Reaction of 3O2 with dihydroflavins. 1. N3,5-Dimethyl-1,5-dihydrolumiflavin and 1,5-dihydroisoalloxazines

The reactivity of flavoenzymes with dioxygen is at the heart of a number of biochemical reactions with far reaching implications for cell physiology and pathology. Flavin-containing monooxygenases are an attractive model system to study flavin-mediated oxygenation. In these enzymes, the NADP(H) cofactor is essential for stabilizing the flavin intermediate, which activates dioxygen and makes it ready to react with the substrate undergoing oxygenation. Our studies combine site-directed mutagenesis with the usage of NADP ؉ analogues to dissect the specific roles of the cofactors and surrounding protein matrix. The highlight of this "double-engineering" approach is that subtle alterations in the hydrogen bonding and stereochemical environment can drastically alter the efficiency and outcome of the reaction with oxygen. This is illustrated by the seemingly marginal replacement of an Asn to Ser in the oxygen-reacting site, which inactivates the enzyme by effectively converting it into an oxidase. These data rationalize the effect of mutations that cause enzyme deficiency in patients affected by the fish odor syndrome. A crucial role of NADP ؉ in the oxygenation reaction is to shield the reacting flavin N5 atom by H-bond interactions. A Tyr residue functions as backdoor that stabilizes this crucial binding conformation of the nicotinamide cofactor. A general concept emerging from this analysis is that the two alternative pathways of flavoprotein-oxygen reactivity (oxidation versus monooxygenation) are predicted to have very similar activation barriers. The necessity of fine tuning the hydrogenbonding, electrostatics, and accessibility of the flavin will represent a challenge for the design and development of oxidases and monoxygenases for biotechnological applications.Flavin-containing monooxygenases (FMOs) 3 form a large family of enzymes present almost ubiquitously among living organisms (1, 2, 3). They are involved in diverse biological processes such as the biosynthesis of various natural products (4) and catabolism of xenobiotics. Humans have five different isozymes that are coded by different genes and represent a key enzymatic system in drug metabolism, whose relevance almost equals that of cytochrome P450s. Among known FMO substrates, there are metabolites directly or indirectly deriving from food digestion such as trimethylamine, drugs such as tamoxifen, and toxic molecules such as nicotine. Mutations in the gene for the isozyme 3 of human FMO (FMO3), which is most abundant in the liver, are directly responsible for trimethylaminuria, also known as fish odor syndrome (TMAU). This genetic disease causes the accumulation of trimethylamine so that the body of the patients tend to have an unpleasant smell (5, 6).Irrespectively of their biological context, FMOs always catalyze the same reaction: the oxygenation of a soft nucleophile using molecular oxygen as oxygen source and NADPH as electron donor (7) (Fig. 1A). At the heart of this very complex reaction is the stabilization of the flavin-hydroperoxide adduct res...

Section: Discussionmentioning

confidence: 99%

Joint Functions of Protein Residues and NADP(H) in Oxygen Activation by Flavin-containing Monooxygenase

Orrù

Pazmiño

Fraaije

et al. 2010

“…/HOO Ϫ . This role has been assigned to a His in glucose oxidase (12,32) and to a Lys in monomeric sarcosine oxidase (36) and in monoamine oxidase (37,38). In DAAO, this would be implemented by the proposed H ϩ relay system.…”

Section: Discussionmentioning

confidence: 99%

“…Although there is agreement on the physical mechanisms of the initial step(s) of electron transfer from the flavin to O 2 (12)(13)(14), key questions regarding the control of O 2 reactivity with the reduced flavin cofactor are still open. There are examples for channels in flavoproteins that guide O 2 to the reaction site (15)(16)(17).…”

mentioning

confidence: 99%

O2 Reactivity of Flavoproteins

Saam

Rosini

Molla

et al. 2010

Molecular dynamics simulations and implicit ligand sampling methods have identified trajectories and sites of high affinity for O 2 in the protein framework of the flavoprotein D-amino-acid oxidase (DAAO). A specific dynamic channel for the diffusion of O 2 leads from solvent to the flavin Si-side (amino acid substrate and product bind on the Re-side). Based on this, amino acids that flank the putative O 2 high affinity sites have been exchanged with bulky residues to introduce steric constraints. In G52V DAAO, the valine side chain occupies the site that in wild-type DAAO has the highest O 2 affinity. In this variant, the reactivity of the reduced enzyme with O 2 is decreased >100-fold and the turnover number ≈1000-fold thus verifying the concept. In addition, the simulations have identified a chain of three water molecules that might serve in relaying a H ؉ from the product imino acid ‫؍‬NH 2 ؉ group bound on the flavin Reside to the developing peroxide on the Si-side. This function would be comparable with that of a similarly located histidine in the flavoprotein glucose oxidase.

“…As summarized in Fig. 5, the reaction of reduced flavin with oxygen is a one-electron transfer process involving formation of a caged radical pair of neutral flavin semiquinone and superoxide anion (47). From this primary complex there are several possible pathways, the formation of flavin C4a-peroxyflavin species, dissociation of the complex into free flavin semiquinone and O 2 .…”

Section: Formation Of Superoxide Anionmentioning

confidence: 99%

The Role of Glutamine 114 in Old Yellow Enzyme

Brown¹,

Hyun²,

Duvvuri³

et al. 2002

Glutamine 114 of OYE1 is a well conserved residue in the active site of the Old Yellow Enzyme family. It forms hydrogen bonds to the O2 and N3 of the flavoprotein prosthetic group, FMN. Glutamine 114 was mutated to asparagine, introducing an R-group that is one methylene group shorter. The resultant enzyme was characterized to determine the effect of the mutation on the mechanistic behavior of the enzyme, and the crystal structure was solved to determine the effect of the mutation on the structure of the protein. The Q114N mutation results in little change in the protein structure, moving the amide group of residue 114 out of H-bonding distance, allowing repositioning of the FMN prosthetic group to form new interactions that replace the lost H-bonds. The mutation decreases the ability to bind ligands, as all dissociation constants for substituted phenols are larger than for the wild type enzyme. The rate constant for the reductive half-reaction with ␤-NADPH is slightly greater, whereas that for the oxidative half-reaction with 2-cyclohexenone is smaller than for the wild type enzyme. Oxidation with molecular oxygen is biphasic and involves formation and reaction with O 2 . , a phenomenon that is more pronounced with this mutation than with wild type enzyme. When superoxide dismutase is added to the reaction, we observe a single-phase reaction typical of the wild type enzyme. Turnover reactions using ␤-NADPH with 2-cyclohexenone and molecular oxygen were studied to further characterize the mutant enzyme.