Download A Novel Activity for Fungal Nitronate Monooxygenase: Detoxification

A Novel Activity for Fungal Nitronate Monooxygenase: Detoxification of the Metabolic
Inhibitor Propionate-3-Nitronate
Kevin Francis‡, Shirley Nishino#, Jim Spain# and Giovanni Gadda‡§
Departments of ‡Chemistry and §Biology, and The Center for Biotechnology and Drug Design, Georgia State
University, Atlanta, GA 30302-4098; #Department of Civil and Environmental Engineering, Georgia Institute of
Technology, Atlanta, GA 30302
Nitronate monooxygenase (NMO; E.C. 1.13.12.16) is an FMN dependent enzyme that
oxidizes alkyl nitronates to their corresponding carbonyl compounds and nitrite. While the
kinetic and mechanistic properties of NMO from Neurospora crassa and Williopsis saturnus var.
Mrakii have been extensively characterized, the physiological role of the enzyme is still
unknown. The current study demonstrates that the enzyme oxidizes propionate-3-nitronate
(P3N), the highly toxic conjugate base form of the plant metabolite 3-nitropropionate (3NPA)
and provides compelling evidence that the physiological role of NMO is detoxification. Thus, the
enzyme appears to play a similar role as propionate-3-nitronate oxidase from Penicillium
atrovenetum (1) and may suggest a general defensive strategy among fungal species The
Michaelis constants of both NMOs were significantly lower with P3N as substrate than with
primary alkyl nitronates. The ability of NMO to detoxify P3N in vivo was tested by measuring
growth of recombinant Escherichia coli cells containing the gene encoding for the enzyme in
either the absence or presence of the nitronate. P3N was toxic to E. coli cells lacking NMO, but
the toxicity was overcome through either expression of the recombinant gene or through addition
of exogenous enzyme to the cultures. The growth of the fungi with 3NPA was also tested to
establish the physiological role of NMO. Both W. saturnus and N. crassa were able to grow in
the presence of up to 20 mM 3NPA, which is within the concentration range naturally produced
in the environment (i.e. ~17 mM in Penicillium (1). A knockout mutant of N. crassa lacking the
gene encoding for NMO could not grow at concentrations above 600 µM, which clearly
establishes a role of the enzyme in detoxification of the nitronate. The results indicate that NMO
functions to protect the fungi against the toxicity of 3NPA and P3N.
1.
Porter, D. J., and Bright, H. J. (1987) Propionate-3-nitronate oxidase from Penicillium
atrovenetum is a flavoprotein which initiates the autoxidation of its substrate by O2, J.
Biol. Chem. 262, 14428-14434.
8th Southeast Enzyme Conference, 04/8/2017