No abstract

Due to the chemical versatility of the flavin cofactor, FMN-dependent ene-reductases and nitroreductases can catalyze or mediate a diverse spectrum of chemical reactions. Among them, two-electron transfer reactions dominate, which may proceed via sequential hydride transfer at the same or at alternate reactive sites. In addition, highly reactive intermediates are often formed, which undergo subsequent spontaneous (non-enzymatic) reactions leading to further enzymatic transformations in a cascade. Besides the well-known reductive processes involving alkenes and nitro groups at the expense of a reduced flavin cofactor, redox-neutral processes including disproportionation and CvC-bond isomerization reactions are catalyzed by OYE homologues. Unusual flavin-dependent biotransformations are reviewed with a special focus on the OYE family of flavoproteins (ene-reductases) and oxygen-insensitive FMN-dependent nitro-reductases.

No abstract

Biuret is an intermediate in the bacterial metabolism of s-triazine ring compounds and is occasionally used as a ruminant feed supplement. We used bioinformatics to identify a biuret hydrolase, an enzyme that has previously resisted efforts to stabilize, purify and characterize. This newly discovered enzyme is a member of the cysteine hydrolase superfamily, a family of enzymes previously not found to be involved in s-triazine metabolism. The gene from Rhizobium leguminosarum bv. viciae strain 3841 encoding biuret hydrolase was synthesized, transformed into Escherichia coli, and expressed. The enzyme was purified and found to be stable. Biuret hydrolase catalyzed the hydrolysis of biuret to allophanate and ammonia. The kcat/KM of 1.7 × 105 M−1s−1 and the relatively low KM of 23 ± 4 μM together suggested that this enzyme acts uniquely on biuret physiologically. This is supported by the fact that of the 34 substrate analogs of biuret tested, only two demonstrated reactivity, both at less than 5% of the rate determined for biuret. Biuret hydrolase does not react with carboxybiuret, the product of the enzyme immediately preceding biuret hydrolase in the metabolic pathway for cyanuric acid. This suggests an unusual metabolic strategy of an enzymatically-produced intermediate undergoing non-enzymatic decarboxylation to produce the substrate for the next enzyme in the pathway.

No abstract

Four NAD(P)H‐dependent non‐flavin ene reductases have been investigated for their ability to reduce activated C=C bonds in an asymmetric fashion by using 20 structurally diverse substrates. In comparison with flavin‐dependent Old Yellow Enzyme homologues, a higher degree of electronic activation was required, because the best activities were obtained with enals and nitroalkenes rather than enones and carboxylic esters. Although FaEO from Fragaria x ananassa (strawberry) and its homologue SlEO from Solanum lycopersicum (tomato) exhibited a narrow substrate spectrum, progesterone 5β‐reductase (At5β‐StR) from Arabidopsis thaliana (thale cress) and leukotriene B4 12‐hydroxydehydrogenase (LTB4DH/PGR) from Rattus norvegicus (rat) appear to be promising candidates, in particular for the asymmetric bioreduction of open‐chain enals, nitroalkenes and α,β‐unsaturated γ‐butyrolactones. Competing nitro reduction and non‐enzymatic Weitz–Scheffer epoxidation were largely suppressed.

An unexpected, redox-neutral C=C bond isomerization of a γ-butyrolactone bearing an exo-methylene unit to the thermodynamically more favoured endo isomer (kcat = 0.076 s−1) catalysed by flavoproteins from the Old Yellow Enzyme family was discovered. Theoretical calculations and kinetic data support a mechanism through which the isomerization proceeds through FMN-mediated hydride addition onto exo-Cβ, followed by hydride abstraction from endo-Cβ′, which is in line with the well-established C=C bond bioreduction of OYEs. This new isomerase activity enriches the catalytic versatility of ene-reductases.

A novel reductive biotransformation pathway for β,β-disubstituted nitroalkenes catalyzed by flavoproteins from the Old Yellow Enzyme (OYE) family was elucidated. It was shown to proceed via enzymatic reduction of the nitro-moiety to furnish the corresponding nitroso-alkene, which underwent spontaneous (non-enzymatic) electrocyclization to form highly strained 1,2-oxazete derivatives. At elevated temperatures the latter lost HCN via a retro-[2 + 2]-cycloaddition to form the corresponding ketones. This pathway was particularly dominant using xenobiotic reductase A, while pentaerythritol tetranitrate-reductase predominantly catalyzed the biodegradation via the Nef-pathway.