Involvement of a protein kinase activity inducer in DNA double strand break repair and radioresistance of Deinococcus radiodurans

Transgenic bacteria producing pyrroloquinoline quinone, a known cofactor for dehydrogenases and an inducer of a periplasmic protein kinase activity, show resistance to both oxidative stress and protection from nonoxidative effects of radiation and DNA-damaging agents. Deinococcus radiodurans R1 encodes an active pyrroloquinoline quinone synthase, and constitutive synthesis of pyrroloquinoline quinone occurred in wild-type bacteria. Disruption of a genomic copy of pqqE resulted in cells that lacked this cofactor. The mutant showed a nearly 3-log decrease in gamma radiation resistance and a 2-log decrease in mitomycin C tolerance compared to wild-type cells. The mutant cells did not show sensitivity to UVC radiation. Expression of pyrroloquinoline quinone synthase in trans showed that there was functional complementation of gamma resistance and mitomycin C tolerance in the pqqE mutant. The sensitivity to gamma radiation was due to impairment or slow kinetics of DNA double strand break repair. Low levels of (32)P incorporation were observed in total soluble proteins of mutant cells compared to the wild type. The results suggest that pyrroloquinoline quinone has a regulatory role as a cofactor for dehydrogenases and an inducer of selected protein kinase activity in radiation resistance and DNA strand break repair in a radioresistant bacterium.     

Excitation energy transfer study of the spatial relationship between the carbonyl and metal cofactors in pig plasma amine oxidase

9-Hydrazinoacridine irreversibly labeled pig plasma amine oxidase by covalent attachment to the active carbonyl cofactor. The visible absorption spectrum of the modified protein displays new absorption bands at 495 and 525 nm. Its emission spectrum exhibited maxima at 415 and 440 nm. In addition, both absorption and emission spectra were insensitive to pH changes between 6 and 10. Phase modulation fluorometry was used to determine fluorescence lifetimes of Zn2+- and Co2+-substituted acridinyl plasma amine oxidase. Energy transfer efficiency was 22%; the distance separating the Co2+ ion (in the copper binding site) and the acridine moiety (the amine substrate binding site) ranges between 11.7 and 14.7 A. This work defines the proximity of the metal and substrate (and hence the carbonyl cofactor) and precludes any direct interaction between Cu2+ and pyrroloquinoline quinone or between Cu2+ and the substrate.     

Pyrroloquinoline quinone as cofactor in galactose oxidase (EC 1.1.3.9)

Galactose oxidase from Dactylium dendroides was shown to contain one molecule of covalently bound pyrroloquinoline quinone (PQQ/enzyme molecule. From the spectroscopic characteristics reported for the enzyme forms, a mechanistic role for PQQ could be deduced. In analogy with other quinoproteins, the initial formation of a PQQ-substrate adduct is proposed. Following internal hydrogen transfer, leading to aldehyde product and reduced pyrroloquinoline quinone, reoxidation of the organic cofactor with molecular oxygen could be mediated by the PQQ-liganded copper ion with concomitant formation of hydrogen peroxide. With PQQ as an additional (two-electron) redox center the occurrence of a "superoxidized" enzyme form must be considered. Possible consequences of this view, in relation to a physiological function of the enzyme and interpretation of ESR data, are discussed.     

On the structure and linkage of the covalent cofactor of methylamine dehydrogenase from the methylotrophic bacterium W3A1

Short amino acid sequences around the two linkage sites of the cofactor of methylamine dehydrogenase are presented. Mass spectral data indicates that the covalently bound cofactor is the tricyclic pyrroloquinoline quinone (PQQ). However, the 3 carboxyl groups characteristic of this o-quinone are absent. A cysteine thioether, via a methylene bridge, and a serine ether link the cofactor to the small subunit of methylamine dehydrogenase.     

A comparison of the local environment of Cu(II) in native and half-Cu-depleted bovine serum amine oxidase

Electron spin-echo envelope modulation spectroscopy has been used to compare the local environment of Cu(II) in native bovine serum amine oxidase, containing two copper atoms/dimer molecule, and in a protein preparation, half depleted of copper, which has little enzymatic activity. For each preparation, two different populations of coordinated imidazoles with inequivalent magnetic coupling to copper could be recognized. In addition, water was shown to be a ligand to copper. No differences in coordinated ligand structures between the native and half-Cu-depleted preparations could be seen. In addition, the amount of ambient, non-coordinated water detected for native and half-Cu-depleted proteins was found to be nearly equivalent. However, the addition of phenylhydrazine, an inhibitor that binds to the pyrroloquinoline quinone cofactor but not to Cu(II) in the native enzyme, displaces ambient water near copper.     

Method of enzymatic determination of pyrroloquinoline quinone

An improved enzymatic method for the determination of pyrroloquinoline quinone, a novel prosthetic group of some important oxidoreductases, has been developed with cytoplasmic membrane of Escherichia coli K-12, in which D-glucose dehydrogenase (EC 1.1.99.17) was completely resolved to apo-enzyme by EDTA treatment. Incubation of the EDTA-treated membrane with exogenous pyrroloquinoline quinone in the presence of magnesium ions gave a quantitative determination of pyrroloquinoline quinone by assaying the restored D-glucose dehydrogenase activity. This novel enzymatic method was confirmed to be highly reproducible up to 10 ng of pyrroloquinoline quinone and could be applied to a routine assay of pyrroloquinoline quinone.     

Adaptation of Escherichia coli to the uncoupler of oxidative phosphorylation 2,4-dinitrophenol.

Escherichia coli was found to adapt to the uncoupler of oxidative phosphorylation 2,4-dinitrophenol. The rates of synthesis of 53 proteins were increased following exposure to 2,4-dinitrophenol. Adaptation was accelerated when the cofactor pyrroloquinoline quinone was provided in the growth medium.     

Electron transfer in quinoproteins

Soluble quinoprotein dehydrogenases oxidize a wide range of sugar, alcohol, amine, and aldehyde substrates. The physiological electron acceptors for these enzymes are not pyridine nucleotides but are other soluble redox proteins. This makes these enzymes and their electron acceptors excellent systems with which to study mechanisms of long-range interprotein electron transfer reactions. The tryptophan tryptophylquinone (TTQ)-dependent methylamine dehydrogenase (MADH) transfers electrons to a blue copper protein, amicyanin. It has been possible to alter the rate of electron transfer by using different redox forms of MADH, varying reaction conditions, and performing site-directed mutagenesis on these proteins. From kinetic and thermodynamic analyses of the reaction rates, it was possible to determine whether a change in rate is due a change in Delta G(0), electronic coupling, reorganization energy or kinetic mechanism. Examples of each of these cases are discussed in the context of the known crystal structures of the electron transfer protein complexes. The pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase transfers electrons to a c-type cytochrome. Kinetic and thermodynamic analyses of this reaction indicated that this electron transfer reaction was conformationally coupled. Quinohemoproteins possess a quinone cofactor as well as one or more c-type hemes within the same protein. The structures of a PQQ-dependent quinohemoprotein alcohol dehydrogenase and a TTQ-dependent quinohemoprotein amine dehydrogenase are described with respect to their roles in intramolecular and intermolecular protein electron transfer reactions.     

Factors affecting the production of pyrroloquinoline quinone by the methylotrophic bacterium W3A1.

Two variants of the methylotrophic bacterium W3A1, designated W3A1-S (slimy) and W3A1-NS (nonslimy), were compared with respect to their ability to grow in batch culture on the C1 substrates methylamine, methanol, and trimethylamine. Substrate utilization, cell density, pH, cellular and soluble polysaccharide production, and concentrations of the enzymes methylamine dehydrogenase, trimethylamine dehydrogenase, and methanol dehydrogenase produced were measured as a function of growth. The ability of the two bacterial variants to excrete the redox cofactor pyrroloquinoline quinone into the growth medium was also investigated. The two variants were similar with respect to all properties measured, except that W3A1-S produced significantly more capsular polysaccharides than variant W3A1-NS. Pyrroloquinoline quinone was excreted when either variant was grown on any of the C1 substrates investigated but was maximally produced when the methylamine concentration was 0.45% (wt/vol). This cofactor is excreted only as bacterial growth enters the stationary phase, a time when the levels of trimethylamine dehydrogenase and the quinoproteins methanol dehydrogenase and methylamine dehydrogenase begin to decline. It is not known whether the pyrroloquinoline quinone found in the medium is made de novo for excretion, derived from the quinoprotein pool, or both. Pyrroloquinoline quinone excretion has been observed with other methylotrophs, but this is the first instance where the excretion was observed with substrates other than methanol.     

Factors affecting the production of pyrroloquinoline quinone by the methylotrophic bacterium W3A1

Two variants of the methylotrophic bacterium W3A1, designated W3A1-S (slimy) and W3A1-NS (nonslimy), were compared with respect to their ability to grow in batch culture on the C1 substrates methylamine, methanol, and trimethylamine. Substrate utilization, cell density, pH, cellular and soluble polysaccharide production, and concentrations of the enzymes methylamine dehydrogenase, trimethylamine dehydrogenase, and methanol dehydrogenase produced were measured as a function of growth. The ability of the two bacterial variants to excrete the redox cofactor pyrroloquinoline quinone into the growth medium was also investigated. The two variants were similar with respect to all properties measured, except that W3A1-S produced significantly more capsular polysaccharides than variant W3A1-NS. Pyrroloquinoline quinone was excreted when either variant was grown on any of the C1 substrates investigated but was maximally produced when the methylamine concentration was 0.45% (wt/vol). This cofactor is excreted only as bacterial growth enters the stationary phase, a time when the levels of trimethylamine dehydrogenase and the quinoproteins methanol dehydrogenase and methylamine dehydrogenase begin to decline. It is not known whether the pyrroloquinoline quinone found in the medium is made de novo for excretion, derived from the quinoprotein pool, or both. Pyrroloquinoline quinone excretion has been observed with other methylotrophs, but this is the first instance where the excretion was observed with substrates other than methanol.     

Specific detection of quinoproteins by redox-cycling staining.

Quinones and related quinonoid substances catalyze redox cycling at an alkaline pH in the presence of excess glycine as reductant. With nitroblue tetrazolium and oxygen present there is concomitant reduction of the tetrazolium to formazan. This property of quinonoid compounds is used for the specific staining of quinoproteins, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose. The dopa-containing vitelline proteins and the 6-hydroxydopa-containing bovine serum amine oxidase are stained with the nitroblue tetrazolium/glycinate reagent. Also, the mammalian quinoproteins, diamine oxidase and lysyl oxidase, purported to contain pyrroloquinoline quinone, tested positive in this procedure. No quinonoid components were detected in three putative pyrroloquinoline quinone-containing quinoproteins, dopamine beta-hydroxylase, lipoxygenase, and peptidylglycine-amidating monoxygenase. Redox-cycling staining therefore confirms the presence of covalently bound quinones in the copper-dependent amine oxidases, but not in two putative quinoprotein oxygenases. Clarification of the biological significance of quinolation should be facilitated by identification of quinoproteins using this approach.     

The role of copper in bovine serum amine oxidase

Summary The role of copper in bovine serum amine oxidase was investigated by studying the effect of copper-binding inhibitors on the reactions of the pyrroloquinoline quinone carbonyl and on the reaction with oxygen. Hydrazines and hydrazides were used as carbonyl reagents and one of the hydrazines, benzylhydrazine, which was found to behave as a pseudo-substrate, was used to probe the reaction with oxygen. The presence ofN,N-diethyldithiocarbamate, a chelator that binds copper irreversibly, did not prevent the reactions at the carbonyl, but slowed down their rate and modified the conformation of the adducts. The same happened to the reaction with oxygen, which was slowed down but not abolished. Copper, which was never seen in the reduced state, thus appears to control all reactions without being directly involved in the binding of either hydrazines or oxygen. The enzyme functionality was in fact preserved upon substitution of copper with cobalt. The specific activity of the cobalt-substituted enzyme was only reduced to about 40% the native amine oxidase value. This is the first case so far in which the role of copper can be performed by a different metal ion.Abbreviations BSAO bovine serum amine oxidase - DDC N,N-diethyldithiocarbamate - PQQ pyrroloquinoline quinone     

Spectroscopic studies of the reaction between bovine serum amine oxidase (copper-containing) and some hydrazides and hydrazines.

The carbonyl cofactor of bovine serum amine oxidase, recently identified as pyrroloquinoline quinone [Ameyama, Hayashi, Matsushita, Shinagawa & Adachi (1984) Agric. Biol. Chem. 48, 561-565; Lobenstein-Verbeek, Jongejan, Frank & Duine (1984) FEBS Lett. 170, 305-309], reacts stoichiometrically and irreversibly with hydrazides of phenylacetic acid and of benzoic acid. With the phenylacetic hydrazides a reversible intermediate step was detected by competition with substrate, carbonylic reagents or phenylhydrazine, a typical inhibitor of the enzyme. All hydrazides form an intense broad band with maximum absorbance in a narrow wavelength range (350-360 nm), irrespective of the acyl group, suggesting that the transition is located on the organic cofactor. A different situation is found with some phenylhydrazines, where extended conjugation can occur between the cofactor and the phenyl pi-electron system via the azo group, as shown by the lower energy and higher intensity of the transition. In this case the transition is sensitive to substituents in the phenyl ring. The c.d. spectrum of the adducts is influenced by the type of hydrazide (derived from phenylacetic acid or benzoic acid), by pH and by NN-diethyldithiocarbamate binding to copper, probably as a result of shifts of equilibria between hydrazone-azo tautomers.     

Pea (Pisum sativum) diamine oxidase contains pyrroloquinoline quinone as a cofactor.

Diamine oxidase was prepared from pea (Pisum sativum) seedlings by a new purification procedure involving two h.p.l.c. steps. We studied the optical and electrochemical properties of the homogeneous enzyme and also analysed the hydrolysed protein by several methods. The data presented here suggest that the carbonyl cofactor of diamine oxidase is firmly bound pyrroloquinoline quinone.     

PQQ and quinoprotein research--the first decade

On the occasion of the first international symposium on pyrroloquinoline quinone (PQQ) and quinoproteins (Delft, September 1988), a review of this novel field in enzymology is presented. Quinoproteins (PQQ-containing enzymes) are widespread, from bacteria to mammalian organisms (including man), and occur in several classes of enzymes. Indications already exist that PQQ is a versatile cofactor, involved not only in oxidation but also in hydroxylation, transamination, decarboxylation and hydration reactions. The current list of quinoproteins shows that it was overlooked in several well-studied enzymes where the presence of a common cofactor had already been established. Up until now, all eukaryotic quinoproteins have covalently bound PQQ (or perhaps pro-PQQ), while free PQQ occurs exclusively in a number of (bacterial) dehydrogenases and in the culture fluid of certain Gram-negative bacteria. Biosynthesis of free PQQ in methylotrophic bacteria starts with tyrosine and glutamic acid as precursors while intermediates in the route have not been detected and the presence of free PQQ is not required for synthesis of the covalently bound form of the cofactor in glutamic acid decarboxylase from Escherichia coli. Therefore, the assembly of covalently bound cofactor might occur in situ, i.e. in the quinoproteins themselves. If the latter also applies to mammalian quinoproteins, this implies that PQQ is not a vitamin. On the other hand, positive effects have been reported upon administration of PQQ to test animals. Methods suited to detach and to detect PQQ with a derivatized o-quinone moiety may answer questions on the uptake and processing of the compound.     

Quinoproteins: structure, function, and biotechnological applications

A new class of oxidoreductase containing an amino acid-derived o-quinone cofactor, of which the most typical is pyrroloquinoline quinone (PQQ), is called quinoproteins, and has been recognized as the third redox enzyme following pyridine nucleotide- and flavin-dependent dehydrogenases. Some quinoproteins include a heme c moiety in addition to the quinone cofactor in the molecule and are called quinohemoproteins. PQQ-containing quinoproteins and quinohemoproteins have a common structural basis, in which PQQ is deeply embedded in the center of the unique superbarrel structure. Increased evidence for the structure and function of quinoproteins has revealed their unique position within the redox enzymes with respect to catalytic and electron transfer properties, and also to physiological and energetic function. The peculiarities of the quinoproteins, together with their unique substrate specificity, have encouraged their biotechnological application in the fields of biosensing and bioconversion of useful compounds, and also to environmental treatment.     

Soybean lipoxygenase-1 is not a quinoprotein

Soybean lipoxygenase-1 was reinvestigated with respect to its quinoprotein nature. It has been reported previously that soybean lipoxygenase-1 contains pyrroloquinoline quinone as the organic cofactor [1]. Because spectroscopie data were found to be inconsistent [2] with the evidence presented in [1], we sought to reproduce the published data by carefully following the procedures described in [1] and supplementing them with new analytical results. The combined data lead us to conclude that soybean lipoxygenase-1 is not a quinoprotein.     

Reassessment of the active site quino-cofactor proposed to occur in the Aspergillus niger amine oxidase AO-I from the properties of model compounds

Quino-cofactors have been found in a wide variety of prokaryotic and eukaryotic organisms. Two variants have, thus far, been demonstrated to derive from tyrosine precursors: these are the 2,4, 5-trihydroxyphenylalanine quinone (topa quinone or TPQ) [Janes, S. M. , et al. (1990) Science 248, 98] and an o-quinone analogue containing the side chain of a lysine residue (lysyltyrosine quinone or LTQ) [Wang, S. Z., et al. (1996) Science 273, 1078]. Additionally, a third variant of the family of tyrosine-derived cofactors has been reported to exist in an Aspergillus niger amine oxidase AO-I. This was described as an o-quinone cross-linked to the side chain of a glutamate residue [Frebort, I. (1996) Biochim. Biophys. Acta 1295, 59]. We have synthesized model compounds related to the proposed structure. Characterization of the redox properties for the model compound and spectral properties of its 4-nitrophenylhydrazine derivative lead us to conclude that the cofactor in A. niger amine oxidase AO-I has been misidentified. A TPQ carboxylate ester is considered an unlikely candidate for a biologically functional quino-cofactor.     

Cytokinin oxidase: Biochemical features and physiological significance

The catabolism of cytokinin in plant tissues appears to be due, in large part, to the activity of a specific enzyme, cytokinin oxidase. This enzyme catalyses the oxidation of cytokinin substrates bearing unsaturated isoprenoid side chains, using molecular oxygen as the oxidant. In general, substrate specificity is highly conserved and cytokinin substrates bearing saturated or cyclic side chains do not serve as substrates for most cytokinin oxidases tested to date. Despite variation in molecular properties of the enzyme from a number of higher plants, oxygen is always required for the reaction. Cytokinin oxidases from several sources have been shown to be glycosylated. Cytokinin oxidase activity appears to be universally inhibited by cytokinin-active urea derivatives. Auxin has been reported to act as an allosteric regulator which increases activity of the enzyme.
Cytokinin oxidase activity is subject to tight regulation. Levels of the enzyme are controlled by a mechanism sensitive to cytokinin supply. The up-regulation of cytokinin oxidase expression in response to exogenous application of cytokinin suggests that the metabolic fate of exogenously applied cytokinins may not accurately mimic that of the endogenous compounds.
Cytokinin oxidase is believed to be a copper-containing amine oxidase (EC 1.4.3.6). Considerable evidence strongly supports a common mechanism for amine oxidases. It is possible that advances in understanding of other amine oxidases could be extrapolated to increase our understanding of cytokinin oxidase at the molecular level. This is discussed with reference to what is currently known about the catalytic mechanism of the enzyme. The possibility of pyrroloquinoline quinone, or a closely related compound, as a redox cofactor of cytokinin oxidase is considered, as are the implications of the glycosylated nature of the enzyme for its regulation and compartmentalisation within the cell.     

Understanding quinone cofactor biogenesis in methylamine dehydrogenase through novel cofactor generation

Cofactors made from constitutive amino acids in proteins are now known to be relatively common. A number of these involve the generation of quinone cofactors, such as topaquinone in the copper-containing amine oxidases, and lysine tyrosylquinone in lysyl oxidase. The biogenesis of the quinone cofactor tryptophan tryptophylquinone (TTQ) in methylamine dehydrogenase (MADH) involves the post-translational modification of two constitutive Trp residues (Trp(beta)(57) and Trp(beta)(108) in Paracoccus denitrificans MADH). The modifications for generating TTQ are the addition of two oxygens to the indole ring of Trp(beta)(57) and the formation of a covalent cross-link between Cepsilon3 of Trp(beta)(57) and Cdelta1 of Trp(beta)(108). The order in which these events occur is unknown. To investigate the role Trp(beta)(108) may play in this process, this residue was mutated to both a His (betaW108H) and a Cys (betaW108C) residue. For each mutant, the majority of the protein that was isolated was inactive and exhibited weaker subunit-subunit interactions than native MADH. Analysis by mass spectrometry suggested that the inactive protein was a biosynthetic intermediate with only one oxygen atom incorporated into Trp(beta)(57) and no cross-link with residue beta108. However, in each mutant preparation, a small percentage of the mutant enzyme was active and appears to possess a functional tryptophylquinone cofactor. In the case of betaW108C, this cofactor may be identical to cysteine tryptophylquinone, recently described in the bacterial quinohemoprotein amine dehydrogenase. In betaW108H, the active cofactor is presumably a histidine tryptophylquinone, which has not been previously described, and represents the synthesis of a novel quinone protein cofactor.