Inflated Kinetic Isotope Effects in the Branched Mechanism of Neurospora crassa 2-Nitropropane Dioxygenase

Abstract

Catalytic turnover of Neurospora crassa 2-nitropropane dioxygenase with nitroethane as substrate occurs through both nonoxidative and oxidative pathways. The pH dependence of the kinetic isotope effects with [1,1-(2)H(2)]nitroethane as substrate was measured in the current study by monitoring the formation of the nitronate product in the nonoxidative pathway. The kinetic isotope effect on the second-order rate constant for nitronate formation, k(cat)/K(m), decreased from an upper limiting value of 23 +/- 1 at low pH to a lower limiting value of 11 +/- 1 at high pH. These kinetic isotope effects are three times larger than those determined previously through measurements of oxygen consumption that occurs in the oxidative pathway of the enzyme [(2006) Biochemistry 45, 13889]. Analytical expressions for the k(cat)/K(m) values determined in each study show that the difference in the kinetic isotope effects arises from the branching of an enzyme-ethylnitronate reaction intermediate through oxidative and nonoxidative turnover. This branching is isotope sensitive due to a kinetic isotope effect on nitronate release rather than on flavin reduction as indicated by the pH-independent (D)k(red) value of 0.99 +/- 0.06 with ethylnitronate as substrate. The kinetic isotope effect on ethylnitronate release arises from the deprotonation of histidine 196, which provides electrostatic interactions with the nitronate to keep it bound in the active site for oxidation. The isotope effect on branching results in an inflation of the kinetic isotope observed for the nonoxidative pathway to values that are larger than the intrinsic values associated with CH bond cleavage.

The biochemistry of the metabolic poison propionate 3‐nitronate and its conjugate acid, 3‐nitropropionate

“…In Arthrinium growing in sugarcane juice, the 3‐NPA concentration is 15 mM , corresponding to an effective [P3N] at equilibrium of 0.3 mM at pH 7.4. Most of the mechanistic knowledge on NMO has been acquired with ethyl nitronate as substrate for the N. crassa enzyme , and recently confirmed using the W. saturnus enzyme with the physiological substrate P3N . The transfer of a single electron from P3N to the enzyme‐bound FMN initiates catalysis, yielding an anionic flavosemiquinone and a nitronate radical (Fig.…”

“…Nitronate monooxygenase (NMO; E.C. 1.13.12.16) from the fungi Neurospora crassa and Williopsis saturnus has been studied for its biochemical and mechanistic properties . NMO was previously known as 2‐nitropropane dioxygenase, until its reclassification in 2010 by the IUBMB.…”

“…The enzyme is a homodimer containing one FMN noncovalently bound on each 39‐kDa subunit . Although NMOs oxidize alkyl nitronates, which allowed for the elucidation of the mechanism of action of these enzymes , alkyl nitronates are not commonly found in nature and are unlikely to be the physiological substrates of the enzyme. It was only in 2012 that the capability of NMO to efficiently oxidize P3N was demonstrated.…”

“…In Arthrinium growing in sugarcane juice, the 3-NPA concentration is 15 mM (34), corresponding to an effective [P3N] at equilibrium of 0.3 mM at pH 7.4. Most of the mechanistic knowledge on NMO has been acquired with ethyl nitronate as substrate for the N. crassa enzyme(45,47,48), and recently confirmed using the W. saturnus enzyme with the physiological substrate P3N…”

Screening of Metagenomic and Genomic Libraries Reveals Three Classes of Bacterial Enzymes That Overcome the Toxicity of Acrylate

“…One of these other groups is exemplified by a fungal 2-nitropropane dioxygenase, which removes the nitro group from various anionic nitroalkanes [35], [36]. Members of the other sub-group have a very different function; these correspond to FabK, first found in Streptococcus and the closely related Enterococcus [37].…”