Purification and properties of methanol dehydrogenase from Hyphomicrobium x.

(1) A method for the isolation of methanol dehydrogenase (alcohol:(acceptor) oxidoreductase, EC 1.1.99.8) from Hyphomicrobium X is decribed. The purified enzyme was resolved by polyacrylamide gel electrophoresis into one main and two minor active bands. Iron and manganese were the only detected metals in the enzyme preparation. (2) The substrate, methanol, was oxidized to formic acid by a stoichiometric amount of artificial electron acceptor. During the reaction, no free formaldehyde could be detected. Other primary alcohols were oxidized to the corresponding aldehydes were a poor substrate or no substrate at all. (3) Some new and efficient one-electron acceptors were found. With these electron acceptors, the enzyme had a high pH optimum and ammonia was still required in the assay system. (4) ESR spectroscopy showed the presence of an enzyme-bound organic free radical. With X-band ESR the signal had a peak-to-peak linewidth of about 0.7 mT. The signal was further resolved by Q-band ESR and the values gparallel = 2.0024 and gperpendicular = 2.0056 were derived. (5) Under denaturing conditions the ESR signal and enzymatic activity disappeared at the same time as fluorescence appeared. Enzymatic activity is not restored when extracted cofactor and apoenzyme are brought together under normal conditions. Some properties of the unusual prosthetic group are presented in a preliminary form.

The prosthetic group of methylamine dehydrogenase from Pseudomonas AM1: evidence for a quinone structure.

The g-value and linewidth of ESR spectra of methylamine dehydrogenase (primary-amine:(acceptor) oxidoreductase (deaminating) EC 1.4.99.-) and methanol dehydrogenase (alcohol:(acceptor) oxidoreductase, EC 1.1.99.8) are very similar. This similarity is also reflected in electron-nuclear double resonance (ENDOR) results, the coupling constants of two protons in one enzyme equalling those in the other. The presence of a third proton in the ENDOR spectrum of methylamine dehydrogenase suggests a different structure or a different kind of interaction which can be related to the finding that the resolved ROSTHETIC GROUP IS PROTEIN-BOUND. The bound prosthetic group has a high redox-potential, supporting the conclusion from the ESR and ENDOR results that it is a quinone derivative.

Crystallization and preliminary crystallographic investigations of the soluble glucose dehydrogenase from Acinetobacter calcoaceticus.

Single crystals of the soluble glucose dehydrogenase (GDH) from Acinetobacter calcoaceticus have been grown by the vapour diffusion method. These crystals diffract to beyond 2.1 A and are suitable for X-ray crystallography. The space group was determined to be P2(1) with unit cell parameters a = 55.5 A, b = 104.5 A, c = 86.5 A and beta = 99.8 degrees. One asymmetric unit contains a dimer of the GDH molecule.

The 1.7 A crystal structure of the apo form of the soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus reveals a novel internal conserved sequence repeat.

The crystal structure of a dimeric apo form of the soluble quinoprotein glucose dehydrogenase (s-GDH) from Acinetobacter calcoaceticus has been solved by multiple isomorphous replacement followed by density modification, and was subsequently refined at 1. 72 A resolution to a final crystallographic R-factor of 16.5% and free R-factor of 20.8% [corrected]. The s-GDH monomer has a beta-propeller fold consisting of six four-stranded anti-parallel beta-sheets aligned around a pseudo 6-fold symmetry axis. The enzyme binds three calcium ions per monomer, two of which are located in the dimer interface. The third is bound in the putative active site, where it may bind and functionalize the pyrroloquinoline quinone (PQQ) cofactor. A data base search unexpectedly showed that four uncharacterized protein sequences are homologous to s-GDH with many residues in the putative active site absolutely conserved. This indicates that these homologs may have a similar structure and that they may catalyze similar PQQ-dependent reactions.A structure-based sequence alignment of the six four-stranded beta-sheets in s-GDH's beta-propeller fold shows an internally conserved sequence repeat that gives rise to two distinct conserved structural motifs. The first structural motif is found at the corner of the short beta-turn between the inner two beta-strands of the beta-sheets, where an Asp side-chain points back into the beta-sheet to form a hydrogen-bond with the OH/NH of a Tyr/Trp side-chain in the same beta-sheet. The second motif involves an Arg/Lys side-chain in the C beta-strand of one beta-sheet, which forms a bidentate salt-bridge with an Asp/Glu in the CD loop of the next beta-sheet. These intra and inter-beta-sheet hydrogen-bonds are likely to contribute to the stability of the s-GDH beta-propeller fold.

L-tyrosine is the precursor of PQQ biosynthesis in Hyphomicrobium X.

A method was developed to study amino acids as possible precursors of PQQ biosynthesis. Cultures of Hyphomicrobium X, growing on [13C]methanol, were supplemented with unlabelled amino acids. Uptake and participation in metabolism were determined via gas chromatography/mass spectrometry of derivatized amino acids, obtained from hydrolyzed cellular protein, by measuring their 12C content. Several amino acids appeared to be incorporated into the protein to a significant extent, without degradation or conversion. Among these were the aromatic amino acids, L-tyrosine and L-phenylalanine. Using the same replacement approach, their incorporation into PQQ was determined by 1H- and 13C-NMR spectroscopy of purified PQQ obtained from the culture medium. It appeared that the complete carbon skeleton of tyrosine was present, forming the o-quinone and pyrrole-2-carboxylic acid moieties in PQQ, while phenylalanine was not incorporated at all. Starting with L-tyrosine, possible biosynthetic routes to PQQ are discussed.

NAD-dependent, PQQ-containing methanol dehydrogenase: a bacterial dehydrogenase in a multienzyme complex.

Cell-free extracts of methanol-grown Nocardia sp. 239 only show significant dye-linked methanol-oxidizing activity when NAD+ is added to the assay mixture. This activity resides in a multienzyme complex which could be resolved into 3 components, namely the methanol dehydrogenase, NAD-dependent aldehyde dehydrogenase and NADH dehydrogenase. In its dissociated form, the methanol dehydrogenase no longer shows dye reduction and although rises in the absorbance values around 340 nm are seen on addition of methanol plus NAD+ to the enzyme, this is not due to NADH production. However, dye reduction (NAD dependent) could be restored on incubating methanol dehydrogenase with the corresponding NADH dehydrogenase, obtained from the enzyme complex. It is concluded that this novel methanol dehydrogenase transfers the reducing equivalents, derived from methanol, directly to its associated NADH dehydrogenase via a mechanism in which NAD+ and PQQ are involved.

Phenylhydrazine as probe for cofactor identification in amine oxidoreductases. Evidence for PQQ as the cofactor in methylamine dehydrogenase.

Homogeneous methylamine dehydrogenase (primary-amine:(acceptor) oxidoreductase (deaminating), EC 1.4.99.3, MADH) from the bacterium Thiobacillus versutus was treated with the inhibitor phenylhydrazine (PH). Derivatization of the cofactor in MADH took place in a fast reaction to give compound I. A different product, compound II, was formed in a slow reaction at high O2 concentrations. The compounds I and II could be removed from the protein by proteolysis with pronase and purified to homogeneity. Products showing identical absorption spectra and chromatographic behaviour were isolated from the reaction mixture after incubating pyrroloquinoline quinone (PQQ) with PH. Upon dissolving in dimethyl sulphoxide, both the enzyme-derived as well as the model-system-derived compounds I and II were nearly quantitatively transformed into PQQ. The conclusion is, therefore, that MADH from T. versutus contains covalently bound PQQ, removable from the protein with pronase, and not a structural analogue of this cofactor without the carboxylic acid groups, as was recently proposed for MADH from Bacterium W3A1 [(1986) Biochem. Biophys. Res. Commun. 141, 562-568]. The properties of compounds I and II suggest that they are the 'azo adduct' and the 'hydrazone adduct' of PH and PQQ at the C(5)-position, respectively. The finding that the reaction of a hydrazine with PQQ can lead to two different products, in enzymes as well as in a model system, has important implications for the interpretation of recent comparative studies aimed at detection of PQQ in amine oxidoreductases with Raman spectroscopy.

Evidence for PQQ as cofactor in 3,4-dihydroxyphenylalanine (dopa) decarboxylase of pig kidney.

Pig kidney 3,4-dihydroxyphenylalanine (dopa) decarboxylase (EC 4.1.1.28) was purified to homogeneity. Treatment of the enzyme with phenylhydrazine (PH) according to a procedure developed for analysis of quinoproteins gave products which were identified as the hydrazone of pyridoxal phosphate (PLP) and the C(5)-hydrazone of pyrroloquinoline quinone (PQQ). This method failed, however, in quantifying the amounts of cofactor. Direct hydrolysis of the enzyme by refluxing with hexanol and concentrated HCl led to detachment of PQQ from the protein in a quantity of 1 PQQ per enzyme molecule. In view of the reactivity of PQQ towards amines and amino acids, we postulate that it participates as a covalently bound cofactor in the catalytic cycle of the enzyme, in interplay with PLP. Since several other enzymes have been reported to show the atypical behaviour of dopa decarboxylase, it seems that the PLP-containing group of enzymes can be subdivided into pyridoxoproteins and pyridoxo-quinoproteins.

On the biosynthesis of free and covalently bound PQQ. Glutamic acid decarboxylase from Escherichia coli is a pyridoxo-quinoprotein.

Analysis of glutamic acid decarboxylase (GDC) (EC 4.1.1.15) from Escherichia coli ATCC 11246 revealed the presence of six pyridoxal phosphates (PLPs) as well as six covalently bound pyrroloquinoline quinones (PQQs) per hexameric enzyme molecule. This is the second example of a pyridoxo-quinoprotein, suggesting that other atypical pyridoxoproteins (PLP-containing enzymes) have similar cofactor composition. Since the organism did not produce free PQQ and its quinoprotein glucose dehydrogenase was present in the apo form, free PQQ is not used in the assemblage of GDC. Most probably, biosynthesis of covalently bound cofactor occurs in situ via a route which is different from that of free PQQ. Thus, organisms previously believed to be unable to synthesize (free) PQQ could in fact be able to produce quinoproteins with covalently bound cofactor. Implications for the role of PQQ in eukaryotic cells are discussed.

Dopamine beta-hydroxylase from bovine adrenal medulla contains covalently-bound pyrroloquinoline quinone.

Treatment of homogeneous dopamine beta-hydroxylase (DBH) preparations from bovine adrenals with the inhibitor phenylhydrazine (PH) changed the structureless absorption spectrum of DBH into spectra with a maximum at 350 nm. A product with this absorption spectrum could be detached with pronase, enabling its isolation. It appeared to be the C(5) hydrazone of pyrroloquinoline quinone (PQQ) and PH, as judged from its properties and the fact that it could be transformed into PQQ itself. From the yield obtained a ratio of 0.85 PQQ per enzyme subunit was calculated. In contrast to copper-quinoprotein amine oxidases (EC 1.4.3.6), hydrazone formation in DBH did not require saturation of the mixture with O2. DBH is the first copper-quinoprotein hydroxylase found so far. The implications of this finding for the current views on mechanism of action and inhibition by hydrazines are discussed. The success of the recently developed 'hydrazine method' [(1987) FEBS Lett. 221, 299-304] for all different types of amine oxidoreductases, suggest that the method could also be applied to other enzymes for which hydrazines are inhibitors and where the identity of the cofactors has not been established or the presence of PQQ is suspected.

N-terminal heterogeneity of methylamine dehydrogenase from Thiobacillus versutus.

The N-terminal processing of MADH from the bacterium T. versutus and the N-terminal heterogeneity of the isolated alpha subunit of the alpha 2 beta 2 protein complex was demonstrated by a combination of Edman sequence analysis of an electroblotted band, in situ digested with pyroglutamate aminopeptidase, and accurate mass determination of the homogeneous subunit by the technique of electrospray ionisation mass spectrometry. From this study, it appears that the corresponding gene of the alpha subunit contains 395 amino acids and that it is preceded by a leader sequence of 31 residues.

Hydrazone formation of 2,4-dinitrophenylhydrazine with pyrroloquinoline quinone in porcine kidney diamine oxidase.

Homogeneous diamine oxidase (EC 1.4.3.6) from porcine kidney was treated with the inhibitor 2,4-dinitrophenylhydrazine (DNPH). The coloured compounds formed were detached with pronase and purified to homogeneity. When the reaction with DNPH was conducted under an O2 atmosphere, the product (obtained in a yield of 55%) was the C(5)-hydrazone of pyrroloquinoline quinone (PQQ) and DNPH, as revealed by its chromatographic behaviour, absorption spectrum and 1H-NMR spectrum. Only 6% of this hydrazone was formed under air, the main product isolated being an unidentified reaction product of DNPH with the enzyme. Porcine kidney diamine oxidase is the second mammalian enzyme shown to have PQQ as its prosthetic group. In view of the requirements for hydrazone formation with DNPH, it is incorrect to assume that inhibition of this type of enzymes with common hydrazines is simply due to blocking of the carbonyl group of its cofactor.

Mycothiol, 1-O-(2'-[N-acetyl-L-cysteinyl]amido-2'-deoxy-alpha-D-glucopyranosyl)-D- myo-inositol, is the factor of NAD/factor-dependent formaldehyde dehydrogenase.

Two different NAD/coenzyme-dependent formaldehyde dehydrogenases exist, the well-known NAD/GSH-dependent (EC 1.2.1.1) and the more recently discovered NAD/Factor-dependent enzyme. The GSH-dependent one has been found in eukaryotes and Gram-negative bacteria, the Factor-dependent one in two different Gram-positive bacteria. Previous work also showed that Factor and GSH are not interchangeable in the enzymatic reactions. Here it is revealed that the Factor is identical to mycothiol (MySH), 1-O-(2'-[N-acetyl-L-cysteinyl]-amido-2'-deoxy-alpha-D-glucopyranosyl)-D- myo-inositol, a thiol compound which has recently been detected in Actinomycetes. Thus, MySH is GSH's companion as it is the coenzyme for the enzyme which henceforth can be indicated as NAD/MySH-dependent formaldehyde dehydrogenase.

Bovine serum amine oxidase: a mammalian enzyme having covalently bound PQQ as prosthetic group.

In addition to the metal ion, copper-containing amine oxidases possess an organic prosthetic group, the nature of which has long been controversial. We show here that in the case of bovine plasma amine oxidase, this second prosthetic group is covalently bound pyrroloquinoline quinone ( PQQ ). Until now the coenzyme PQQ has been found in several bacterial dehydrogenases. Thus the finding reported here is the first example of a quinoprotein oxidoreductase discovered in a eukaryotic organism.

The role of His117 in the redox reactions of azurin from Pseudomonas aeruginosa.

The electron-transfer properties of H117G- and wild-type azurin were compared by applying both as electron acceptors in the conversion of 4-ethylphenol by 4-ethylphenol methylenehydroxylase (4-EPMH). The reactivity of H117G-azurin was determined in the absence and presence of imidazoles, which can substitute the missing fourth ligand. In the absence of imidazoles, H117G-azurin reacted directly with 4-ethylphenol; this reaction was abolished in the presence of imidazoles. The enzymatic reduction of H117G-azurin by 4-EPMH was 40 times slower than that of wild-type azurin. The rate of this reaction was enhanced by some imidazoles, diminished by others. In all cases the reduction of H117G-azurin was irreversible. These results demonstrate that His117 is vital for electron transfer and effectively protects the copper site against aspecific reactions.

The redox-cycling assay is not suited for the detection of pyrroloquinoline quinone in biological samples.

Based on the results of the so-called redox-cycling assay it has been claimed that various common foods and beverages as well as mammalian body fluids and tissues contain substantial quantities (microM) of free PQQ [M. Paz et al. (1989) in: PQQ and Quinoproteins (J.A. Jongejan and J.A. Duine, eds.) Kluwer Academic Publishers, Dordrecht, pp. 131-143 and J. Killgore et al. (1989) Science 245, 850-852]. However, by investigating samples from such sources with a biological assay of nM sensitivity, we could not confirm these claims. Analysis of the samples with procedures that proved adequate for the detection of PQQ adducts and conjugates gave equally negative results. To account for the positive response in the redox-cycling assay, as opposed to the negative results obtained by other methods, a search was made for those substances in these samples that caused the false-positive reactions. It was found that a number of commonly occurring biochemicals like ascorbic and dehydroascorbic acid, riboflavin and to a lesser extent pyridoxal phosphate, gave a positive response in the redox-cycling assay. The amounts of these interfering substances that were determined in the samples by independent methods could well explain the response. In separate experiments it was found that the effect of PQQ added to biological samples was obscured over an appreciable range of concentrations. For these reasons it must be concluded that the redox-cycling assay is not suited for the detection of PQQ in these samples. Any claims that are based on the results of this method should be disregarded.

Status of the cofactor identity in copper oxidative enzymes.

Much conflicting data have appeared in the literature regarding the nature of the active site structures responsible for catalysis in three classes of copper enzymes: the copper amine oxidases, dopamine beta-monooxygenase and galactose oxidase. Although pyrroloquinoline quinone has been proposed to be the active site cofactor in each instance, new findings indicate this is not the case. Instead, recently available data indicate a spectrum of strategies for substrate activation, which range from direct metal catalysis (dopamine beta-monooxygenase) to the involvement of protein-derived radicals (galactose oxidase) and protein-derived quinones (copper amine oxidases).

Crystallographic investigations of the tryptophan-derived cofactor in the quinoprotein methylamine dehydrogenase.

A model of tryptophan tryptophylquinone (TTQ), recently proposed by McIntire et al. (Science (1991) 252, 817-824) to be the prosthetic group of the quinoprotein methylamine dehydrogenase, has been compared with electron density maps of this dehydrogenase from Thiobacillus versutus and Paracoccus denitrificans. The comparison shows that the TTQ model can be neatly accommodated, providing strong supportive evidence that TTQ is indeed the cofactor for this group of quinoproteins.

A novel combination of prosthetic groups in a fungal laccase; PQQ and two copper atoms.

Extracellular laccase (benzenediol: oxygen oxidoreductase EC 1.10.3.2) from the lignin-degrading fungus, Phlebia radiata, was shown to contain a novel combination of electron carriers as its prosthetic groups. In addition to two copper atoms per enzyme molecule, one molecule of PQQ was included as a cofactor. The EPR spectrum exhibits features of type 1 and type 2 copper atoms. In the enzymatic reaction 4 molecules of lignin model compound, coniferyl alcohol, are oxidized per molecule of oxygen reduced to water. During the reaction coniferyl alcohol is transformed to dilignols.

Preliminary studies on quinoprotein glucose dehydrogenase under extreme conditions of temperature and pressure.

The kinetics of the reduction of the quinoprotein glucose dehydrogenase by substrate were studied as a function of 3 parameters: pressure (1-1000 bar), temperature (down to -25 degrees C) and solvent (water and 40% dimethyl sulfoxide, DMSO) using a high-pressure low-temperature stopped-flow apparatus. A 2-step formation of the reduced enzyme by its substrate (xylose), was observed. A rapid equilibrium described by the constant K1 was followed by a slower process described by the constants k2 and k-2. By using the transition state theory, the thermodynamic quantities delta V (activation volumes) were determined for these various kinetics constants under different experimental conditions. The results are discussed in terms of conformational change and solvation effect on the protein shell, and compared with results obtained for other systems as the 2-step formation of horseradish peroxidase compound I.