Versatility and specificity in flavoenzymes: Control mechanisms of flavin reactivity

Miura, Retsu

doi:10.1002/tcr.1007

Cited by 108 publications

(91 citation statements)

References 30 publications

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Section: Resultsmentioning

confidence: 96%

“…The symmetric structure of the latter suggests that it corresponds to a flavin charge-transfer (CT) band rather than of a neutral semiquinone (23). The shape and amplitude of the Q 1 SADS is generally similar to that of anionic semiquinone The latter has a narrow absorption peak with a maximum at Ϸ500 nm and a broad CT band with a maximum at Ϸ700 nm (23,24). Given that the absorption maximum of flavin CT bands may vary widely from one flavoprotein to another and depend on specific interactions between flavin and protein, we identify the intermediate species Q 1 as a CT͞anionic semiquinone FAD •Ϫ species.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Hydrogen-bond switching through a radical pair mechanism in a flavin-binding photoreceptor

Gauden

Stokkum²,

Key³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

216

435

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BLUF (blue light sensing using FAD) domains constitute a recently discovered class of photoreceptor proteins found in bacteria and eukaryotic algae, where they control a range of physiological responses including photosynthesis gene expression, photophobia, and negative phototaxis. Other than in well known photoreceptors such as the rhodopsins and phytochromes, BLUF domains are sensitive to light through an oxidized flavin rather than an isomerizable cofactor. To understand the physicochemical basis of BLUF domain photoactivation, we have applied femtosecond transient absorption spectroscopy to the Slr1694 BLUF domain of Synechocystis PCC6803. We show that photoactivation of BLUF domains proceeds by means of a radical-pair mechanism, driven by electron and proton transfer from the protein to the flavin, resulting in the transient formation of anionic and neutral flavin radical species that finally result in the long-lived signaling state on a 100-ps timescale. A pronounced deuteration effect is observed on the lifetimes of the intermediate radical species, indicating that proton movements underlie their molecular transformations. We propose a photoactivation mechanism that involves a successive rupture of hydrogen bonds between a conserved tyrosine and glutamine by light-induced electron transfer from tyrosine to flavin and between the glutamine and flavin by subsequent protonation at flavin N5. These events allow a reorientation of the conserved glutamine, resulting in a switching of the hydrogen-bond network connecting the chromophore to the protein, followed by radicalpair recombination, which locks the glutamine in place. It is suggested that the redox potential of flavin generally defines the light sensitivity of flavin-binding photoreceptors.flavoprotein ͉ light sensing ͉ photochemistry ͉ reaction mechanism ͉ spectroscopy

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Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

Hydrogen-bond switching through a radical pair mechanism in a flavin-binding photoreceptor

Gauden

Stokkum²,

Key³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

216

435

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“…Flavin cofactors undergo both one-electron and two-electron chemistry (44)(45)(46), so either reaction manifold could be involved in halogen transfer. An FAD-O-Cl intermediate could react via a one-electron mechanism to form • Cl as the chlorinating species.…”

Section: Discussionmentioning

confidence: 99%

Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis

Yeh

Garneau²,

Walsh³

2005

Proc. Natl. Acad. Sci. U.S.A.

253

278

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The indolocarbazole antitumor agent rebeccamycin is modified by chlorine atoms on each of two indole moieties of the aglycone scaffold. These halogens are incorporated during the initial step of its biosynthesis from conversion of L-Trp to 7-chlorotryptophan. Two genes in the biosynthetic cluster, rebF and rebH, are predicted to encode the flavin reductase and halogenase components of an FADH 2-dependent halogenase, a class of enzymes involved in the biosynthesis of numerous halogenated natural products. Here, we report that, in the presence of O 2, chloride ion, and L-Trp as cosubstrates, purified RebH displays robust regiospecific halogenating activity to generate 7-chlorotryptophan over at least 50 catalytic cycles. Halogenation by RebH required the addition of RebF, which catalyzes the NADH-dependent reduction of FAD to provide FADH 2 for the halogenase. Maximal rates were achieved at a RebF͞RebH ratio of 3:1. In air-saturated solutions, a k cat of 1.4 min ؊1 was observed for the RebF͞RebH system but increased at least 10-fold in low-pO 2 conditions. RebH was also able to use bromide ions to generate monobrominated Trp. The demonstration of robust chlorinating activity by RebF͞RebH sets up this system for the probing of mechanistic questions regarding this intriguing class of enzymes.enzymology ͉ natural product biosynthesis ͉ flavoenzyme

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“…Based on the activestate IPP-IDI red structure, the N-5 nitrogen of FMN, which is located 3.60 Ϯ 0.10 Å from the C4 carbon of IPP (3.65 Ϯ 0.09 Å from the E-methyl carbon of DMAPP in DMAPP-IDI red ), appeared to be the most promising candidate. The N-1 nitrogen is another likely candidate, because it is located 3.81 Ϯ 0.09 Å from C-4 of IPP in the same structure (3.73 Ϯ 0.05 Å from the E-methyl carbon of DMAPP in DMAPP-IDI red ) and because the pK a of the N-1 proton of reduced flavin was reported to be ϳ7, which is in the physiological pH range (14,15). However, if FMN N-1 is the general acid-base catalyst, the strict specificity of deprotonation from the E-methyl group of DMAPP cannot be explained because the nitrogen is located nearly equidistance from C-4 and C-5 of IPP, which correspond to E-methyl and Z-methyl of DMAPP, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…The N-5 nitrogen of FMN must be integrally involved in both mechanisms. Although the pK a values of reduced flavins are within the physiological pH range (14,15), there has been no definitive report on the flavoenzymes in which flavin only acts as a general acid-base catalyst without having any redox function. Recent studies of type 2 IDI using substrate analogues such as 3,4-epoxy-3-methylbutyl diphosphate (eIPP), which form adducts with FMN, suggest the possibility that FMN N-5 may act as a general acid or base (10,16,17).…”

mentioning

confidence: 99%

New Role of Flavin as a General Acid-Base Catalyst with No Redox Function in Type 2 Isopentenyl-diphosphate Isomerase

Unno

Yamashita

Ikeda

et al. 2009

Journal of Biological Chemistry

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Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.Flavins are generally regarded as redox coenzymes because their primary function in redox-catalyzing flavoenzymes is donation and/or acceptance of electrons (1). As summarized in a review article (2), the redox activities of flavins also take part in the flavoenzymes that catalyze reactions with no net redox change. Most of these enzymes are thought to have redoxbased mechanisms, whereas flavins have only structural or stabilizing roles in a few exceptions. Recently, however, a report on UDP-galactopyranose mutase unexpectedly showed that flavin can act as a nucleophilic catalyst (3, 4). In UDP-galactopyranose mutase, a sugar carbon undergoes nucleophilic attack by the N-5 nitrogen of reduced FMN, concomitantly with the dissociation of UDP, forming an adduct intermediate. In this reaction, the flavin cofactor has no redox function because it is continuously in the reduced state.Type 2 isopentenyl-diphosphate isomerase (IDI) 4 is the flavoenzyme that catalyzes the interconversion between isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) that occurs with no net change in redox status (5). Both compounds are fundamental units for the biosynthesis of isoprenoids, a diverse family of Ͼ50,000 metabolites (6). Type 2 IDI requires FMN, NAD(P)H, and Mg 2ϩ to be active; however, NAD(P)H is used only for the reduction of FMN and can be replaced with Na 2 S 2 O 4 (7-9). The observation that reduced FMN is required for type 2 IDI activity allowed for development of various plausible reaction mechanisms, including redoxbased mechanisms. Based on the traditional interpretation of the results from the experiments that used cofactor analogues such as 5-deaza-FMN, radical-mediated mechanisms were proposed at first, negating both the hydride transfer mechanism and the mer...

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Versatility and specificity in flavoenzymes: Control mechanisms of flavin reactivity

Cited by 108 publications

References 30 publications

Hydrogen-bond switching through a radical pair mechanism in a flavin-binding photoreceptor

Hydrogen-bond switching through a radical pair mechanism in a flavin-binding photoreceptor

Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis

New Role of Flavin as a General Acid-Base Catalyst with No Redox Function in Type 2 Isopentenyl-diphosphate Isomerase

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