Genes involved in the biosynthesis of PQQ fromAcinetobacter calcoaceticus

From a gene bank of theAcinetobacter calcoaceticus genome a plasmid was isolated that complements four different classes of PQQ^- mutants. Subclones of this plasmid revealed that the four corresponding PQQ genes are located on a fragment of 5 kilobases. The nucleotide sequence of this 5 kb fragment was determined and by means of Tn5 insertion mutants the reading frames of the PQQ genes could be identified. Three of the PQQ genes code for proteins of M_r 29700 (gene I), M_r 10800 (gene II) and M_r 43600 (gene III) respectively. In the DNA region where gene IV was mapped however the largest possible reading frame encodes for a polypeptide of only 24 amino acids. A possible role for this small polypeptide will be discussed. Finally we show that expression of the four PQQ genes inAcinetobacter lwoffi andEscherichia coli lead to the synthesis of the coenzyme in these organisms.     

Nitrogen assimilation in the facultative methylotroph Hyphomicrobium X

A survey of the possible nitrogen assimilation pathways in Hyphomicrobium X showed that when the nitrogen source was satisfied by ammonium sulphate or methylamine and the supply was in excess, NADPH-dependent glutamate dehydrogenase was used to assimilate nitrogen. When the nitrogen supply was limited the cells expressed high levels of glutamine synthetase and NADH-dependent glutamine:2-oxoglutamate aminotransferase activity whilst the activity of the glutamate dehydrogenase was lower. When nitrate was the N-source, the glutamine synthetase/glutamine oxoglutamate aminotransferase pathway was utilised irrespective of the nitrogen concentration in the medium. Evidence was obtained to suggest that the glutamine synthetase activity was regulated by adenylylation/deadenylylation. Carbon-limited chemostat cultures showed low glutamine synthetase activity levels but the synthesis of the enzyme was derepressed when the cultures became N-limited.     

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.     

L-arginine is a precursor for nitrate biosynthesis in humans

Nitrogen from L-arginine was incorporated into urinary nitrate in human subjects. Two subjects given an oral dose of [15N2]L-arginine excreted 24 and 17 umol [15N]nitrate/24 hr, respectively, in their urine in the 24 hr period following the dose. This work demonstrates that L-arginine, a nitrogen source for biosynthesized nitrate in cultured cells and research animals, is a precursor for endogenously synthesized nitrate in humans.     

Cloning of Klebsiella pneumoniae pqq genes and PQQ biosynthesis in Escherichia coli

We have cloned genes from Klebsiella pneumoniae which are required for pyrroloquinoline quinone (PQQ) biosynthesis. The cloned 6.7 kb fragment can complement several chromosomal pqq mutants. Escherichia coli strains are unable to synthesize PQQ but E. coli strains containing the cloned 6.7 kb K. pneumoniae fragment can synthesize PQQ in large amounts and E. coli pts mutants can be complemented on minimal glucose medium by this clone.     

Obligatory biosynthesis of L-tyrosine via the pretyrosine branchlet in coryneform bacteria.

Species of coryneform bacteria (Corynebacterium glutamicum, Brevibacterium flavum, and B. ammoniagenes) utilize pretyrosine [beta-(1-carboxy-4-hydroxy-2,5-cyclohexadien-1-yl) alanine] as an intermediate in L-tyrosine biosynthesis. Pretyrosine is formed from prephenate via the activity of at least one species of aromatic aminotransferase which is significantly greater with prephenate as substrate than with either phenylpyruvate or 4-hydroxyphenylpyruvate. Pretyrosine dehydrogenase, capable of converting pretyrosine to L-tyrosine, has been partially purified from all three species. Each of the three pretyrosine dehydrogenases is catalytically active with either nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate as cofactors. The Km values for nicotinamide adenine dinucleotide phosphate in C. glutamicum and B. flavum are 55 microM and 14.2 microM, respectively, and corresponding Km values for nicotinamide adenine dinucleotide are 350 microM and 625 microM, respectively. The molecular weights of pretyrosine dehydrogenase in C. glutamicum and in B. flavum are both about 158,000, compared with 68,000 moleculr weitht in B. ammoniagenes. In all three species the enzyme is not feedback inhibited by L-tyrosine. Results obtained with various auxotropic mutants, which were used to manipulate internal concentrations of L-tyrosine, suggest that pretyrosine dehydrogenase is expressed constitutively. Pretyrosine dehydrogenase is quite sensitive to p-hydroxymercuribenzoic acid, complete inhibition being achieved at 10 to 25 microM concentrations. This inhibition is readily reversed by thiol reagents such as 2-mercaptoethanol. Coryneform organisms, like species of blue-green bacteria, appear to lack the 4-hydroxyphenylpyruvate pa thway of L-tyrosine synthesis altogether. The loss of pretyrosine dehydrogenase in extracts prepared from a tyrosine auxotroph affirms the exclusive role of pretyrosine dehydrogenase in L-tyrosine biosynthesis. Other reports in the literature, in which the presence in these organisms of prephenate dehydrogenase is described, appear to be erroneous.     

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.     

Identification of the prosthetic groups of dimethylamine dehydrogenase from Hyphomicrobium X.

Trimethylamine dehydrogenase (TMADH) and dimethylamine dehydrogenase (DMADH) were purified from Hyphomicrobium X. The absorbance spectra of the two enzymes were similar with λmax = 443 nm for TMADH and 440 nm for DMADH. DMADH had an apparent molecular weight of 138,000 daltons and was composed of two subunits of similar molecular weights. DMADH contained 3.91 atoms S and 4.55 atoms Fe per mole of the enzyme. Both DMADH and TMADH contained a covalently bound yellow coenzyme. The coenzyme-peptides obtained from DMADH and TMADH of Hyphomicrobium X by tryptic-chymotryptic digestion were partially purified and found to differ electrophoretically and chromatographically from the coenzyme-peptide obtained similarly from TMADH of bacterium W₃A₁. After digestion with aminopeptidase M the aminoacyl-coenzymes from the three enzymes had identical spectral, electrophoretic and chromatographic properties. DMADH is only the second enzyme yet found to contain 6-S-cysteinyl-FMN as coenzyme. Dissimilarities between the coenzyme-peptides of DMADH and TMADH from either Hyphomicrobium X or bacterium W₃A₁ are consequently located in the peptide component.     

Proteolytic precursor processing in the biosynthesis of mitochondria

Essentially all polypeptides synthesized in the cytoplasm and imported into either the matrix or into the inner or outer membrane of mitochondria are made as larger molecular weight precursors. All known examples of in vivo or in vitro synthesized precursors are summarized. Little information on the nature of the proteolytic enzymes involved in the processing of the larger precursor polypeptides exists. The biosynthesis of rat liver cytochrome c oxidase is discussed in detail. In contrast to reported data, the cytoplasmic subunits of rat liver cytochrome c oxidase are synthesized as larger molecular weight precursors and not as a polyprotein. Precursors to subunits IV and V show an extra-peptide sequence of about 3000 daltons. Evidence against the existence of a polyprotein precursor was also obtained, when messenger RNAs for the individual subunits IV and V were isolated and analyzed in respect to their size. A length of 990 +/- 80 and 830 +/- 70 nucleotides was estimated for the poly(A)+-RNA of cytochrome c oxidase subunits IV and V, respectively. In experiments on the site of synthesis, it was found that cytochrome c oxidase subunits IV and V are made on free, loosely and tightly membrane-bound polyribosomes.     

NAD-linked, GSH- and factor-independent aldehyde dehydrogenase of the methylotrophic bacterium, Hyphomicrobium X

Cell-free extracts of Hyphomicrobium X showed NAD-dependent aldehyde dehydrogenase activity, provided that NAD addition preceded that of aldehyde. Activity was lost rather rapidly, especially during purification attempts, but this could be partially masked by including a time-dependent restoration step with thiol compounds in the protocol. The nature of the assay buffer appeared to be critical and stimulation occurred on incorporation of K+ ions in the mixture. An even higher specific activity could be achieved by 1,4-dithiothreitol (DTT) treatment of the preparation, followed by removal of DTT, and assaying in the absence of thiol compounds under anaerobic conditions. Exposure of such a preparation to O2 led to a significant decrease in activity within a couple of hours. Immediate inactivation occurred on addition of H2O2, but this could be prevented completely by prior addition of NAD. Since GSH does not participate in the reaction and no stimulating factor was detected, the role of thiol compounds is most probably confined to restoration or prevention of damage to an O2-sensitive, necessary thiol group. Since the same features were found for cell-free extract as for the partially purified enzyme, only one enzyme type seems to be present. Although the enzyme is a general aldehyde dehydrogenase, the kinetic parameters and the specific activity of the cell-free extract for formaldehyde indicate that it may play a role in formaldehyde dissimilation by Hyphomicrobium X. The NAD-linked, GSH- and factor-independent aldehyde dehydrogenase described here appears to be different in several respects from the formaldehyde dehydrogenase of Pseudomonas putida (EC 1.2.1.46) (despite showing similar behavior toward coenzymes and factors) but resembles the aldehyde dehydrogenase from baker's yeast (EC 1.2.1.5).     

Identification of Pantoea ananatis gene encoding membrane pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase and pqqABCDEF operon essential for PQQ biosynthesis

Pantoea ananatis accumulates gluconate during aerobic growth in the presence of glucose. Computer analysis of the P. ananatis SC17(0) sequenced genome revealed an ORF encoding a homologue (named gcd) of the mGDH (EC 1.1.99.17) apoenzyme from Escherichia coli and a putative pyrroloquinoline quinone (PQQ) biosynthetic operon homologous to pqqABCDEF from Klebsiella pneumoniae. Construction of Δgcd and Δpqq mutants of P. ananatis confirmed the proposed functions of these genetic elements. The P. ananatis pqqABCDEF was cloned in vivo and integrated into the chromosomes of P. ananatis and E. coli according to the Dual In/Out strategy. Introduction of a second copy of pqqABCDEF to P. ananatis SC17(0) doubled the accumulation of PQQ. Integration of the operon into E. coli MG1655ΔptsGΔmanXY restored the growth of bacteria on glucose. The obtained data show the essential role of pqqABCDEF in PQQ biosynthesis in P. ananatis and E. coli. We propose that the cloned operon could be useful for an efficient phosphoenolpyruvate-independent glucose consumption pathway due to glucose oxidation and construction of E. coli strains with the advantage of phosphoenolpyruvate-derived metabolite production.     

A heme peroxidase with a functional role as an L-tyrosine hydroxylase in the biosynthesis of anthramycin

We report the first characterization and classification of Orf13 (S. refuineus) as a heme-dependent peroxidase catalyzing the ortho-hydroxylation of L-tyrosine to L-DOPA. The putative tyrosine hydroxylase coded by orf13 of the anthramycin biosynthesis gene cluster has been expressed and purified. Heme b has been identified as the required cofactor for catalysis, and maximal L-tyrosine conversion to L-DOPA is observed in the presence of hydrogen peroxide. Preincubation of L-tyrosine with Orf13 prior to the addition of hydrogen peroxide is required for L-DOPA production. However, the enzyme becomes inactivated by hydrogen peroxide during catalysis. Steady-state kinetic analysis of L-tyrosine hydroxylation revealed similar catalytic efficiency for both L-tyrosine and hydrogen peroxide. Spectroscopic data from a reduced-CO(g) UV-vis spectrum of Orf13 and electron paramagnetic resonance of ferric heme Orf13 are consistent with heme peroxidases that have a histidyl-ligated heme iron. Contrary to the classical heme peroxidase oxidation reaction with hydrogen peroxide that produces coupled aromatic products such as o,o'-dityrosine, Orf13 is novel in its ability to catalyze aromatic amino acid hydroxylation with hydrogen peroxide, in the substrate addition order and for its substrate specificity for L-tyrosine. Peroxygenase activity of Orf13 for the ortho-hydroxylation of L-tyrosine to L-DOPA by a molecular oxygen dependent pathway in the presence of dihydroxyfumaric acid is also observed. This reaction behavior is consistent with peroxygenase activity reported with horseradish peroxidase for the hydroxylation of phenol. Overall, the putative function of Orf13 as a tyrosine hydroxylase has been confirmed and establishes the first bacterial class of tyrosine hydroxylases.     

Malonate as a precursor in the biosynthesis of aflatoxins.

Incorporation of [I-14C]acetate and [2-14C]malonate into aflatoxins by resting mycelia of Aspergillus parasiticus resuspended in different buffers was studied. A decrease in pH from 5-8 to 2-8, as well as addition of EDTA, markedly stimulated the incorporation of malonate but the effect on acetate incorporation was less pronounced. Mycelia took up comparatively more acetate than malonate, but more malonate (4-3%) entering mycelia was incorporated into aflatoxins than was acetate (1-6%). Furthermore, the addition of unlabelled acetate reduced the incorporation of label from [I-14C]acetate by 75% but from [2-14C]malonate by only 25%. These results suggest that malonate is an intermediate in aflatoxin synthesis and that is can be incorporated without prior conversion to acetate.     

Methylglyoxal is an intermediate in the biosynthesis of 6-deoxy-5-ketofructose-1-phosphate: a precursor for aromatic amino acid biosynthesis in Methanocaldococcus jannaschii

A biosynthetic pathway is proposed for creating 6-deoxy-5-ketofructose-1-phosphate (DKFP), a precursor sugar for aromatic amino acid biosynthesis in Methanocaldococcus jannaschii. First, two possible routes were investigated to determine if a modified, established biosynthetic pathway could be responsible for generating 6-deoxyhexoses in M. jannaschii. Both the nucleoside diphosphate mannose pathway and a pathway involving nucleoside diphosphate derivatives of fructose-1-P, fructose-2-P, or fructose-1,6-bisP were tested and eliminated. The established pathways did not produce the expected intermediates nor did the anticipated enzymes have the predicted enzymatic activities. Because neither anticipated pathway could produce DKFP, M. jannaschii glucose-6-P metabolism was studied in detail to establish exactly how glucose-6-P is converted into DKFP. This detailed analysis showed that methylglyoxal and a fructose-1-P- or fructose-1,6-bisP-derived dihydroxyacetone-P fragment are key intermediates in DKFP production. Glucose-6-P readily converts to fructose-6-P, which in turn converts to fructose-1,6-bisP. Fructose-6-P and fructose-1,6-bisP convert into glyceraldehyde-3-P (Ga-P-3), which converts into methylglyoxal by a 2,3-elimination of phosphate. The MJ1585-derived enzyme catalyzes the condensation of methylglyoxal with a dihydroxyacetone-P fragment, which is derived from fructose-1-P and/or fructose-1,6-bisP, generating DKFP. The elimination of phosphate from Ga-P-3 proceeds by both enzymatic and chemical routes in cell extracts, producing sufficient concentrations of methylglyoxal to support the reaction. This work is the first report of methylglyoxal functioning in central metabolism.     

The non-mevalonate pathway of isoprenoid precursor biosynthesis

The recently discovered non-mevalonate biosynthetic route to isoprenoid precursors is an essential metabolic pathway in plants, apicomplexan parasites, and many species of bacteria. The pathway relies on eight enzymes exploiting different cofactors and metal ions. Structural and mechanistic data now exist for most components of the pathway though there remain some gaps in our knowledge. The individual enzymes represent new, validated targets for broad spectrum antimicrobial drug and herbicide development. Detailed knowledge of the pathway may also be exploited to genetically modify microorganisms and plants to produce compounds of agricultural and medical interest.