TY - JOUR
T1 - Mechanism of the Flavoprotein l -Hydroxynicotine Oxidase
T2 - Kinetic Mechanism, Substrate Specificity, Reaction Product, and Roles of Active-Site Residues
AU - Fitzpatrick, Paul F.
AU - Chadegani, Fatemeh
AU - Zhang, Shengnan
AU - Roberts, Kenneth M.
AU - Hinck, Cynthia S.
N1 - Funding Information:
This work was supported in part by the National Institutes of Health (Grant R01 GM058698) and The Welch Foundation (AQ-1245). The NMR Spectroscopy Core at the University of Texas Health Science Center at San Antonio is supported in part by National Institutes of Health Grant P30 NCI/CA054174. Support for the Center for NMR Spectroscopy from the Office of the Vice President for Research at the University of Texas Health Science Center at San Antonio is gratefully acknowledged.
PY - 2016/2/2
Y1 - 2016/2/2
N2 - The flavoprotein l-hydroxynicotine oxidase (LHNO) catalyzes an early step in the bacterial catabolism of nicotine. Although the structure of the enzyme establishes that it is a member of the monoamine oxidase family, LHNO is generally accepted to oxidize a carbon-carbon bond in the pyrrolidine ring of the substrate and has been proposed to catalyze the subsequent tautomerization and hydrolysis of the initial oxidation product to yield 6-hydroxypseudooxynicotine [Kachalova, G., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 4800-4805]. Analysis of the product of the enzyme from Arthrobacter nicotinovorans by nuclear magnetic resonance and continuous-flow mass spectrometry establishes that the enzyme catalyzes the oxidation of the pyrrolidine carbon-nitrogen bond, the expected reaction for a monoamine oxidase, and that hydrolysis of the amine to form 6-hydroxypseudooxynicotine is nonenzymatic. On the basis of the kcat/Km and kred values for (S)-hydroxynicotine and several analogues, the methyl group contributes only marginally (∼0.5 kcal/mol) to transition-state stabilization, while the hydroxyl oxygen and pyridyl nitrogen each contribute ∼4 kcal/mol. The small effects on activity of mutagenesis of His187, Glu300, or Tyr407 rule out catalytic roles for all three of these active-site residues.
AB - The flavoprotein l-hydroxynicotine oxidase (LHNO) catalyzes an early step in the bacterial catabolism of nicotine. Although the structure of the enzyme establishes that it is a member of the monoamine oxidase family, LHNO is generally accepted to oxidize a carbon-carbon bond in the pyrrolidine ring of the substrate and has been proposed to catalyze the subsequent tautomerization and hydrolysis of the initial oxidation product to yield 6-hydroxypseudooxynicotine [Kachalova, G., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 4800-4805]. Analysis of the product of the enzyme from Arthrobacter nicotinovorans by nuclear magnetic resonance and continuous-flow mass spectrometry establishes that the enzyme catalyzes the oxidation of the pyrrolidine carbon-nitrogen bond, the expected reaction for a monoamine oxidase, and that hydrolysis of the amine to form 6-hydroxypseudooxynicotine is nonenzymatic. On the basis of the kcat/Km and kred values for (S)-hydroxynicotine and several analogues, the methyl group contributes only marginally (∼0.5 kcal/mol) to transition-state stabilization, while the hydroxyl oxygen and pyridyl nitrogen each contribute ∼4 kcal/mol. The small effects on activity of mutagenesis of His187, Glu300, or Tyr407 rule out catalytic roles for all three of these active-site residues.
UR - http://www.scopus.com/inward/record.url?scp=84957107058&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84957107058&partnerID=8YFLogxK
U2 - 10.1021/acs.biochem.5b01325
DO - 10.1021/acs.biochem.5b01325
M3 - Article
C2 - 26744768
AN - SCOPUS:84957107058
VL - 55
SP - 697
EP - 703
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 4
ER -