Robert A. van der Meer

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 incubatng 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.

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.

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

Treatment of homogeneous dopamine β‐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.

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.

Pyrroloquinoline quinone (PQQ) is the organic cofactor in soybean lipoxygenase-1

Treatment of soybean lipoxygenase‐1 (SLO) with phenylhydrazine (PH) induced inactivation and a maximum (350 nm) in the absorption spectrum of the enzyme. To detach the product having this absorption maximum, proteolysis was required. The product appeared to be the C(5) phenylhydrazone of pyrroloquinoline quinone (PQQ). Quantification showed that one covalently bound PQQ is present per enzyme molecule. Inspection of published spectroscopic data leads to the conclusion that the established cofactor of SLO, Fe, present as one ion per enzyme molecule, interacts with PQQ, the latter functioning as a terdentate ligand for the Fe ion (the N atom and COOH group of the quinoline ring together with the hydrated C(5) carbonyl group). If it is assumed that the substrate has two sites of interaction with this two‐cofactor complex, namely with the Fe ion and the C(5) carbonyl group of PQQ, the electron‐relay system generated very well explains the reported mechanistic features as well as inhibition effects of substrates with extended conjugation systems. The finding of PQQ in SLO is an additional indication that this cofactor is very versatile with respect to involvement in different types of redox reaction. A further implication might be that PQQ has been overlooked in other well‐known enzymes with an established cofactor but where the presence of this cofactor is unable to explain the mechanistic and spectroscopic features.

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.

Determination of PQQ in quinoproteins with covalently bound cofactor and in PQQ‐derivatives

Application of the so‐called hexanol extraction procedure for PQQ determination, originally based on detachment of the cofactor from quinoproteins and conversion into PQQ‐5,5‐dihexyl ketal, leads in several cases to a number of products due to uncontrollable esterification. The present modified procedure, detaching the covalently bound cofactor and converting it into 4‐hydroxy‐5‐hexoxy‐pyrroloquinoline, was tested on a number of proteins. Only the expected product was obtained for the known quinoproteins, in a quantitative yield, as revealed by comparison with the values determined with the hydrazine method. Thus this independent method confirmed that bovine serum amine oxidase, porcine kidney diamine oxidase, dopamine β‐hydroxylase from bovine adrenal medulla, methylamine dehydrogenase from Thiobacillus inversutus, glutamate decarboxylase from Escherichia coli, and 3,4‐dihydroxyphenylalanine decarboxylase from pig kidney are really quinoproteins. Quantitative conversion was also achieved for condensation and addition products of PQQ (PQQ‐acetone, PQQH2, PQQ‐oxazole, PQQ‐dinitrophenylhydrazone, and PQQ‐tryptophan). In view of this conversion and the fact that catalytic activity of PQQ is not required, the method seems suited to investigate the distribution of the cofactor in eukaryotes, especially in mammals where it is almost certain that PQQ occurs only in derivatized form. Finally, just like the hydrazine method, the hexanol extraction procedure seems unable to keep the structure of the cofactor as it exists in the active site, intact, as demonstrated for the pro‐PQQ cofactor of methylamine dehydrogenase.

Primary structure of a pyrroloquinoline quinone (PQQ) containing peptide isolated from porcine kidney diamine oxidase

After treating porcine kidney diamine oxidase (PKDAO, EC 1.4.3.6) with the inhibitor 2,4-dinitrophenylhydrazine (DNPH), the enzyme was subjected to proteolysis with trypsin. The hydrolysate contained a peptide to which the C(5) hydrazone of PQQ and DNPH (PQQ-DNPH) was bound. The peptide was purified to homogeneity after which the amino acid sequence was determined. It appeared to consist of 11 amino acids, with PQQ bound to number eight. Further proteolysis of the peptide with aminopeptidase and carboxypeptidase gave a compound which was identical to a product prepared from coupling of PQQ-DNPH to lysine. Therefore, the cofactor in PKDAO has most probably an amide bond between one of its carboxylic acid groups with the epsilon-NH2 group of a lysine residue. Possibilities for attachment of the cofactor to the protein chain are discussed.

The redox-cycling assay is not suited for the detection of pyrroquinoline 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 (μM) 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 occuring 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.

Identification and Quantification of PQQ

PQQ can occur in free form, as a C5-addition compound, as a condensation product with amino acids (e.g. an oxazole) and in an extractable or covalently bound form. Free, underivatized PQQ can be quantified by several chromatographic methods and biological assays. However, alternative analytical procedures had to be developed for the other cases. The so-called hydrazine method has proven to be a powerful tool in the detection and determination of PQQ in enzymes where it is covalently bound. The conditions established also give some insight into the inhibition mechanism of quinoproteins with hydrazines and allow isolation of peptides to which PQQ is still attached in a non-reactive form. On the other hand, the so-called hexanol extraction procedure is suited to detect PQQ in enzymes for which the hydrazine method fails, and in its condensation products. Since PQQ is very reactive with nucleophilic compounds, the occurrence of free PQQ in complex biological systems is very unlikely so that the latter method could be very useful in studies on the biosynthesis and the possible vitamin role of PQQ in mammals.