Complementation of Methylobacterium organophilum mutants affected in pyrroloquinoline quinone biosynthesis genes pqqE and pqqF by cloned Escherichia coli chromosomal DNA

The hybrid plasmid pBGT3, a derivative of pLA2917 containing a 7.8-kb fragment of Escherichia coli DNA, was found to complement pqqE and pqqF mutants of Methylobacterium organophilum, both impaired in PQQ biosynthesis. The cloned fragment of E. coli DNA did not hybridize with DNA fragments containing pqqE or pqqF previously cloned from M. organophilum. Yet, in M. organophilum mutants, expression of pqqE and pqqF genes from E. coli resulted in a PQQ production estimated at 9-16% of the production observed in M. organophilum wild-type. The growth rate in methanol medium of the complemented M. organophilum mutants was about 60% of that of the wild-type.     

Genetic and physical analyses of Methylobacterium organophilum XX genes encoding methanol oxidation.

When allyl alcohol was used as a suicide substrate, spontaneous mutants and UV light- and nitrous acid-generated mutants of Methylobacterium organophilum XX were selected which grew on methylamine but not on methanol. There was no detectable methanol dehydrogenase (MDH) activity in crude extracts of these mutants, yet Western blots revealed that some mutants still produced MDH protein. Complementation of 50 mutants by a cosmid gene bank of M. organophilum XX demonstrated that three major regions of the genome, each of which was separated by a minimum of 40 kilobases, were required for expression of active MDH. By subcloning and Tn5 insertion mutagenesis of subcloned fragments, at least 11 genes clustered within these three regions were subsequently identified. The identity of the MDH structural gene, which was initially determined by hybridization to the structural gene of Methylobacterium sp. strain AM1, was confirmed by Western blot analysis of an MDH-beta-galactosidase fusion protein.     

Localization of a pyrroloquinoline quinone biosynthesis gene near the methanol dehydrogenase structural gene in Methylobacterium organophilum DSM 760

A partial Sau3AI genomic bank of Methylobacterium organophilum DSM 760 was constructed in the cosmid pSUP106 and moxF, the structural gene for methanol dehydrogenase, was isolated. In M. organophilum, pSUP106 behaves as a suicide plasmid. This property was used to insert Tn5 into the bacterial chromosome, in the vicinity of moxF, by marker exchange. Mobilization of the Tn5-labelled chromosomal region by a broad-host-range plasmid, pJB3J1 (an R68-45 derivative), allowed the selection of several large R' hybrid plasmids in Escherichia coli HB101. Most of them were able to complement both mutants of the moxF region and mutant MTM1, the first mutant of the pyrroloquinoline quinone (PQQ) biosynthesis pathway in M. organophilum. The gene involved, pqqA, was subcloned and localized.     

甲基营养菌MP688甲醇脱氢酶基因mpq1818的敲除及功能研究

目的:研究甲醇脱氢酶基因mpq1818在甲基营养菌MP688生长代谢中的作用。方法:利用同源重组原理构建中间为庆大霉素抗性基因Gmr、两侧mpq1818基因上下游序列同源的敲除载体pAK0-up-Gmr-down,接合转移导入MP688,通过庆大霉素抗性和组合PCR方法筛选基因敲除菌,并检测其生长、甲醇脱氢酶活性、甲醇利用及吡咯喹啉醌(PQQ)生物合成能力等方面的差异。结果:抗性和PCR验证显示mpq1818缺失株构建成功;与野生菌相比,缺失株的甲醇脱氢酶活力及利用甲醇的能力降低,而且菌株的生长和PQQ产量也有显著下降。结论:基因mpq1818的缺失影响菌株前期生长与PQQ合成。     

The role of the MxaD protein in the respiratory chain of Methylobacterium extorquens during growth on methanol

The largest of the gene clusters coding for proteins involved in methanol oxidation is the cluster mxaFJGIR(S)ACKLDEHB. Disruption of most of these genes leads to lack of growth on methanol. The previous results showed that the mutant lacking MxaD grows on methanol although at a low rate. This is explained by the low rate of methanol oxidation by whole cells. The specific activity of methanol dehydrogenase (MDH) is higher in the mutant but its electron acceptor (cytochrome c(L)) is unchanged. Using the purified proteins, it was shown that the rate of interaction of MDH and cytochrome c(L) was higher in the wild-type MDH containing some MxaD proteins, which was absent in the mutant MDH. It is suggested that the gene mxaD codes for the 17-kDa periplasmic protein that directly or indirectly stimulates the interaction between MDH and cytochrome c(L); its absence leads to a lower rate of respiration with methanol and therefore a lower growth rate on this substrate.     

甲基营养菌甲醇脱氢酶基因的克隆及表达

目的:从甲基营养菌MP681中扩增甲醇脱氢酶(MDH)基因,在大肠杆菌中表达并检测其活性,同时在MP681中考察该基因对吡咯喹啉醌(PQQ)产生的影响。方法:根据MP681基因组序列设计引物,PCR扩增靶基因mdh,构建表达载体,考察活性,利用接合转移转化至MP681,考察PQQ的合成。结果:扩增得到甲基营养菌MP681甲醇脱氢酶基因,在大肠杆菌中的表达产物能够催化甲醇脱氢;将携带mdh基因的质粒转入MP681后,PQQ产量略有提高。结论:获得编码MDH的基因,该基因能够在大肠杆菌中表达,且表达产物具有生物活性;甲醇脱氢酶基因表达对宿主菌的PQQ合成可能有一定影响。     

Determination of enzyme mechanisms by molecular dynamics: studies on quinoproteins, methanol dehydrogenase, and soluble glucose dehydrogenase

Molecular dynamics (MD) simulations have been carried out to study the enzymatic mechanisms of quinoproteins, methanol dehydrogenase (MDH), and soluble glucose dehydrogenase (sGDH). The mechanisms of reduction of the orthoquinone cofactor (PQQ) of MDH and sGDH involve concerted base-catalyzed proton abstraction from the hydroxyl moiety of methanol or from the 1-hydroxyl of glucose, and hydride equivalent transfer from the substrate to the quinone carbonyl carbon C5 of PQQ. The products of methanol and glucose oxidation are formaldehyde and glucolactone, respectively. The immediate product of PQQ reduction, PQQH- [-HC5(O-)-C4(=O)-] and PQQH [-HC5(OH)-C4(=O)-] converts to the hydroquinone PQQH2 [-C5(OH)=C4(OH)-]. The main focus is on MD structures of MDH * PQQ * methanol, MDH * PQQH-, MDH * PQQH, sGDH * PQQ * glucose, sGDH * PQQH- (glucolactone, and sGDH * PQQH. The reaction PQQ-->PQQH- occurs with Glu 171-CO2- and His 144-Im as the base species in MDH and sGDH, respectively. The general-base-catalyzed hydroxyl proton abstraction from substrate concerted with hydride transfer to the C5 of PQQ is assisted by hydrogen-bonding to the C5=O by Wat1 and Arg 324 in MDH and by Wat89 and Arg 228 in sGDH. Asp 297-COOH would act as a proton donor for the reaction PQQH(-)-->PQQH, if formed by transfer of the proton from Glu 171-COOH to Asp 297-CO2- in MDH. For PQQH-->PQQH2, migration of H5 to the C4 oxygen may be assisted by a weak base like water (either by crystal water Wat97 or bulk solvent, hydrogen-bonded to Glu 171-CO2- in MDH and by Wat89 in sGDH).     

Methanol metabolism in thermotolerant methylotrophic Bacillus strains involving a novel catabolic NAD-dependent methanol dehydrogenase as a key enzyme

The enzymology of methanol utilization in thermotolerant methylotrophic Bacillus strains was investigated. In all strains an immunologically related NAD-dependent methanol dehydrogenase was involved in the initial oxidation of methanol. In cells of Bacillus sp. C1 grown under methanol-limiting conditions this enzyme constituted a high percentage of total soluble protein. The methanol dehydrogenase from this organism was purified to homogeneity and characterized. In cell-free extracts the enzyme displayed biphasic kinetics towards methanol, with apparent K _m values of 3.8 and 166 mM. Carbon assimilation was by way of the fructose-1,6-bisphosphate aldolase cleavage and transketolase/transaldolase rearrangement variant of the RuMP cycle of formaldehyde fixation. The key enzymes of the RuMP cycle, hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), were present at very high levels of activity. Failure of whole cells to oxidize formate, and the absence of formaldehyde-and formate dehydrogenases indicated the operation of a non-linear oxidation sequence for formaldehyde via HPS. A comparison of the levels of methanol dehydrogenase and HPS in cells of Bacillus sp. C1 grown on methanol and glucose suggested that the synthesis of these enzymes is not under coordinate control.Abbreviations RuMP ribulose monophosphate - HPS hexulose-6-phosphate synthase - HPI hexulose-6-phosphate isomerase - MDH methanol dehydrogenase - ADH acohol dehydrogenase - PQQ pyrroloquinoline, quinone - DTT dithiothreitol - NBT nitrobluetetrazolium - PMS phenazine methosulphate - DCPIP dichlorophenol indophenol     

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     

Environmental regulation of alcohol metabolism in thermotolerant methylotrophic Bacillus strains

The thermotolerant methylotroph Bacillus sp. C1 possesses a novel NAD-dependent methanol dehydrogenase (MDH), with distinct structural and mechanistic properties. During growth on methanol and ethanol, MDH was responsible for the oxidation of both these substrates. MDH activity in cells grown on methanol or glucose was inversely related to the growth rate. Highest activity levels were observed in cells grown on the C₁-substrates methanol and formaldehyde. The affinity of MDH for alcohol substrates and NAD, as well as V _max, are strongly increased in the presence of a M _r 50,000 activator protein plus Mg²⁺-ions [Arfman et al. (1991) J Biol Chem 266: 3955–3960]. Under all growth conditions tested the cells contained an approximately 18-fold molar excess of (decameric) MDH over (dimeric) activator protein. Expression of hexulose-6-phosphate synthase (HPS), the key enzyme of the RuMP cycle, was probably induced by the substrate formaldehyde. Cells with high MDH and low HPS activity levels immediately accumulated (toxic) formaldehyde when exposed to a transient increase in methanol concentration. Similarly, cells with high MDH and low CoA-linked NAD-dependent acetaldehyde dehydrogenase activity levels produced acetaldehyde when subjected to a rise in ethanol concentration. Problems frequently observed in establishing cultures of methylotrophic bacilli on methanol- or ethanol-containing media are (in part) assigned to these phenomena.Abbreviations MDH NAD-dependent methanol dehydrogenase - ADH NAD-dependent alcohol dehydrogenase - A1DH CoA-linked NAD-dependent aldehyde dehydrogenase - HPS hexulose-6-phosphate synthase - G6Pdh glucose-6-phosphate dehydrogenase     

The structure and mechanism of methanol dehydrogenase

This is a review of recent work on methanol dehydrogenase (MDH), a pyrroloquinoline quinone (PQQ)-containing enzyme catalysing the oxidation of methanol to formaldehyde in methylotrophic bacteria. Although it is the most extensively studied of this class of dehydrogenases, it is only recently that there has been any consensus about its mechanism. This is partly due to recent structural studies on normal and mutant enzymes and partly due to more definitive work on the mechanism of related alcohol and glucose dehydrogenases. This work has also led to conclusions about the subsequent path of electrons and protons during the reoxidation of the reduced quinol form of the prosthetic group.     

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.     

Structural requirements of pyrroloquinoline quinone dependent enzymatic reactions

On the basis of crystal structures of the pyrroloquinoline quinone (PQQ) dependent enzymes methanol dehydrogenase (MDH) and soluble glucose dehydrogenase (s-GDH), different catalytic mechanisms have been proposed. However, several lines of biochemical and kinetic evidence are strikingly similar for both enzymes. To resolve this discrepancy, we have compared the structures of these enzymes in complex with their natural substrates in an attempt to bring them in line with a single reaction mechanism. In both proteins, PQQ is located in the center of the molecule near the axis of pseudo-symmetry. In spite of the absence of significant sequence homology, the overall binding of PQQ in the respective active sites is similar. Hydrogen bonding interactions are made with polar protein side chains in the plane of the cofactor, whereas hydrophobic stacking interactions are important below and above PQQ. One Arg side chain and one calcium ion are ligated to the ortho-quinone group of PQQ in an identical fashion in either active site, in agreement with their proposed catalytic function of polarizing the PQQ C5-O5 bond. The substrates are bound in a similar position above PQQ and within hydrogen bond distance of the putative general bases Asp297 (MDH) and His144 (s-GDH). On the basis of these similarities, we propose that MDH and s-GDH react with their substrates through an identical mechanism, comprising general base-catalyzed hydride transfer from the substrate to PQQ and subsequent tautomerization of the PQQ intermediate to reduced PQQ.     

Cyclopropanol in the exploration of bacterial alcohol oxidation

Abstract Cyclopropanol selectively inhibits bacterial alcohol oxidation proceeding via NAD-independent, quinoprotein alcohol dehydrogenases. Thus, for instance, alcohol oxidation by Pseudomonas aeruginosa , grown on ethanol, was inhibited for about 50% by cyclopropanol treatment. Accordingly, cell-free extracts of untreated cells had nearly equal activities of quinoprotein and NAD-dependent alcohol dehydrogenases, whereas only the latter enzyme activity was found in cell-free extracts of cyclopropanol-treated cells. Upon incubation of Hyphomicrobium X with cyclopropanol, oxidation of alcohols was blocked while formaldehyde oxidation was not. Therefore, methanol dehydrogenase in this organism is not specifically involved in formaldehyde oxidation. The examples show that cyclopropanol-derived substrates are potential tools in revealing the physiological role of bacterial alcohol dehydrogenases.     

The quinoprotein dehydrogenases for methanol and glucose

This review summarises our current understanding of two of the main types of quinoprotein dehydrogenase in which pyrroloquinoline quinone (PQQ) is the only prosthetic group. These are the soluble methanol dehydrogenase and the membrane glucose dehydrogenase (mGDH). The membrane GDH has an additional N-terminal domain by which it is tightly anchored to the membrane, and a periplasmic domain whose structure has been modelled on the X-ray structure of the alpha-subunit of MDH which contains PQQ in the active site. This review discusses their structures and mechanisms, concentrating particularly on the pathways for electron transfer from the reduced PQQ, through the protein, to their electron acceptors. In MDH, this is the specific cytochrome c(L), the electron transfer pathway probably involving the unique disulphide ring in the active site. By contrast, mGDH contains a permanently bound ubiquinone, which acts as a single electron carrier, mediating electron transfer through the protein to the membrane ubiquinone.     

Functions of the membrane-associated and cytoplasmic malate dehydrogenases in the citric acid cycle of Corynebacterium glutamicum

Like many other bacteria, Corynebacterium glutamicum possesses two types of L-malate dehydrogenase, a membrane-associated malate:quinone oxidoreductase (MQO; EC 1.1.99.16) and a cytoplasmic malate dehydrogenase (MDH; EC 1.1.1.37) The regulation of MDH and of the three membrane-associated dehydrogenases MQO, succinate dehydrogenase (SDH), and NADH dehydrogenase was investigated. MQO, MDH, and SDH activities are regulated coordinately in response to the carbon and energy source for growth. Compared to growth on glucose, these activities are increased during growth on lactate, pyruvate, or acetate, substrates which require high citric acid cycle activity to sustain growth. The simultaneous presence of high activities of both malate dehydrogenases is puzzling. MQO is the most important malate dehydrogenase in the physiology of C. glutamicum. A mutant with a site-directed deletion in the mqo gene does not grow on minimal medium. Growth can be partially restored in this mutant by addition of the vitamin nicotinamide. In contrast, a double mutant lacking MQO and MDH does not grow even in the presence of nicotinamide. Apparently, MDH is able to take over the function of MQO in an mqo mutant, but this requires the presence of nicotinamide in the growth medium. It is shown that addition of nicotinamide leads to a higher intracellular pyridine nucleotide concentration, which probably enables MDH to catalyze malate oxidation. Purified MDH from C. glutamicum catalyzes oxaloacetate reduction much more readily than malate oxidation at physiological pH. In a reconstituted system with isolated membranes and purified MDH, MQO and MDH catalyze the cyclic conversion of malate and oxaloacetate, leading to a net oxidation of NADH. Evidence is presented that this cyclic reaction also takes place in vivo. As yet, no phenotype of an mdh deletion alone was observed, which leaves a physiological function for MDH in C. glutamicum obscure.     

Effects of multiple ligand binding on kinetic isotope effects in PQQ-dependent methanol dehydrogenase

The reaction of PQQ-dependent methanol dehydrogenase (MDH) from Methylophilus methylotrophus has been studied by steady-state and stopped-flow kinetic methods, with particular reference to multiple ligand binding and the kinetic isotope effect (KIE) for PQQ reduction. Phenazine ethosulfate (PES; an artificial electron acceptor) and cyanide (a suppressant of endogenous activity), but not ammonium (an activator of MDH), compete for binding at the catalytic methanol-binding site. Cyanide does not activate turnover in M. methylotrophus MDH, as reported previously for the Paracoccus denitrificans enzyme. Activity is dependent on activation by ammonium but is inhibited at high ammonium concentrations. PES and methanol also influence the stimulatory and inhibitory effects of ammonium through competitive binding. Reaction profiles as a function of ammonium and PES concentration differ between methanol and deuterated methanol, owing to force constant effects on the binding of methanol to the stimulatory and inhibitory ammonium binding sites. Differential binding gives rise to unusual KIEs for PQQ reduction as a function of ammonium and PES concentration. The observed KIEs at different ligand concentrations are independent of temperature, consistent with their origin in differential binding affinities of protiated and deuterated substrate at the ammonium binding sites. Stopped-flow studies indicate that enzyme oxidation is not rate-limiting at low ammonium concentrations (<4 mM) during steady-state turnover. At higher ammonium concentrations (>20 mM), the low effective concentration of PES in the active site owing to the competitive binding of ammonium lowers the second-order rate constant for enzyme oxidation, and the oxidative half-reaction becomes more rate limiting. A sequential stopped-flow method is reported that has enabled, for the first time, a detailed study of the reductive half-reaction of MDH and comparison with steady-state data. The limiting rate of PQQ reduction (0.48 s(-1)) is less than the steady-state turnover number, and the observed KIE in stopped-flow studies is unity. Although catalytically active, we propose reduction of the oxidized enzyme generated in stopped-flow analyses is gated by conformational change or ligand exchange. Slow recovery from this trapped state on mixing with methanol accounts for the slow reduction of PQQ and a KIE of 1. This study emphasizes the need for caution in using inflated KIEs, and the temperature dependence of KIEs, as a probe for hydrogen tunneling.     

Site-directed mutagenesis and X-ray crystallography of the PQQ-containing quinoprotein methanol dehydrogenase and its electron acceptor, cytochrome c(L)

Two proteins specifically involved in methanol oxidation in the methylotrophic bacterium Methylobacterium extorquens have been modified by site-directed mutagenesis. Mutation of the proposed active site base (Asp303) to glutamate in methanol dehydrogenase (MDH) gave an active enzyme (D303E-MDH) with a greatly reduced affinity for substrate and with a lower activation energy. Results of kinetic and deuterium isotope studies showed that the essential mechanism in the mutant protein was unchanged, and that the step requiring activation by ammonia remained rate limiting. No spectrally detectable intermediates could be observed during the reaction. The X-ray structure, determined to 3 A resolution, of D303E-MDH showed that the position and coordination geometry of the Ca2+ ion in the active site was altered; the larger Glu303 side chain was coordinated to the Ca2+ ion and also hydrogen bonded to the O5 atom of pyrroloquinoline quinone (PQQ). The properties and structure of the D303E-MDH are consistent with the previous proposal that the reaction in MDH is initiated by proton abstraction involving Asp303, and that the mechanism involves a direct hydride transfer reaction. Mutation of the two adjacent cysteine residues that make up the novel disulfide ring in the active site of MDH led to an inactive enzyme, confirming the essential role of this remarkable ring structure. Mutations of cytochrome c(L), which is the electron acceptor from MDH was used to identify Met109 as the sixth ligand to the heme.     

On the mechanism of inhibition of methanol dehydrogenase by cyclopropane-derived inhibitors

Extraction of cyclopropanol-inactivated methanol dehydrogenase (MDH) gave a mixture of two interconverting compounds. The same compounds could be prepared from 2,7,9-tricarboxy-1H-pyrrolo[2,3-f]quinoline-4,5-dione (PQQ) and cyclopropanol using a metal oxide (e.g. Ag2O) as a catalyst. Structure elucidation revealed that a C5 3-propanal adduct of PQQ is formed which is present in the extract as a diastereoisomeric mixture of the ring-closed form. Cyclopropanone gave an analogous product, while cyclopropylmethanol behaved as a substrate and was oxidized by the enzyme without ring-opening. From the work described, several arguments can be derived to reject the idea that inactivation proceeds via formation of a pair of free radicals. The mechanism probably consists of a concerted proton abstraction, rearrangement of the cyclopropoxy anion to a ring-opened carbanion and attack of the latter on the electrophilic C5 of PQQ. The measured rate of inactivation (3.7 s-1) is in agreement with such a mechanism. The role of the metal oxide and the enzyme in this process is the catalysis of the addition step and possibly a positioning of the reactants. As only a sole type of quinoprotein alcohol dehydrogenase becomes inhibited, the cyclopropane derivatives studied here can be regarded as mechanism-based inhibitors. The modified PQQ in cyclopropanone-inactivated MDH is fluorescent. A fluorescent intermediate was also observed in the catalytic cycle of MDH with methanol as a substrate. Its rate of formation and decay and the strongly decreased level of fluorescence in the presence of activator are in accordance with the view that the fluorescing species is the previously found oxidized-MDH.substrate (MDHox.S) complex. Since the decomposition of this complex requires activator and model studies have failed so far to mimic the enzyme, it seems that the combination of enzyme and activator is essential for the oxidation of the alcohol substrate.