydrolyzed to anthranilate and L-alanine (Momany et al, 2004;Lima et al, 2007). Temperature, pH and substrate concentration were optimized.…”

“…1); T. cruzi kynureninase shared the highest identity (50-66%) with sequences assigned as kynureninases in the genomes of other Trypanosomatids. T. cruzi kynureninase contains the conserved Asp-241, expected for an aminotransferase fold, and most of the active site residues conserved among kynureninases (Momany et al, 2004): and Arg-441. The residue in position 267, which binds to PLP and is a Lys in all kynureninases known to date, is replaced by an Arg in T. cruzi kynureninase.…”

“…Two functional orthologs of kynureninase are known. One of them, described as an inducible enzyme, preferentially hydrolyzes Lkynurenine and is mainly found in prokaryotic organisms (Momany et al, 2004). The other one, primarily eukaryotic, is described as a constitutive kynureninase and preferentially uses 3-hydroxy-Lkynurenine as substrate (Lima et al, 2007).…”