A Pitcher-and-Catcher Mechanism Drives Endogenous Substrate Isomerization by a Dehydrogenase in Kynurenine Metabolism.
Aldehyde dehydrogenase typically performs oxidation of aldehydes to their corresponding carboxylic acid while reducing NAD(P)+to NAD(P)H via covalent catalysis mediated by an active-site cysteine residue. One member of this superfamily, the enzyme 2-aminomuconate-6-semialdehyde dehydrogenase (AMSDH), is a component of the kynurenine pathway, which catabolizes tryptophan in mammals and certain bacteria. AMSDH catalyzes the NAD+-dependent oxidation of 2-aminomuconate semialdehyde to 2-aminomuconate. We recently determined the first crystal structure of AMSDH and several catalytic cycle intermediates. A conserved asparagine in the oxyanion hole, Asn-169, is found to be H-bonded to substrate-derived intermediates in the active site of AMSDH during catalysis, including both the covalently bound thiohemiacetal and thioacyl intermediates. To better interrogate the significance of the hydrogen bond provided by Asn-169 to the reaction mechanism of AMSDH, we created Ala, Ser, Asp, and Gln mutants and studied them using biochemical, kinetic, crystallographic, and computational studies. The in crystallo chemical reaction of the primary substrate with the co-crystalized complex of the N169D mutant and NAD+led to the successful trapping of a new catalytic intermediate that was not previously seen. The structural and computational data are consistent with a substrate imine/enol tautomer intermediate being formed prior to the formation of the covalent bond between the substrate and the active-site cysteine. Thus, AMSDH surprisingly includes an isomerization process within its known catalytic mechanism. These data establish a hidden intrinsic isomerization activity of the dehydrogenase and allow us to propose a pitcher-catcher type of catalytic mechanism for the isomerization.
From the Department of Chemistry, University of Texas, San Antonio, Texas 78249 and.
Yu
Y
Yang
From the Department of Chemistry, University of Texas, San Antonio, Texas 78249 and.|the Department of Chemistry, Georgia State University, Atlanta, Georgia 30303.
Ian
I
Davis
the Department of Chemistry, Georgia State University, Atlanta, Georgia 30303.
Uyen
U
Ha
From the Department of Chemistry, University of Texas, San Antonio, Texas 78249 and.
Yifan
Y
Wang
From the Department of Chemistry, University of Texas, San Antonio, Texas 78249 and.
Inchul
I
Shin
From the Department of Chemistry, University of Texas, San Antonio, Texas 78249 and Feradical@utsa.edu.|the Department of Chemistry, Georgia State University, Atlanta, Georgia 30303.
Aimin
http://orcid.org/0000-0002-4182-8176
A
Liu
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007737:Kynurenine / Q000737:chemistry / Q000378:metabolism*; D020125:Mutation, Missense; D009243:NAD / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011551:Pseudomonas fluorescens / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D009243:NAD; D007737:Kynurenine; D010088:Oxidoreductases
bifunctional enzyme; biodegradation; crystal structure; decarboxylase; dehydrogenase; enzyme mechanism; enzyme structure; isomerization; kynurenine; metabolic pathway; tryptophan