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.

A search for intermediates in the bacterial biosynthesis of PQQ.

Studies on the biosynthesis of pyrroloquinoline quinone (PQQ) were performed with Acinetobacter calcoaceticus PQQ- -mutants belonging to genetically different complementation groups. All mutants were unable to grow on L-arabinose, the conversion of this substrate by the organism only occurring via membrane-bound quinoprotein (PQQ-containing) glucose dehydrogenase. In general, the same observation and conclusion applied to shikimate and quinate, requiring active quinoprotein quinate dehydrogenase (EC 1.1.99.--), although some mutants appeared to be leaky with respect to PQQ biosynthesis under this condition. A number of mutants were unable to grow on anthranilate and accumulated this compound when the growth medium was supplemented with L-kynurenine. Combined with other observations, it strongly suggests that these are deletion mutants, missing a gene for synthesis of anthranilate hydroxylase (EC 1.14.12.1) as well as nearby located genes for the biosynthesis of PQQ. Supplementation of the growth media with amino acids did not result in stimulation of PQQ biosynthesis. Also cross-feeding experiments, using normal and permeabilized cells with extensive variation in combination and conditions, resulted in neither stimulation nor reconstitution of PQQ synthesis. Under conditions optimal for PQQ production in the wild-type strain, as well as under stress conditions using a limiting amount of added cofactor, excretion of intermediates by PQQ- -mutants could not be detected. Similar results were obtained with PQQ- -mutants from Methylobacterium organophilum and Pseudomonas aureofaciens. A tentative explanation, accounting for the absence of detectable intermediates in the biosynthetic route, is given.

Bacterial NAD(P)-independent quinate dehydrogenase is a quinoprotein.

Acinetobacter calcoaceticus LMD 79.41 produced significant amounts of pyrrolo-quinoline quinone (PQQ) in its culture medium when grown on quinic acid or shikimic acid. Studies with LMD 79.41 and PQQ- -mutants of this strain demonstrated that this organism contains an NAD(P)-independent quinate dehydrogenase (QDH) (EC 1.1.99.-), catalyzing the first degradation step of these compounds, and that the enzyme contains PQQ as a cofactor, i.e. is a quinoprotein. Synthesis of QDH was induced by protocatechuate and the enzyme appeared to be particle-bound. Acinetobacter Iwoffi RAG-1 produced a quinoprotein QDH apoenzyme since growth on quinic acid only occurred in the presence of PQQ. The results obtained with the PQQ- -mutants of strain LMD 79.41 also provided some insight into the regulation of PQQ biosynthesis and assemblage of quinoprotein enzymes in the periplasmic space. Since two species of Pseudomonas also contained a quinoprotein QDH, it is assumed that bacterial NAD(P)-independent quinate dehydrogenase is a quinoprotein.

Factors relevant in bacterial pyrroloquinoline quinone production.

Quinoprotein content and levels of external pyrroloquinoline quinone (PQQ) were determined for several bacteria under a variety of growth conditions. From these data and those from the literature, a number of factors can be indicated which are relevant for PQQ production. Synthesis of PQQ is only started if synthesis of a quinoprotein occurs, but quinoprotein synthesis does not depend on PQQ synthesis. The presence of quinoprotein substrates is not necessary for quinoprotein and PQQ syntheses. Although the extent of PQQ production was determined by the type of organism and quinoprotein produced, coordination between quinoprotein and PQQ syntheses is loose, since underproduction and overproduction of PQQ with respect to quinoprotein were observed. The results can be interpreted to indicate that quinoprotein synthesis depends on the growth rate whereas PQQ synthesis does not. In that view, the highest PQQ production can be achieved under limiting growth conditions, as was shown indeed by the much higher levels of PQQ produced in fed-batch cultures compared with those produced in batch experiments. The presence of nucleophiles, especially amino acids, in culture media may cause losses of PQQ due to transformation into biologically inactive compounds. Some organisms continued to synthesize PQQ de novo when this cofactor was administered exogenously. Most probably PQQ cannot be taken up by either passive diffusion or active transport mechanisms and is therefore not able to exert feedback regulation on its biosynthesis in these organisms.

Cytochrome b-562 from Acinetobacter calcoaceticus L.M.D. 79.41. Its characteristics and role as electron acceptor for quinoprotein glucose dehydrogenase.

A soluble cytochrome b was purified from Acinetobacter calcoaceticus L.M.D. 79.41. On the basis of the alpha-band maximum of a reduced preparation, measured at 25 degrees C, it is designated as cytochrome b-562. This cytochrome is a basic monomeric protein (pI 10.2; Mr 18,000), containing one protohaem group per molecule. The reduced form, at 25 degrees C, showed absorption bands at 428, 532 and 562 nm. At 77 K the alpha-band shifted to 560 nm (with a shoulder at 558 nm). The reduced cytochrome did not react with CO. Cytochrome b-562 is most probably (loosely) attached to the outside of the cytoplasmic membrane, since substantial amounts of it, equimolar to quinoprotein glucose dehydrogenase (GDH), were present in the culture medium when cells were grown in the presence of low concentrations of Triton X-100. The midpoint potential at pH 7.0 was found to be +170 mV, a value that was lowered to +145 mV by the presence of GDH. Since the GDH was shown to have a midpoint potential of +50 mV, cytochrome b-562 could function as the natural primary electron acceptor. Arguments to substantiate this view and to propose a role of ubiquinone-9 as electron acceptor for cytochrome b-562 are presented.

Factors relevant in the reaction of pyrroloquinoline quinone with amino acids. Analytical and mechanistic implications.

In order to reveal the stability of pyrroloquinoline quinone (PQQ) in complex samples, its reaction on incubation with amino acids was followed spectrophotometrically by monitoring oxygen consumption, and with a biological assay. For several alpha-amino acids, the formation of a yellow coloured compound (lambda max = 420 nm) was accompanied by oxygen uptake and disappearance of biological activity from the reaction mixture. The yellow product appeared to be an oxazole of PQQ, the exact structure depending on the amino acid used. Oxazole formation also occurred under anaerobic conditions with concomitant formation of PQQH2, suggesting that PQQ is able to oxidize the presumed oxazoline to the oxazole. Besides the condensation reaction, there is also a catalytic cycle in which an aldimine adduct of PQQ and the amino acid is converted into the aminophenol form of the cofactor and an aldehyde resulting from oxidative decarboxylation of the amino acid. Addition of NH4+ salts, as well as that of certain divalent cations, greatly stimulated both the cyclic and the linear reaction. With basic amino acids, oxazole formation scarcely occurred. However, as oxygen consumption was observed (provided that certain divalent cations were present), conversion of these compounds took place. A reaction scheme is proposed accounting for the products formed and the effects observed. Since NH4+ ions activate several quinoproteins (PQQ-containing enzymes) and divalent cations (Ca2+, Fe2+, and Cu2+) are additional (co)factors in certain metallo quinoproteins, the effects of metal ions observed here could be related to the mechanistic features of these enzymes. Although all oxazoles were converted to PQQ by acid hydrolysis, PQQ was not detected when hydrolysis was carried out in the presence of tryptophan, a compound which appeared to have a deleterious effect on the cofactor under this condition. The results here described explain why analysis methods for free PQQ in complex samples fail in certain cases, or are not quantitative.

Quinohaemoprotein alcohol dehydrogenase apoenzyme from Pseudomonas testosteroni.

Cell-free extracts of Pseudomonas testosteroni, grown on alcohols, contain quinoprotein alcohol dehydrogenase apoenzyme, as was demonstrated by the detection of dye-linked alcohol dehydrogenase activity after the addition of PQQ (pyrroloquinoline quinone). The apoenzyme was purified to homogeneity, and the holoenzyme was characterized. Primary alcohols (except methanol), secondary alcohols and aldehydes were substrates, and a broad range of dyes functioned as artificial electron acceptor. Optimal activity was observed at pH 7.7, and the presence of Ca2+ in the assay appeared to be essential for activity. The apoenzyme was found to be a monomer (Mr 67,000 +/- 5000), with an absorption spectrum similar to that of oxidized cytochrome c. After reconstitution to the holoenzyme by the addition of PQQ, addition of substrate changed the absorption spectrum to that of reduced cytochrome c, indicating that the haem c group participated in the enzymic mechanism. The enzyme contained one haem c group, and full reconstitution was achieved with 1 mol of PQQ/mol. In view of the aberrant properties, it is proposed to distinguish the enzyme from the common quinoprotein alcohol dehydrogenases by using the name 'quinohaemoprotein alcohol dehydrogenase'. Incorporation of PQQ into the growth medium resulted in a significant shortening of lag time and increase in growth rate. Therefore PQQ appears to be a vitamin for this organism during growth on alcohols, reconstituting the apoenzyme to a functional holoenzyme.

Detection of the cofactor pyrroloquinoline quinone.

In order to demonstrate the presence or absence of a pyrroloquinoline quinone (PQQ) synthesizing capacity in microorganisms, we have found that media and equipment must be treated to remove contaminating PQQ. Procedures are described which appear to be effective for that purpose. These have been used with Acinetobacter calcoaceticus PQQ- strains to develop a sensitive and reliable assay for PQQ. They also have been used to show that under our conditions of growth Escherichia coli does not synthesize PQQ. Fluorescence spectroscopy is not selective enough to detect PQQ in a protein hydrolysate due to background fluorescence in the same spectral regions as PQQ. In addition, PQQ reacts with amino acids to give products that cannot be detected by either fluorescence spectroscopy or biological assay. In this regard, claims that several materials originating from plants or animals contain PQQ should be reexamined. Moreover, PQQ cannot be detected with these methods in hydrolysates of enzymes containing covalently bound PQQ.

PQQ: biosynthetic studies in Methylobacterium AM1 and Hyphomicrobium X using specific 13C labeling and NMR.

Using 13C labeling and NMR spectroscopy we have determined biosynthetic precursors of pyrroloquinoline quinone (PQQ) in two closely related serine-type methylotrophs, Methylobacterium AM1 and Hyphomicrobium X. Analysis of the 13C-labeling data revealed that PQQ is constructed from two amino acids: the portion containing N-6,C-7, 8, 9 and the two carboxylic acid groups, C-7' and 9', is derived-intact -from glutamate. The remaining portion is derived from tyrosine; the phenol side chain provides the six carbons of the ring containing the orthoquinone, whereas internal cyclization of the amino acid backbone forms the pyrrole-2-carboxylic acid moiety. This is analogous to the cyclization of dopaquinone to form dopachrome. Dopaquinone is a product of the oxidation of tyrosine (via dopa) in reactions catalyzed by monophenol monooxygenase (EC 1.14.18.1). Starting with tyrosine and glutamate, we will discuss possible biosynthetic routes to PQQ.

Cloning, sequencing and expression studies of the genes encoding amicyanin and the beta-subunit of methylamine dehydrogenase from Thiobacillus versutus.

The genes encoding amicyanin and the beta-subunit of methylamine dehydrogenase (MADH) from Thiobacillus versutus have been cloned and sequenced. The organization of these genes makes it likely that they are coordinately expressed and it supports earlier findings that the blue copper protein amicyanin is involved in electron transport from methylamine to oxygen. The amino acid sequence deduced from the nucleotide sequence of the amicyanin-encoding gene is in agreement with the published protein sequence. The gene codes for a pre-protein with a 25-amino-acid-long signal peptide. The amicyanin gene could be expressed efficiently in Escherichia coli. The protein was extracted with the periplasmic fraction, indicating that pre-amicyanin is translocated across the inner membrane of E. coli. Sequence studies on the purified beta-subunit of MADH confirm the amino acid sequence deduced from the nucleotide sequence of the corresponding gene. The latter codes for a pre-protein with an unusually long (56 amino acids) leader peptide. The sequencing results strongly suggest that pyrroloquinoline quinone (PQQ) or pro-PQQ is not the co-factor of MADH.