Para-hydroxybenzoate hydroxylase (EC 1.14.13.2) is a flavoprotein involved in degradation of aromatic compounds, and it has become a model for enzymes involved in the oxygenation of a substrate. The chemical and kinetic mechanisms of this enzyme are described and integrated with an outline of the structure of the protein from crystallographic analysis. The structure is unusual because there is no recognizable domain for the binding of NADPH involved in the reaction. Recently, mechanistic studies of site-directed mutants, combined with structural analyses, have provided some exciting discoveries about protein function. The substrate during catalysis is largely isolated from solvent in the active site, a necessary condition for successful product formation. The flavin ring structure moves substantially in the active site, probably to enable substrate and product exchange into this site and possibly to regulate the reduction of the flavin by NADPH. A chain of H-bonds can connect p-hydroxy-benzoate in the active site of the enzyme with the protein surface. This chain is responsible for the reversible formation of substrate phenolate anion observed in the active site and partly responsible for the reactivity of this substrate.
By mutating the target residue of covalent flavinylation in vanillyl-alcohol oxidase, the functional role of the histidyl-FAD bond was studied. Three His 422 mutants (H422A, H422T, and H422C) were purified, which all contained tightly but noncovalently bound FAD. Steady state kinetics revealed that the mutants have retained enzyme activity, although the turnover rates have decreased by 1 order of magnitude. Stopped-flow analysis showed that the H422A mutant is still able to form a stable binary complex of reduced enzyme and a quinone methide product intermediate, a crucial step during vanillyl-alcohol oxidase-mediated catalysis. The only significant change in the catalytic cycle of the H422A mutant is a marked decrease in reduction rate. Redox potentials of both wild type and H422A vanillylalcohol oxidase have been determined. During reduction of H422A, a large portion of the neutral flavin semiquinone is observed. Using suitable reference dyes, the redox potentials for the two one-electron couples have been determined: ؊17 and ؊113 mV. Reduction of wild type enzyme did not result in any formation of flavin semiquinone and revealed a remarkably high redox potential of ؉55 mV. The marked decrease in redox potential caused by the missing covalent histidyl-FAD bond is reflected in the reduced rate of substrate-mediated flavin reduction limiting the turnover rate.Elucidation of the crystal structure of the H422A mutant established that deletion of the histidyl-FAD bond did not result in any significant structural changes. These results clearly indicate that covalent interaction of the isoalloxazine ring with the protein moiety can markedly increase the redox potential of the flavin cofactor, thereby facilitating redox catalysis. Thus, formation of a histidyl-FAD bond in specific flavoenzymes might have evolved as a way to contribute to the enhancement of their oxidative power.Until now, several hundred flavin-containing enzymes have been described. Most of these enzymes contain a dissociable FAD or FMN cofactor. However, it has been shown that in several cases the flavin is covalently linked to an amino acid of the polypeptide chain. In fact, in humans 10% of the cellular FAD is covalently bound to enzymes like e.g. succinate dehydrogenase and monoamine oxidase (1). Within the group of covalent flavoproteins, five different types of covalent flavinylation have been identified. Except for a few examples of cysteinyl-or tyrosyl-linked flavins, tethering to a histidine is by far the most favored binding mode, since it has been observed in about 20 isolated flavoenzymes (for a recent review, see Ref. 2).Although the first covalent flavoprotein, succinate dehydrogenase, was already identified in 1955 (3), the rationale for covalent flavinylation is still unresolved. Only recently, a clear influence of the covalent bond on the reactivity of the cofactor has been observed in trimethylamine dehydrogenase. Unlike the wild type enzyme, mutants of trimethylamine dehydrogenase containing dissociable FMN (4, 5) are inactivated b...
Abstract:Baeyer±Villiger monooxygenases (BVMOs) are flavoenzymes that catalyze a remarkably wide variety of oxidative reactions such as regioand enantioselective Baeyer±Villiger oxidations and sulfoxidations. Several of these conversions are difficult to achieve using chemical approaches. Due to their selectivity and catalytic efficiency, BVMOs are highly valuable biocatalysts for the synthesis of a broad range of fine chemicals. For a long time, only one member of this class of flavin-containing biocatalysts had been cloned and overexpressed which has limited their application for synthetic processes. Recently a number of new genes that encode BVMOs have been sequenced and overexpressed. In this paper the biocatalytic properties of recently cloned BVMOs are reviewed. Furthermore, the potential for obtaining novel BVMOs from sequenced genomes will be discussed.
A flavodoxin from Azotobacter vinelandii is chosen as a model system to study the folding of a/P doubly wound proteins. The guanidinium hydrochloride induced unfolding of apoflavodoxin is demonstrated to be reversible. Apoflavodoxin thus can fold in the absence of the FMN cofactor. The unfolding curves obtained for wild-type, C69A and C69S apoflavodoxin as monitored by circular dichroism and fluorescence spectroscopy do not coincide. Apoflavodoxin unfolding occurs therefore not via a simple two-state mechanism. The experimental data can be described by a three-state mechanism of apoflavodoxin equilibrium unfolding in which a relatively stable intermediate is involved. The intermediate species lacks the characteristic tertiary structure of native apoflavodoxin as deduced from fluorescence spectroscopy, but has significant secondary structure as inferred from circular dichroism spectroscopy. Both spectroscopic techniques show that thermally-induced unfolding of apoflavodoxin also proceeds through formation of a similar molten globule-like species. Thermal unfolding of apoflavodoxin is accompanied by anomalous circular dichroism characteristics: the negative ellipticity at 222 nM increases in the transition zone of unfolding. This effect is most likely attributable to changes in tertiary interactions of aromatic side chains upon protein unfolding. From the presented results and hydrogen/deuterium exchange data, a model for the equilibrium unfolding of apoflavodoxin is presented.Keywords: apoflavodoxin; circular dichroism; equilibrium unfolding; fluorescence; molten globule-like species; NMR; protein stability; three-state model In contrast to most protein folds, the flavodoxin-like fold is shared by many (i.e., nine) superfamilies (Brenner, 1997 a broad range of unrelated proteins with different functions like catalases, chemotactic proteins, lipases, esterases, and flavodoxins. They are all characterized by a five-stranded parallel P-sheet surrounded by a-helices at either side of the sheet. Of these proteins, a flavodoxin is chosen by us as a model system to study protein folding and stability. General rules governing protein folding are only beginning to emerge and there is a strong need for additional well-characterized protein systems to obtain a better understanding of the fundamental rules describing protein folding. By studying the folding of flavodoxin, we extend the number of proteins under investigation and expect to learn the rules by which other, if not all, proteins with a flavodoxin-like topology fold.Flavodoxins are a group of small flavoproteins that function as low-potential one-electron carriers and contain a noncovalently bound FMN cofactor (Mayhew & Tollin, 1992). The protein investigated by us is flavodoxin 11 from Azotobacter vinelandii (strain ATCC 478), henceforth designated flavodoxin. Its gene has been cloned in Escherichia coli and brought to high expression in our laboratory. The protein consists of 179 amino acid residues and belongs to the class of "long-chain" flavodoxins (Ta...
Of all NMR observable isotopes 19F is the one perhaps most convenient for studies on biodegradation of environmental pollutants. The reasons underlying this potential of 19F NMR are discussed and illustrated on the basis of a study on the biodegradation of fluorophenols by four Rhodococcus strains. The results indicate marked differences between the biodegradation pathways of fluorophenols among the various Rhodococcus species. This holds not only for the level and nature of the fluorinated biodegradation pathway intermediates that accumulate, but also for the regioselectivity of the initial hydroxylation step. Several of the Rhodococcus species contain a phenol hydroxylase that catalyses the oxidative defluorination of ortho-fluorinated di- and trifluorophenols. Furthermore, it is illustrated how the 19F NMR technique can be used as a tool in the process of identification of an accumulated unknown metabolite, in this case most likely 5-fluoromaleylacetate. Altogether, the 19F NMR technique proved valid to obtain detailed information on the microbial biodegradation pathways of fluorinated organics, but also to provide information on the specificity of enzymes generally considered unstable and, for this reason, not much studied so far.
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