Crystal structure of the Homo sapiens kynureninase-3-hydroxyhippuric acid inhibitor complex: insights into the molecular basis of kynureninase substrate specificity.
Homo sapiens kynureninase is a pyridoxal-5'-phosphate dependent enzyme that catalyzes the hydrolytic cleavage of 3-hydroxykynurenine to yield 3-hydroxyanthranilate and L-alanine as part of the tryptophan catabolic pathway leading to the de novo biosynthesis of NAD(+). This pathway results in quinolinate, an excitotoxin that is an NMDA receptor agonist. High levels of quinolinate have been correlated with the etiology of neurodegenerative disorders such as AIDS-related dementia and Alzheimer's disease. We have synthesized a novel kynureninase inhibitor, 3-hydroxyhippurate, cocrystallized it with human kynureninase, and solved the atomic structure. On the basis of an analysis of the complex, we designed a series of His-102, Ser-332, and Asn-333 mutants. The H102W/N333T and H102W/S332G/N333T mutants showed complete reversal of substrate specificity between 3-hydroxykynurenine and L-kynurenine, thus defining the primary residues contributing to substrate specificity in kynureninases.
Lima
Santiago
S
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
Kumar
Sunil
S
Gawandi
Vijay
V
Momany
Cory
C
Phillips
Robert S
RS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018360:Crystallography, X-Ray; D017931:DNA Primers; D006626:Hippurates / Q000037:antagonists & inhibitors* / Q000737:chemistry; D006801:Humans; D006860:Hydrogen Bonding; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D006626:Hippurates; C535238:3-hydroxyhippuric acid; D006867:Hydrolases; C021480:kynureninase