Old yellow enzyme: Reduction of nitrate esters, glycerin trinitrate, and propylene 1,2-dinitrate

Abstract

The reaction of the old yellow enzyme and reduced flavins with organic nitrate esters has been studied. Reduced flavins have been found to react readily with glycerin trinitrate (GTN ) (nitroglycerin) and propylene dinitrate, with rate constants at pH 7.0, 25°C of 145 M ؊1 s ؊1 and 5.8 M ؊1 s ؊1 , respectively. With GTN, the secondary nitrate was removed reductively 6 times faster than the primary nitrate, with liberation of nitrite. With propylene dinitrate, on the other hand, the primary nitrate residue was 3 times more reactive than the secondary residue. In the old yellow enzyme-catalyzed NADPH-dependent reduction of GTN and propylene dinitrate, ping-pong kinetics are displayed, as found for all other substrates of the enzyme. Rapid-reaction studies of mixing reduced enzyme with the nitrate esters show that a reduced enzyme-substrate complex is formed before oxidation of the reduced flavin. The rate constants for these reactions and the apparent Kd values of the enzyme-substrate complexes have been determined and reveal that the rate-limiting step in catalysis is reduction of the enzyme by NADPH. Analysis of the products reveal that with the enzymecatalyzed reactions, reduction of the primary nitrate in both GTN and propylene dinitrate is favored by comparison with the freeflavin reactions. This preferential positional reactivity can be rationalized by modeling of the substrates into the known crystal structure of the enzyme. In contrast to the facile reaction of free reduced flavins with GTN, reduced 5-deazaflavins have been found to react some 4 -5 orders of magnitude slower. This finding implies that the chemical mechanism of the reaction is one involving radical transfers.

‘New uses for an Old Enzyme’ – the Old Yellow Enzyme family of flavoenzymes

“…Similar enzymes are responsible for nitrate ester degradation by Agrobacterium radiobacter (Snape et al, 1997) -' nitrate ester reductase ' -and by strains of Pseudomonas fluorescens and Pseudomonas putida (Blehert et al, 1999) -' xenobiotic reductases'. OYE also catalyses the cleavage of nitrate esters, albeit at a slower rate than these bacterial enzymes, probably via a radical-based mechanism (Meah et al, 2001).…”

Characterization of Glycerol Trinitrate Reductase (NerA) and the Catalytic Role of Active-Site Residues

“…All four enzymes exhibit similar properties in that they are oxidoreductases that convert GTN to glycerol dinitrate and nitrite by using either NADH or NADPH as a reducing agent, and they are members of the old yellow enzyme (OYE) family of oxidoreductases (27). Crystallization of OYE in the presence of an inhibitor, p-hydroxybenzaldehyde, and subsequent mutagenesis studies have shown that are involved in OYE's activity toward its substrates, including substituted phenols, cyclohexanone, and GTN (6,8,11,15). The aim of the work presented here was to establish unequivocally the involvement of flavin mononucleotide (FMN) as a prosthetic group in the action of NerA on GTN, the kinetic form of the reaction, and the effect on the kinetics of mutations at the corresponding His, Asn, and Tyr residues.…”

Structure of anAspergillus fumigatusold yellow enzyme (EasA) involved in ergot alkaloid biosynthesis

“…The catalytic residues found in OYE are conserved in EasA, as was predicted from sequence alignments (Cheng et al, 2010a). A mechanism for the reduction of chanoclavine-I aldehyde to dihydrochanoclavine aldehyde has been proposed (Cheng et al, 2010a) based on the extensive literature for old yellow enzymes (Brown et al, 1998(Brown et al, , 2002Fox & Karplus, 1994;Karplus et al, 1995;Kohli & Massey, 1998;Meah et al, 2001;Williams & Bruce, 2002;Xu et al, 1999). In this mechanism, the reductive half reaction allows reduction of the FMN by NADPH.…”