Monomeric Flavoprotein Research Articles

Extremophile DNA Photolyases: DNA Repair under Extreme Conditions

Adaptation to extreme environments is a seminal characteristic of life on Earth. Nowhere is this property more strongly evident than in single-celled organisms. Bacteria, archaea, and eukaryotes all have representatives that thrive in cold or hot environments. Many of these extremophile enzymes share significant amino acid sequence homology in spite of very different optimal growth temperatures (TG). Based on a simple amino acid sequence analysis it is unclear what adaptive changes are required to tailor homologous enzymes to function in extreme environments.A case in point is DNA photolyase (PL), a monomeric flavoprotein that binds UV-damaged DNA and repairs it by blue light activated picosecond electron transfer from a conserved flavin adenine dinucleotide cofactor (FADH¯) to the tightly bound cyclobutylpyrimidine dimer DNA lesion. These enzymes also include a second light-harvesting cofactor. Here we present a comparative analysis of three recombinant CPD photolyases, hyperthermophilic Sulfolobus solfataricus (TG=353K, rSsPL), mesophilic E. coli (TG=310K, rEcPL), and psychrophilic Colwellia psychrerythraea (TG=281K, rCpPL).All PLs utilize FADH¯ bound in a highly conserved site for repair. In addition, each PL demonstrates different properties for its 2nd cofactor. We used a variety of biochemical, physical, and molecular biological tools to compare these extremophile proteins with regard to repair yield, cofactor reduction potential and excited state properties. The difference in the stability of the redox state of the purified protein suggests different structural adaptations of each PL to their respective thermal environments. Denaturation studies reveal that rSsPL has a very stable structure, whereas rCpPL is extremely sensitive to its thermal and aqueous environments. A comparison of cofactor absorption and emission spectra reveal significant differences in how the flavin cofactor is bound in the binding pocket, in spite of significant amino acid sequence homology for this cavity across all proteins.

Agaricus meleagris pyranose dehydrogenase: Influence of covalent FAD linkage on catalysis and stability

Pyranose dehydrogenase (PDH) is a monomeric flavoprotein belonging to the glucose–methanol–choline (GMC) family of oxidoreductases. It catalyzes the oxidation of free, non-phosphorylated sugars to the corresponding keto sugars. The enzyme harbors an FAD cofactor that is covalently attached to histidine 103 via an 8α-N(3) histidyl linkage. Our previous work showed that variant H103Y was still able to bind FAD (non-covalently) and perform catalysis but steady-state kinetic parameters for several substrates were negatively affected. In order to investigate the impact of the covalent FAD attachment in Agaricus meleagris PDH in more detail, pre-steady-state kinetics, reduction potential and stability of the variant H103Y in comparison to the wild-type enzyme were probed. Stopped-flow analysis revealed that the mutation slowed down the reductive half-reaction by around three orders of magnitude whereas the oxidative half-reaction was affected only to a minor degree. This was reflected by a decrease in the standard reduction potential of variant H103Y compared to the wild-type protein. The existence of an anionic semiquinone radical in the resting state of both the wild-type and variant H103Y was demonstrated using electron paramagnetic resonance (EPR) spectroscopy and suggested a higher mobility of the cofactor in the variant H103Y. Unfolding studies showed significant negative effects of the disruption of the covalent bond on thermal and conformational stability. The results are discussed with respect to the role of covalently bound FAD in catalysis and stability.

Biochemical Studies of asoxF-Encoded Monomeric Flavoprotein Purified from the Green Sulfur BacteriumChlorobaculum tepidumThat Stimulatesin VitroThiosulfate Oxidation

In the green sulfur bacterium Chlorobaculum tepidum, three sulfur oxidizing enzyme system (Sox) proteins, SoxAXK, SoxYZ, and SoxB (the core TOMES, thiosulfate oxidizing multi-enzyme system) are essential to in vitro thiosulfate oxidation. We purified monomeric flavoprotein SoxF from this bacterium, which had sulfide dehydrogenase activity. SoxF enhanced the thiosulfate oxidation activity of the purified core TOMES with various cytochromes as electron acceptors to different degrees without any change in the affinity for thiosulfate. The apparent reaction rates with 50 microM- C. tepidum cytochrome c-554 were slightly higher than with horse-heart cytochrome c, and the addition of 0.5 microM- SoxF increased the rate by 92%. The rates with 50 microM- horse-heart cytochrome c and 50 muM- horse-heart cytochrome c plus 0.5 muM- cytochrome c-554 were increased by SoxF by 31% and 120% respectively. We conclude that SoxF mediates electron transfer between the components of core TOMES and externally added cytochromes.

Export of functional Streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis

Streptomyces coelicolor A3(2) alditol oxidase (AldO) is a soluble monomeric flavoprotein in which the flavin cofactor is covalently linked to the polypeptide chain. AldO displays high reactivity towards different polyols such as xylitol and sorbitol. These characteristics make AldO industrially relevant, but full biotechnological exploitation of this enzyme is at present restricted by laborious and costly purification steps. To eliminate the need for enzyme purification, this study describes a whole-cell AldO biocatalyst system. To this end, we have directed AldO to the periplasm or cell surface of Escherichia coli. For periplasmic export, AldO was fused to endogenous E. coli signal sequences known to direct their passenger proteins into the SecB, signal recognition particle (SRP), or Twin-arginine translocation (Tat) pathway. In addition, AldO was fused to an ice nucleation protein (INP)-based anchoring motif for surface display. The results show that Tat-exported AldO and INP-surface-displayed AldO are active. The Tat-based system was successfully employed in converting xylitol by whole cells, whereas the use of the INP-based system was most likely restricted by lipopolysaccharide LPS in wild-type cells. It is anticipated that these whole-cell systems will be a valuable tool for further biological and industrial exploitation of AldO and other cofactor-containing enzymes.

Discovery, Characterization, and Kinetic Analysis of an Alditol Oxidase from Streptomyces coelicolor

A gene encoding an alditol oxidase was found in the genome of Streptomyces coelicolor A3(2). This newly identified oxidase, AldO, was expressed at extremely high levels in Escherichia coli when fused to maltose-binding protein. AldO is a soluble monomeric flavoprotein with subunits of 45.1 kDa, each containing a covalently bound FAD cofactor. From sequence alignments with other flavoprotein oxidases, it was found that AldO contains a conserved histidine (His(46)) that is typically involved in covalent FAD attachment. Covalent FAD binding is not observed in the H46A AldO mutant, confirming its role in covalent attachment of the flavin cofactor. Steady-state kinetic analyses revealed that wild-type AldO is active with several polyols. The alditols xylitol (K(m) = 0.32 mm, k(cat) = 13 s(-1)) and sorbitol (K(m) = 1.4 mm, k(cat) = 17 s(-1)) are the preferred substrates. From pre-steady-state kinetic analyses, using xylitol as substrate, it can be concluded that AldO mainly follows a ternary complex kinetic mechanism. Reduction of the flavin cofactor by xylitol occurs at a relatively high rate (99 s(-1)), after which a second kinetic event is observed, which is proposed to represent ring closure of the formed aldehyde product, yielding the hemiacetal of d-xylose. Reduced AldO readily reacts with molecular oxygen (1.7 x 10(5) m(-1) s(-1)), which confirms that the enzyme represents a true flavoprotein oxidase.

Sulfide Dehydrogenase Activity of the Monomeric Flavoprotein SoxF of Paracoccus pantotrophus,

Flavocytochrome c-sulfide dehydrogenases (FCSDs) are complexes of a flavoprotein with a c-type cytochrome performing hydrogen sulfide-dependent cytochrome c reduction in vitro. The amino acid sequence analysis revealed that the phylogenetic relationship of different flavoproteins reflected the relationship of sulfur-oxidizing bacteria. The flavoprotein SoxF of Paracoccus pantotrophus is 29-67% identical to the flavoprotein subunit of FCSD of phototrophic sulfur-oxidizing bacteria. Purification of SoxF yielded a homogeneous emerald-green monomeric protein of 42 797 Da. SoxF catalyzed sulfide-dependent horse heart cytochrome c reduction at the optimum pH of 6.0 with a k(cat) of 3.9 s(-1), a K(m) of 2.3 microM for sulfide, and a K(m) of 116 microM for cytochrome c, as determined by nonlinear regression analysis. The yield of 1.9 mol of cytochrome c reduced per mole of sulfide suggests sulfur or polysulfide as the product. Sulfide dehydrogenase activity of SoxF was inhibited by sulfur (K(i) = 1.3 microM) and inactivated by sulfite. Cyanide (1 mM) inhibited SoxF activity at pH 6.0 by 25% and at pH 8.0 by 92%. Redox titrations in the infrared spectral range from 1800 to 1200 cm(-1) and in the visible spectral range from 400 to 700 nm both yielded a midpoint potential for SoxF of -555 +/- 10 mV versus Ag/AgCl at pH 7.5 and -440 +/- 20 mV versus Ag/AgCl at pH 6.0 (-232 mV versus SHE') and a transfer of 1.9 electrons. Electrochemically induced FTIR difference spectra of SoxF as compared to those of free flavin in solution suggested a strong cofactor interaction with the apoprotein. Furthermore, an activation/variation of SoxF during the redox cycles is observed. This is the first report of a monomeric flavoprotein with sulfide dehydrogenase activity.

Cis-syn thymidine dimer repair by DNA photolyase in real time.

DNA photolyase (PL) is a monomeric flavoprotein that repairs cyclobutylpyrimidine dimers (CPDs) via photoinduced electron transfer from a reduced flavin adenine dinucleotide cofactor (FADH(-)) to the bound CPD. We have used subpicosecond UV transient absorption spectroscopy to measure the electron-transfer and repair kinetics of Anacystis nidulans DNA photolyase with dimeric and pentameric oligothymidine substrates. Here we show that the electron-transfer lifetime is 32 +/- 20 ps for the pentameric substrate. Repair of the carbon-carbon double bonds (C=C) in the CPD is initiated in approximately 60 ps, and bond scission appears to be completed by 1500 ps. This suggests that the repair of the two C=C bonds proceeds sequentially and that the first bond scission has a much lower activation barrier than the second. Our experiments also suggest that the semiquinone FADH(*) cofactor is not reduced to its catalytically active FADH(-) state by substrate after repair but remains in the semiquinone state. In contrast to the longer substrate, the dinucleotide substrate produced a mixture of kinetics representing bound and unbound substrate.

NADH:Fe(III)-chelate reductase of maize roots is an active cytochrome b5 reductase

Microsomal NADH:Fe(III)-chelate reductase (NFR) of maize roots has been purified as a monomeric flavoprotein of 32 kDa with non-covalently bound FAD. In the presence of NADH, NFR efficiently reduced the physiological iron-chelate Fe(III)-citrate ( K cat/ K m(Fe(III)-citrate)=6.0×10 6 M −1 s −1) with a sequential reaction mechanism. Purified NFR was totally inhibited by the sulfhydryl reagent PHMB at 10 −9 M, and it could use cyt b 5 as alternative electron acceptor with a maximal reduction rate as high as with Fe(III)-citrate. We conclude that in maize roots the reduction of Fe(III)-citrate is chiefly performed by a cytochrome b 5 reductase, mostly associated with intracellular membranes and in part with the plasma membrane.

A H2O-producing NADH oxidase from the protozoan parasite Giardia duodenalis.

We describe the purification of a H2O-producing NADH oxidase from the protozoan parasite Giardia duodenalis. The enzyme is a monomeric flavoprotein containing flavin adenine dinucleotide in a 1:1 molar ratio with the polypeptide. The NADH oxidase has an apparent molecular mass of 46 kDa and was homogenous as determined by denaturing gel electrophoresis and N-terminal amino acid sequencing. NADPH could substitute for NADH as an electron donor with a K(m) value of 4.2 microM for NADH and 16 microM for NADPH (pH 7.8 at room temperature). With oxygen as the primary electron acceptor under aerobic conditions, the pure enzyme did not produce O.-2 nor H2O2 as stoichiometric products of oxygen reduction, implicating H2O as the end product and obviating the need for superoxide dismutase. The ability to utilise oxygen explains the apparent respiration of the amitochondrial fermentative metabolism of Giardia. Mercurials, flavoantagonists and heavy metals (Cu2+ and Zn2+) inhibited this activity. Under anaerobic conditions the enzyme catalysed electron transfer at lower efficiencies to other electron acceptors including nitroblue tetrazolium, potassium ferricyanide, FAD and FMN, using either NADH or NADPH as electron donors. NADPH, however, was a more efficient electron donor. Cytochrome c was not reduced under any assay conditions used. The enzyme reduced the nitrofuran drugs, furazolidone (an antigiardial) and nitrofurantoin, to their toxic radical forms as determined by EPR. Metronidazole, a nitroimidazole, was not reduced. Pure NADH oxidase did not demonstrate ferredoxin:NAD(P)1 oxidoreductase activity since it could not accept electrons from reduced ferredoxin to regenerate NAD(P)H. The G. duodenalis NADH oxidase may, therefore, function as a terminal oxidase, similar to the mitochondrial cytochrome oxidase, and in the maintenance of an optimum intracellular redox ratio. This report of a flavoenzyme from Giardia places Giardia close to the anaerobic bacteria in evolutionary terms.

Flavodoxin from Wolinella succinogenes.

A monomeric flavoprotein (18.8 kDa) was isolated from the soluble cell fraction of Wolinella succinogenes and was identified as a flavodoxin based on its N-terminal sequence, FMN content, and redox properties. The midpoint potentials of the flavodoxin (Fld) at pH 7. 5 were measured as -95 mV (Fldox/Flds) and -450 mV (Flds/Fldred) relative to the standard hydrogen electrode. The cellular flavodoxin content [0.3 micromol (g protein)-1] was the same in bacteria grown with fumarate or with polysulfide as the terminal acceptor of electron transport. The flavodoxin did not accept electrons from hydrogenase or formate dehydrogenase, the donor enzymes of electron transport to fumarate or polysulfide. Pyruvate:flavodoxin oxidoreductase activity [180 U (g cellular protein)-1] was detected in the soluble cell fraction of W. succinogenes grown with fumarate or polysulfide. The enzyme was equally active with Fldox or Flds at high concentrations. The Km for Flds (80 microM) was larger than that for Fldox and for the ferredoxin isolated from W. succinogenes (15 microM). We conclude that flavodoxin serves anabolic rather than catabolic functions in W. succinogenes.

Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2.

A mixed microbial culture capable of metabolizing the explosive pentaerythritol tetranitrate (PETN) was obtained from soil enrichments under aerobic and nitrogen-limiting conditions. A strain of Enterobacter cloacae, designated PB2, was isolated from this culture and was found to use PETN as a sole source of nitrogen for growth. Growth yields suggested that 2 to 3 mol of nitrogen was utilized per mol of PETN. The metabolites pentaerythritol dinitrate, 3-hydroxy-2,2-bis-[(nitrooxy)methyl]propanal, and 2,2-bis-[(nitrooxy)methyl]-propanedial were identified by mass spectrometry and 1H-nuclear magnetic resonance. An NADPH-dependent PETN reductase was isolated from cell extracts and shown to liberate nitrite from PETN, producing pentaerythritol tri- and dinitrates which were identified by mass spectrometry. PETN reductase was purified to apparent homogeneity by ion-exchange and affinity chromatography. The purified enzyme was found to be a monomeric flavoprotein with a M(r) of approximately 40,000, binding flavin mononucleotide noncovalently.

NADH oxidase ofThermus thermophilus HB8 overproduced fromEscherichia coli

An NADH oxidase purified from the extreme thermophileThermus thermophilus HB8 is a monomeric flavoprotein with a 1 ∶ 1 ratio of flavin-adenine dinucleotide (FAD) to the polypeptide chain. It catalyzes in vitro the oxidation of reduced NADH or NADPH with the formation of H2O2. The gene encoding the NADH oxidase fromT. thermophilus HB8 was cloned, and its nucleotide sequence was determined. The molecular mass of 22,749 Da, as deduced from thenox gene, agrees with that of the purified NADH oxidase fromT. thermophilus HB8, as estimated by mass spectrometry. Thenox gene does not contain a GX4GK consensus sequence typical for nucleotide binding proteins. Thenox gene was overexpressed inEscherichia coli, and a protocol for the rapid purification of theE. coli-borneT. thermophilus NADH oxidase or its His6-tagged analogue was developed by using thermal denaturation step and affinity chromatography.

Purification and properties of tobacco ferredoxin-dependent glutamate synthase, and isolation of corresponding cDNA clones

Ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) was purified to electrophoretic homogeneity from leaves of tobacco (Nicotiana tabacum L.). The holoenzyme is a monomeric flavoprotein with a molecular weight of 164 kDa. Polyclonal rabbit antibodies against the purified enzyme were used to isolate a 450-bp Fd-GOGAT cDNA clone (C16) from a tobacco λgt11 expression library. A longer Fd-GOGAT cDNA clone (C35) encoding about 70% of the amino acids of tobacco Fd-GOGAT was isolated from a tobacco λgt10 cDNA library using C16 as the probe. The amino-acid sequence of the protein encoded by the Fd-GOGAT cDNA clone C35 was delineated. It is very likely that Fd-GOGAT is encoded by two genes in the amphidiploid genome of tobacco while only a single Fd-GOGAT gene appears to be present in the diploid genome of Nicotiana sylvestris. Two Fd-GOGAT isoenzymes could be distinguished in extracts of tobacco leaf protein. In contrast, a single Fd-GOGAT protein species was detected in leaves of Nicotiana sylvestris speg. et Comes. In tobacco leaves, the 6-kb Fd-GOGAT mRNA is about 50-fold less abundant than chloroplastic glutamine synthetase (EC 6.3.1.2) mRNA. Both Fd-GOGAT mRNA and Fd-GOGAT protein accumulated during greening of etiolated tobacco leaves, and a concomitant increase in Fd-GOGAT activity was observed. These results indicate that tobacco Fd-GOGAT gene expression is light-inducible. Levels of Fd-GOGAT mRNA in tobacco organs other than leaves were below the detection limit of our Northern-blot analysis. Polypeptides of Fd-GOGAT were present in tobacco leaves and, to a lesser extent, in pistils and anthers, but not in corollas, stems and roots. These results support organ specificity in tobacco Fd-GOGAT gene expression.

The Detection and Characterization of Several Conformational States of Cytochrome b5 Aporeductase during Reversible Denaturation

Abstract The reversible denaturation of cytochrome b5 reductase at pH 2.0 has been described. During the relatively slow process of recombination, several protein species have been detected by spectral and fluorescence measurements of nucleotide binding, by protein fluorescence changes, and by alterations in the number of reactive sulfhydryl groups. Mercuribenzoate and mersalyl were used to prepare stable derivatives of several forms of the aporeductase that appear during recombination. This series of reactions, and in particular the stable mercurial derivatives, thus provide an ideal system for examining the dependence of the tertiary structure of a monomeric flavoprotein upon its primary structure and chemical environment.