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.     

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.     

Regulation by carbon source of enzyme expression and slime production in bacterium W3A1.

Slime production by bacterium W3A1 was greatly enhanced during growth on methanol and, to a lesser extent, during growth on trimethylamine. Of the major dehydrogenases synthesized, trimethylamine and methylamine dehydrogenases were induced to different levels by certain carbon sources, while methanol dehydrogenase was expressed during growth on all carbon sources.     

Steady-state kinetic analysis for the reaction of ammonium and alkylammonium ions with methylamine dehydrogenase from bacterium W3A1

The steady-state kinetic mechanism for the reaction of n-alkylamines and phenazine ethosulfate (PES) or phenazine methosulfate (PMS) with methylamine dehydrogenase from bacterium W3A1 is found to be of the ping-pong type. This conclusion is based on the observations that 1/v versus 1/[methylamine] or 1/[butylamine] plots, at various constant concentrations of an oxidizing substrate, and 1/v versus 1/[PES] or 1/[PMS] plots, at various constant concentrations of a reducing substrate, are parallel. Additionally, the values of kcat/Km for four n-alkylamines are identical when PES is the oxidizing substrate, as were the kcat/Km values for four reoxidizing substrates when methylamine was the reducing substrate. Last, analysis of steady-state kinetic data obtained when methylamine and propylamine are presented to the enzyme simultaneously and PES and PMS are used simultaneously also supports the involvement of a ping-pong mechanism. The enzymic reaction with either methylamine or PES is dependent on the ionic strength, and the data indicate that each interacts with an anionic site on methylamine dehydrogenase. The presence of ammonium ion at low concentration activates the enzyme, but at high concentration this ion is a competitive inhibitor in the reaction involving methylamine and the enzyme. A complete steady-state mechanism describing these ammonia effects is presented and is discussed in light of the nature of the pyrroloquinoline quinone cofactor covalently bound to the enzyme.     

Organization of the methylamine utilization (mau) genes in Methylophilus methylotrophus W3A1-NS.

The organization of genes involved in utilization of methylamine (mau genes) was studied in Methylophilus methylotrophus W3A1. The strain used was a nonmucoid variant termed NS (nonslimy). The original mucoid strain was shown to be identical to the NS strains on the basis of chromosomal digest and hybridization patterns. An 8-kb PstI fragment of the chromosome from M. methylotrophus W3A1-NS encoding the mau genes was cloned and a 6,533-bp region was sequenced. Eight open reading frames were found inside the sequenced area. On the basis of a high level of sequence identity with the Mau polypeptides from Methylobacterium extorquens AM1, the eight open reading frames were identified as mauFBEDAGLM. The mau gene cluster from M. methylotrophus W3A1 is missing two genes, mauC (amicyanin) and mauJ (whose function is unknown), which have been found between mauA and mauG in all studied mau gene clusters. Mau polypeptides sequenced so far from five different bacteria show considerable identity. A mauA mutant of M. methylotrophus W3A1-NS that was constructed lost the ability to grow on all amines as sources of nitrogen but still retained the ability to grow on trimethylamine as a source of carbon. Thus, unlike M. extorquens AM1 and Methylobacillus flagellatum KT, M. methylotrophus W3A1-NS does not have an additional methylamine dehydrogenase system for amine oxidation. Using a promoter-probe vector, we identified a promoter upstream of mauF and used it to construct a potential expression vector, pAYC229.     

Localization of the major dehydrogenases in two methylotrophs by radiochemical labeling.

The localization of prominent proteins in intact cells of two methylotrophic bacteria, Hyphomicrobium sp. strain X and bacterium W3A1, was investigated by radiochemical labeling with [14C]isethionyl acetimidate. In bacterium W3A1, trimethylamine dehydrogenase was not labeled by the reagent and is, therefore, an intracellular protein, whereas the periplasmic location of the methylamine and methanol dehydrogenases was evidenced by being readily labeled in intact cells. Similarly, an intracellular location of the trimethylamine and dimethylamine dehydrogenases in Hyphomicrobium sp. strain X was indicated, whereas methanol dehydrogenase was periplasmic.     

Inhibition by trimethylamine of methylamine oxidation by Paracoccus denitrificans and bacterium W3A1

Trimethylamine, a common substrate for methylotrophic growth, specifically inhibited methylamine-dependent respiration by Paracoccus denitrificans and bacterium W3A1. These effects were caused by the specific inhibition by trimethylamine of the periplasmic quinoprotein methylamine dehydrogenase. Steady-state kinetic analysis of the effect of trimethylamine on methylamine oxidation by methylamine dehydrogenase indicated that the inhibition was a mixed type. Apparent Ki values for trimethylamine of 1.1 mM and 4.7 mM, respectively, were obtained for the P. denitrificans and bacterium W3A1 enzymes. Methylamine-dependent oxygen consumption by each bacterium was inhibited either by preincubation of cells with trimethylamine prior to the addition of substrate or by addition of trimethylamine to actively respiring cells. Formate-dependent respiration was not inhibited by trimethylamine. A scheme is proposed which describes a regulatory role for trimethylamine in the metabolism and dissimilation of methylamine by methylotrophic bacteria.     

Intestinal absorption and tissue distribution of [14C]pyrroloquinoline quinone in mice.

Pyrroloquinoline quinone (PQQ) functions as a cofactor for prokaryotic oxidoreductases, such as methanol dehydrogenase and membrane-bound glucose dehydrogenase. In animals fed chemically defined diets, PQQ improves reproductive outcome and neonatal growth. Consequently, the present study was undertaken to determine the extent to which PQQ is absorbed by the intestine, its tissue distribution, and route of excretion. About 28 micrograms of PQQ (0.42 microCi/mumol), labeled with 14C derived from L-tyrosine, was administered orally to Swiss-Webster mice (18-20 g) to estimate absorption. PQQ was readily absorbed (62%, range 19-89%) in the lower intestine, and was excreted by the kidneys (81% of the absorbed dose) within 24 hr. The only tissues that retained significant amounts of [14C]PQQ at 24 hr were skin and kidney. For kidney, it was assumed that retention of [14C]PQQ represented primarily PQQ destined for excretion. For skin, the concentration of [14C]PQQ increased from 0.3% of the absorbed dose at 6 hr to 1.3% at 24 hr. Furthermore, most of the [14C]PQQ in blood (greater than 95%) was associated with the blood cell fraction, rather than plasma.     

Identification and characterization of the pqqDGC gene cluster involved in pyrroloquinoline quinone production in an obligate methylotroph Methylobacillus flagellatum

Abstract Pyrroloquinoline quinone is a prosthetic group of bacterial methanol dehydrogenases as well as some alcohol and glucose dehydrogenases. Genes involved in pyrroloquinoline quinone production have previously been cloned from the representatives of the α and γ subdivisions of the Proteobacteria. We report identification and the sequence of the pqqDGC gene cluster in the obligate methylotroph, Methylobacillus flagellatum , which belongs to the β subdivision. The deduced products of the pqq genes from M. flagellatum appear to be more similar to their counterparts from non-methylotrophic species of the γ subdivision than to a facultative methylotroph of the a subdivision. A non-polar mutation in pqqG was constructed and resulted in a strain impaired in growth on methanol. This mutant accumulated a detectable amount of intracellular pyrroloquinoline quinone, but in contrast to the wild type, did not excrete pyrroloquinoline quinone into the culture medium. The possible role of PqqG is discussed.     

Apparent oxygen-dependent inhibition by superoxide dismutase of the quinoprotein methanol dehydrogenase.

Methanol dehydrogenase activity, when assayed with phenazine ethosulfate (PES) as an electron acceptor, was inhibited by superoxide dismutase (SOD) and by Mn2+ only under aerobic conditions. Catalase, formate, and other divalent cations did not inhibit the enzyme. The enzyme also exhibited significantly higher levels of activity when assayed with PES under anaerobic conditions relative to aerobic conditions. The oxygen- and superoxide-dependent effects on methanol dehydrogenase were not observed when either Wurster's Blue or cytochrome c-55li was used as an electron acceptor. Another quinoprotein, methylamine dehydrogenase, which possesses tryptophan tryptophylquinone (TTQ) rather than pyrroloquinoline quinone (PQQ) as a prosthetic group, was not inhibited by SOD or Mn2+ when assayed with PES as an electron acceptor. Spectroscopic analysis of methanol dehydrogenase provided no evidence for any oxygen- or superoxide-dependent changes in the redox state of the enzyme-bound PQQ cofactor of methanol dehydrogenase. To explain these data, a model is presented in which this cofactor reacts reversibly with oxygen and superoxide, and in which oxygen is able to compete with PES as an electron acceptor for the reduced species.     

Does the intestinal microflora synthesize pyrroloquinoline quinone?

Pyrroloquinoline quinone (PQQ) functions as a cofactor for prokaryotic oxidoreductases, such as methanol dehydrogenase and glucose dehydrogenase. When chemically-defined diets without PQQ are fed to animals, lathyritic changes are observed. In previous studies, it was assumed that PQQ was produced by the intestinal microflora; consequently, antibiotics were routinely added to diets. In the present study this assumption is tested further in mice by: (i) examining the effects of dietary antibiotics on fecal PQQ excretion, (ii) isolating the intestinal flora to identify bacteria known to synthesize PQQ and (iii) determining in vitro if the intestinal microflora synthesizes PQQ from radio-chemically labeled precursors. The results of these experiments indicate that little if any PQQ is synthesized by the intestinal microflora. Rather, when PQQ is present in the intestine, the diet is a more obvious source.     

依赖PQQ甲醇脱氢酶对底物催化专一性差的原因分析

采用甲基营养杆菌NO .2为实验菌株 ,经超声波破细胞 ,酸处理 ,DEAE 纤维素和CM 纤维素柱层析等改进的纯化程序 ,可得到比活力为 12 .5u/mg的甲醇脱氢酶 (MDH)样品。该酶在测活系统中除能氧化甲醇等醇类化合物外 ,还能以较大速率氧化氯化铵、甲胺、脲等物质 ,MDH对不同底物亲和力的差异性主要取决于其辅基吡咯喹啉醌 (PQQ)与底物的结合力。甲醇脱氢酶与底物结合前后在特定区域的光谱有一定的差异性     

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.     

Complex formation between methylamine dehydrogenase and amicyanin from Paracoccus denitrificans

Two proteins isolated from Paracoccus denitrificans, the copper-containing electron carrier amicyanin and the pyrroloquinoline quinone-containing enzyme methylamine dehydrogenase, have been shown to form a complex. Complex formation between methylamine dehydrogenase and either oxidized or reduced amicyanin resulted in alterations in the absorbance spectrum of the pyrroloquinoline quinone prosthetic group of methylamine dehydrogenase. Binding of amicyanin to the enzyme exhibited positive cooperativity. Complex formation with methylamine dehydrogenase shifted the oxidation-reduction midpoint potential of amicyanin by 73 mV, from +294 to +221 mV, making electron transfer from amicyanin to cytochrome c551 (Em = +190 mV) thermodynamically possible.     

Steady-state kinetic analysis of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans.

A steady-state kinetic analysis was performed of the reaction of methylamine and phenazine ethosulphate (PES) with the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans. Experiments with methylamine and PES as varied-concentration substrates produced a series of parallel reciprocal plots, and when the concentrations of these substrates were varied in a constant ratio a linear reciprocal plot of initial velocity against PES concentration was obtained. Nearly identical values of V/Km of PES were obtained with four different n-alkylamines. These data suggest that this reaction proceeds by a ping-pong type of mechanism. The enzyme reacted with a variety of n-alkylamines but not with secondary, tertiary or aromatic amines or amino acids. The substrate specificity was dictated primarily by the Km value exhibited by the particular amine. A deuterium kinetic isotope effect was observed with deuterated methylamine as a substrate. The enzyme exhibited a pH optimum for V at pH 7.5. The absorbance spectrum of the pyrroloquinoline quinone prosthetic group of this enzyme was also effected by pH at values greater than 7.5. The enzyme was relatively insensitive to changes in ionic strength, and exhibited a linear Arrhenius plot over a range of temperatures from 10 degrees C to 50 degrees C with an energy of activation 46 kJ/mol (11 kcal/mol).     

Resonance Raman spectroscopy of methylamine dehydrogenase from bacterium W3A1

Resonance Raman spectroscopy has been used to probe the structure of the covalently bound quinone cofactor in methylamine dehydrogenase from the bacterium W3A1. Spectra were obtained on the phenylhydrazine and 2-pyridylhydrazine derivatives of the native enzyme, on the quinone-containing subunit labeled with phenylhydrazine, and on an active-site peptide also labeled with phenylhydrazine. Comparisons of these spectra to the corresponding spectra of copper-containing amine oxidase derivatives indicate that the quinones in these two classes of quinoproteins are not identical. The resonance Raman spectra of the native enzyme and small subunit have also been measured. 16O/18O exchange permitted the carbonyl modes of the quinone to be identified in the resonance Raman spectrum of oxidized methylamine dehydrogenase: a band at 1614 cm-1, together with a shoulder at 1630 cm-1, are assigned as modes containing substantial C = O stretching character. D2O/H2O exchange has pronounced effects on the resonance Raman spectrum of the oxidized enzyme, suggesting that the quinone may have numerous hydrogen bonds to the protein or that it is unusually sensitive to the local environment. Resonance Raman spectra of the isolated small subunit, and its phenylhydrazine derivative, are considerably different from the corresponding spectra of the intact protein. An attractive explanation for these observations is that the quinone cofactor in methylamine dehydrogenase from W3A1 is located at the interface between the large and small subunits, as found for the enzyme from Thiobacillus versutus [Vellieux, F. M. D., Huitema, F., Groendijk, H., Kalk, K. H., Frank, J. Jzn., Jongejan, J. A., & Duine, J. A. (1989) EMBO J. 8, 2171-2178].(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning, sequencing, and mutation of a gene for azurin in Methylobacillus flagellatum KT.

The gene cluster for methylamine utilization (mau genes) has been cloned from the obligate methylotrophic bacterium Methylobacillus flagellatum KT. Partial sequence data showed that the organization of these genes was similar to that found in Methylophilus methylotrophus W3A1-NS, including the lack of a gene for amicyanin, which had been thought to be the electron acceptor for methylamine dehydrogenase in M. flagellatum KT. However, a gene encoding azurin was discovered at the 3' end of the mau gene cluster, transcribed in the opposite orientation. A mutant with a defect in this gene showed impaired growth on methylamine, suggesting that azurin is involved in methylamine oxidation in M. flagellatum KT.     

Electron transfer flavoprotein from Methylophilus methylotrophus: properties, comparison with other electron transfer flavoproteins, and regulation of expression by carbon source.

When grown on methylated amines as a carbon source, Methylophilus methylotrophus synthesizes an electron transfer flavoprotein (ETF) which is the natural electron acceptor of trimethylamine dehydrogenase. It is composed of two dissimilar subunits of 38,000 and 42,000 daltons and 1 mol of flavin adenine dinucleotide. It was reduced by trimethylamine dehydrogenase to a stable anionic semiquinone form, which could not be converted, either enzymatically or chemically, to the fully reduced dihydroquinone. This ETF exhibited spectral properties which were nearly identical to ETFs from bacterium W3A1, Paracoccus denitrificans, and pig liver mitochondria. M. methylotrophus ETF cross-reacted immunologically and enzymatically with the ETF of bacterium W3A1 but not with the other two ETFs. In M. methylotrophus and bacterium W3A1, ETF and trimethylamine dehydrogenase were each expressed during growth on trimethylamine and were each absent during growth on methanol.     

Physiological properties of a pyrroloquinoline quinone mutant of Methylobacterium organophilum

Abstract The grwoth of MTMl, a mutant of methylobacterium organophilum) blocked in the use of methanol as a carbon and energy source, was restored by addition of pyrroloquinoline quinone (PQQ) in the culture medium. No PQQ could be detected in crude medium. No PQQ could be of MTMl. Therefore, MTMl can be regarded as a mutant blocked in the biosynthesis of PQQ. Under the conditions of growth employed, growth rates of MTMl on methanol, comparable to those of the wild type, occured at a PQQ concentration of 1 μM. Since lower amounts of methanol dehydrogenase (MDH) wer found in cell-free extracts of PQQ-supplemented MTMl, the wild type strain synthesizes a surplus of MDH under these conditions. Growth of M. organophilum on ethanol proceeds via MDH as a catalyst for the first step, since (NAD(P) -dependent etanol. dehydrogenase was absent in cell-free extracts and growth of MTMl on ethanol only took place in the presence of PQQ. On the hand, growth of MTMl on mthylamine was unimpaired. This is in accordance with the fact that methylamine dehydrogenase was absent and N -methylamine mate dehydrogenase was present in cell-free extracts