Flavoenzymes: versatile catalysts in biosynthetic pathways

Abstract

Riboflavin-based coenzymes, tightly bound to enzymes catalyzing substrate oxidations and reductions, enable an enormous range of chemical transformations in biosynthetic pathways. Flavoenzymes catalyze substrate oxidations involving amine and alcohol oxidations and desaturations to olefins, the latter setting up Diels-Alder cyclizations in lovastatin and solanapyrone biosyntheses. Both C(4a) and N(5) of the flavin coenzymes are sites for covalent adduct formation. For example, the reactivity of dihydroflavins with molecular oxygen leads to flavin-4a-OOH adducts which then carry out a diverse range of oxygen transfers, including Baeyer-Villiger type ring expansions, olefin epoxidations, halogenations via transient HOCl generation, and an oxidative Favorskii rerrangement during enterocin assembly.

Fig. 1

Two redox half reactions in flavoenzyme catalysis: (a) flavin reductive half reaction; (b) dihydroflavin reoxidative half reaction.

Fig. 2

Three kinetically and thermodynamically accessible redox states: oxidized flavin (Fl_ox); the one-electron reduced semiquinone (FlH•); the two-electron reduced dihydroflavin (FlH₂).

Fig. 3

N₅ and bridgehead C_4a as sites of electron entry and exit from the tricyclic isoalloazine ring of flavin coenzymes: covalent 4a-thiol and 4a-hydroperoxide adducts.

Fig. 4

Scope of flavoenzyme redox transformations: (a) E-Fl_ox ⇔E-FlH₂; (b) reoxidative half reaction of FlH₂ with O₂ via one-electron transfer and recombination of FLH• and O ⁻₂•.

Fig. 5

Selective action of the in trans enoyl reductase LovC during elongation of the triketide to heptaketide intermediate in lovastatin biosynthesis. The Diels-Alder cyclization to the decalin scaffold occurs at the triene-containing hexaketide stage.

Fig. 6

Activation and desaturation of a tethered glutamine by the oxidase domain of the NRPS module that makes the purple pigment indigoidine.

Fig. 7

Thiazoline and oxazoline formation: (a) schematic for cyclodehydration of Cys and Ser residues in nascent proteins to thiazoline and oxazoline rings; (b) the V. cholerae siderophore vibriobactin with methyloxazoline rings; the Y. pestis siderophore yersiniabactin with thiazoline and thiazole rings. The dihydroaromatic thiazolines and methyoxazolines are part of the ferric iron chelation set.

Fig. 8

(a) Generation of methylthiazolyl-S-thiolation domain intermediate at the start of the epothilone biosynthetic assembly line from acetyl- and Cys thioesters. The FAD-dependent oxidase domain converts methylthiazoline to methylthiazole; (b) tandem conversion of adjacent Cys residues to the bithiazolyl unit in the DNA-damaging antitumor antibiotic bleomycin.

Fig. 9

Three microbial peptides that have been posttranslationally converted into heterocyclic-containing mature products: Microcin B17 targets bacterial DNA Gyrase; Goadsporin is a morphogen for Streptomyces development (“goads spore formation”); Plantazolicin A, with two sets of five tandem heterocycles, is an antibiotic active against B. anthracis.

Fig. 10

Steps in assembly of the pentacyclic fungal toxin cyclopiazonic acid A(α-CPA): (a) conversion of tryptophan to the tetramic acid cycloacetyl tryptophan (cAATrp) by action of a hybrid NRPS-PKS assembly line; (b) mechanistic proposal for the flavoenzyme CpaO-mediated-conversion of β-CPA to α-CPA with formation of the final two rings of the mature toxin.

Fig. 11

Conversion of proline units to pyrroles: (a) selected natural products where the pyrrole moieties arise from flavoenzyme desaturation of prolyl-S-thiolation domains; (b) scheme for activation and desaturation of proline monomers on NRPS modules; (c) proposal for generation of pyridine carboxylate by iterative desaturation in collismycin A biosynthesis.

Fig. 12

Oxidative dimerization of two tryptophans to indolocarbazole scaffolds: (a) action of RebO and RebD to generate chromopyrroli c acid: (b) action of RebP and RebC to make the final C-C bond in the indolocarbazole scaffold and control the redox state in the oxopyrrole ring.

Fig. 13

Biosynthetic pathway to fumiquinazoline C (FQC). The last step in the pathway involves oxidation of the secondary amine in fumiquinazoline A by the FAD-enzyme Af12070, followed by enzymatic intramolecular conversion of the nascent imine to the heptacyclic hemiaminal in FQC (path a) or nonenzymatic intramolecular conversion to the aminal FQD (path b).

Fig. 14

Flavoprotein alcohol oxidases carrying out net four electron oxidations: (a) structures of glycopeptides antibiotics vancomy cin, teicoplanin, and acetylglucosamine residue of A40926 in two discrete steps, via the aldehyde, A40926; (b) the flavoprotein oxidase Dbv29 oxidizes the C₆-OH of the N-generating the GlcNAc-6-carboxylate product; capture of the aldehyde intermediate by long chain amines and oxidation of the hemiaminal adduct produces amide variant products with two long hydrophobic substituents that confer distinct antibiotic properties; (c) Aclacinomycin oxidase acts first as an alcohol to ketone oxidase on the terminal rhodinose sugar to yield cinerulose and then carries out an α,β-desaturation to the enone functionality in the terminal sugar L-aculose.

Fig. 15

Flavoenzyme mediated conversion of uridine to uridine-5′-aldehyde, an intermediate in several peptidyl nucleoside antibiotic pathways.

Fig. 16

In solanapyrone biosynthesis the FAD-enzyme Sol5 oxidizes the alcohol group in prosolanapyrone II to the aldehyde which enables a [4 + 2] cyclization of the triene to the decalin ring in solanapyrone A.

Fig. 17

Three flavoprotein dithiol to disulfide oxidases as the last steps in the biosynthesis of holomycin, gliotoxin, and FK228.

Fig. 18

Flavoenzymes coupling thiol oxidation with decarboxylation: (a) the pantotheinyl cysteine decarboxylase in the coenzyme A biosynthetic pathway effects decarboxylation via reversible redox at the CH₂-SH side chain of substrate; (b) maturation of some lantipeptides involves decarboxylation and crosslinking to yield an aminovinyl cysteine unit; (c) four flavoenzymes proposed to catalyze net eight-electron oxidation of a Cys-Cys precursor to holomycin.

Fig. 19

Nitroalkane oxidase catalysis involves a covalent nitroalkyl-N₅ flavin adduct.

Fig. 20

Carbanion intermediate preceeds N₅ adduct formation to setup elimination of the 2-O-acyl substituent in formation of alkyl DHAP lipids.

Fig. 21

(a) The FAD-enzyme MurB uses NADPH as hydride transfer agent to generate FADH₂ in the reductive half reaction; (b) in the reoxidative half reaction a hydride is transferred from N₅ of FADH₂ to the olefinic terminus of UDP-enolpyruvyl-GlcNAc, generating the UDP-muramic acid product.

Fig. 22

Flavoprotein monooxygenases: Transfer of the distal oxygen from Fl-4a-OOH to phenol to generate catechol.

Fig. 23

Hydroxylation of C₁₂ in angucycline biosynthesis on the way to gaudimycin C.

Fig. 24

Baeyer-Villiger oxygenases act via Fl-4a-OO⁻ as nucleophile: (a) schematic for oxygen insertion into cyclohexanone with ring expansion to the 7 member lactone; (b) conversion of the tetracyclic scaffold of premithramycin B to the tricyclic scaffold of mithramycin DK starts with a Baeyer-Villiger ring expansion of ketone to lactone, followed by lactone hydrolysis and β-keto acid decarboxylation; (c) conversion of angucycline framework to twisted spiroketal in griseorhodin involves flavoprotein catalysis and can be formulated to involve Baeyer-Villiger enzymology; (d) Baeyer-Villigerase generation of neo-pentalenolactone D.

Fig. 25

Flavoenzyme epoxidases: (a) squalene 2,3-epoxidase; (b) Af12060 converts fumiquinazoline F to fumiquinazoline A by way of an epoxyindole/hydroxyiminium ion intermediate; (c) indole epoxidation en route from notoamide E to notoamides C and D; (d) regioselective epoxidation by Lsd18 and tandem cyclization by Lsd19 in the lasalocid pathway.

Fig. 26

Oxidative cleavage of uracil to Z-3-ureidoacrylate: proposed involvement of Fl-4a-peroxyanion in ring-opening step.

Fig. 27

Halogenases utilizing FADH₂ and O₂ to generate a “Cl⁺” chlorinating species for electron rich cosubstrates: (a) generation of nascent HOCl in halogenase active sites and proposed conversion to a Lys-N₆-Cl chloramine as proximal halogenating species by tryptophan 7-halogenase; (b) sequential chlorination of pyrrolyl-S-carrier protein at C_β and C_α during pyoluteorin biosynthesis; (c) tandem chlorination on C_β of acetyl CoA to generate the dichloroacetyl group during chloramphenicol assembly.

Fig. 28

The role of EncM in enterocin biosynthesis: oxidation of a polyketonic intermediate to an 11,12,13-triketo species that undergoes a Favorskii rearrangement and then two regiospecific aldol condensations.

Fig. 29

Creating the dimethylbenzimidazole ligand for B12 from FMN: (a) vitamin B12 with the dimethylbenzimidazole (DMB) as bottom axial ligand to the cobalt atom; (b) BluB acts as a flavin “destructase”, generating DMB and erythrose-4-P from FMN; (c) possible mechanism involving a Fl-4a-OOH and an internal Baeyer-Villiger reaction on the pyrimidine ring of FMN.

Fig. 30

Proposed role of FlH₂ in the type II isopentenyl diphosphate isomerase: N₅ as proton transfer catalysts for the 1,3-allylic isomerization.

Fig. 31

Proposed role of FlH₂ in the UDP-galactopyranose mutase reaction: Covalent FlH₂-N₅-galactose-C₁ adduct as reaction intermediate.