Biology of the molybdenum cofactor

The transition element molybdenum (Mo) is an essential micronutrient for plants where it is needed as a catalytically active metal during enzyme catalysis. Four plant enzymes depend on molybdenum: nitrate reductase, sulphite oxidase, xanthine dehydrogenase, and aldehyde oxidase. However, in order to gain biological activity and fulfil its function in enzymes, molybdenum has to be complexed by a pterin compound thus forming the molybdenum cofactor. In this article, the path of molybdenum from its uptake into the cell, via formation of the molybdenum cofactor and its storage, to the final modification of the molybdenum cofactor and its insertion into apo-metalloenzymes will be reviewed.     

Assay of molybdenum cofactor of barley

Conditions for assay of molybdenum cofactor in barley shoot extracts in the presence of molybdate (25 mM N₂MoO₄) and the sulphydryl-group protector, reduced glutathione (5 mM) were optimized. Both total Mo-cofactor (assayed after heat-treatment of cell-free extracts) and ‘free’ Mo-cofactor (assayed in untreated cell-free extracts) were assayed. Compared to control plants grown in the absence of an exogenous nitrogen source total Mo-cofactor levels increased around 70 % when plants were grown for 4 days in the presence of either 15 mM KNO₃ or 15 mM NH₄NO₃. Growth in the presence of 15 mM (NH₄)₂SO₄ did not affect the Mo-cofactor level. Very similar results were seen when plants were transferred to these nitrogen sources for 24 hr after previous growth in the absence of an exogenous nitrogen source. In contrast ‘free’ Mo-cofactor levels of both KNO₃ and NH₄NO₃-treated plants were increased 2-3-fold over untreated controls. Growth in the presence of (NH₄)₂SO₄ did not affect the ‘free’ Mo-cofactor level.     

Different forms of molybdenum cofactor inVicia faba seeds: The presence of molybdenum cofactor carrier protein and its purification

There were significant differences in the contents of molybdenum cofactor (Mo-co), both in a low-molecular-mass form (free Mo-co) and in a protein-bound form, in seeds of sevenVicia faba genotypes. Low-molecular-mass Mo-co species present in the extracts were detected by their ability to reactivate, through a dialysis membrane, aponitrate reductase from theNeurospora crassa nit-1 mutant. In extracts of all genotypes tested, the amount of Mo-co capable of directly reactivating nitrate reductase of theN. crassa nit-1 mutant was always much higher than that of low-molecular-mass Moco. These data cannot be explained by considering, as traditionally, that Mo-co detected directly, i.e. without any previous treatment for its release from Mo-coproteins, corresponds to free low-molecular mass Mo-co. A protein which bound Mo-co was purified to electrophoretic homogeneity. This protein consisted of a single 70-kDa polypeptide chain and carried a Mo-co that could be efficiently released when in contact with aponitrate reductase.Abbreviations CP carrier protein - Mo-co molybdenum cofactor - NR nitrate reductase - XO xanthine oxidase     

Purification,cofactor analysis,and site-directed mutagenesis of Synechococcus ferredoxin-nitrate reductase

The narB gene of the cyanobacterium Synechococcus sp. strain PCC 7942 encodes an assimilatory nitrate reductase that uses photosynthetically reduced ferredoxin as the physiological electron donor. This gene was expressed in Escherichia coli and electrophoretically pure preparations of the enzyme were obtained using affinity chromatography with either reduced-ferredoxin or NarB antibodies. The electronic absorption spectrum of the oxidized enzyme showed a shoulder at around 320 nm and a broad absorption band between 350 and 500 nm. These features are indicative of the presence of an iron-sulfur centre(s) and accordingly metal analysis showed ca. 3 atoms of Fe per molecule of protein that could represent a [3Fe-4S] cluster. Further analysis indicated the presence of 1 atom of Mo and 2 molecules of ribonucleotide-conjugated molybdopterin per molecule of protein. This, together with the requirement of a mobA gene for production of an active enzyme, strongly suggests the presence of Mo in the form of the bis-MGD (bis-molybdopterin guanine dinucleotide) cofactor in Synechococcusnitrate reductase. A model for the coordination of the Mo atom to the enzyme is proposed. Four conserved Cys residues were replaced by site-directed mutagenesis. The effects of these changes on the enzyme activity and electronic absorption spectra support the participation of those residues in iron-sulfur cluster coordination. This revised version was published online in June 2006 with corrections to the Cover Date.     

Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling.

An unresolved key issue in the mechanism of protein folding assisted by the molecular chaperone GroEL is the nature of the substrate protein bound to the chaperonin at different stages of its reaction cycle. Here we describe the conformational properties of human dihydrofolate reductase (DHFR) bound to GroEL at different stages of its ATP-driven folding reaction, determined by hydrogen exchange labeling and electrospray ionization mass spectrometry. Considerable protection involving about 20 hydrogens is observed in DHFR bound to GroEL in the absence of ATP. Analysis of the line width of peaks in the mass spectra, together with fluorescence quenching and ANS binding studies, suggest that the bound DHFR is partially folded, but contains stable structure in a small region of the polypeptide chain. DHFR rebound to GroEL 3 min after initiating its folding by the addition of MgATP was also examined by hydrogen exchange, fluorescence quenching, and ANS binding. The results indicate that the extent of protection of the substrate protein rebound to GroEL is indistinguishable from that of the initial bound state. Despite this, small differences in the quenching coefficient and ANS binding properties are observed in the rebound state. On the basis of these results, we suggest that GroEL-assisted folding of DHFR occurs by minor structural adjustments to the partially folded substrate protein during iterative cycling, rather than by complete unfolding of this protein substrate on the chaperonin surface.     

Redox reactions of the pyranopterin system of the molybdenum cofactor

This work provides the first extensive study of the redox reactivity of the pyranopterin system that is a component of the catalytic site of all molybdenum and tungsten enzymes possessing molybdopterin. The pyranopterin system possesses certain characteristics typical of tetrahydropterins, such as a reduced pyrazine ring; however, it behaves as a dihydropterin in redox reactions with oxidants. Titrations using ferricyanide and dichloroindophenol (DCIP) prove a 2e^–/2H⁺ stoichiometry for pyranopterin oxidations. Oxidations of pyranopterin by Fe(CN)₆ ^3– or DCIP are slower than tetrahydropterin oxidation under a variety of conditions, but are considerably faster than observed for oxidations of dihydropterin. The rate of pyranopterin oxidation by DCIP was studied in a variety of media. In aqueous buffered solution the pyranopterin oxidation rate has minimal pH dependence, whereas the rate of tetrahydropterin oxidation decreases 100-fold over the pH range 7.4–8.5. Although pyranopterin reacts as a dihydropterin with oxidants, it resists further reduction to a tetrahydropterin. No reduction was achieved by catalytic hydrogenation, even after several days. The reducing ability of the commonly used biological reductants dithionite and methyl viologen radical cation was investigated, but experiments showed no evidence of pyranopterin reduction by any of these reducing agents. This study illustrates the dual personalities of pyranopterin and underscores the unique place that the pyranopterin system holds in the spectrum of pterin redox reactions. The work presented here has important implications for understanding the biosynthesis and reaction chemistry of the pyranopterin cofactor in molybdenum and tungsten enzymes.Abbreviations DCIP dichloroindophenol - H₄DMP 6,7-dimethyltetrahydropterin - MV⁺ methyl viologen radical cation     

Presence of molybdenum cofactor in seeds of wheat and barley

Molybdenum cofactor (Mo-co) was determined in seeds of wheat and barley by its ability to restore nitrate reductase (NR) activity in extracts of nitrate reductase-deficient mutants. Its activity was compared with that of wheat roots and leaves. Conditions for assay of Mo-co from different sources in the presence of molybdate and reduced glutathione (GSH) were optimised. The effect of heat-treatment of cell-free extracts from seeds, roots and leaves was also investigated. Mutant extracts of Neurospora crassa nit-1 and Nicotiana tabacum CnxA68, used as apoprotein source for in vitro complementation, were shown to give comparable results. The Mo-co activity, extracted from wheat and barley seeds, could be separated into two peaks by gel chromatography.     

ADP ribosylation factor-like protein 2 (Arl2) regulates the interaction of tubulin-folding cofactor D with native tubulin

The ADP ribosylation factor-like proteins (Arls) are a family of small monomeric G proteins of unknown function. Here, we show that Arl2 interacts with the tubulin-specific chaperone protein known as cofactor D. Cofactors C, D, and E assemble the alpha/beta- tubulin heterodimer and also interact with native tubulin, stimulating it to hydrolyze GTP and thus acting together as a beta-tubulin GTPase activating protein (GAP). We find that Arl2 downregulates the tubulin GAP activity of C, D, and E, and inhibits the binding of D to native tubulin in vitro. We also find that overexpression of cofactors D or E in cultured cells results in the destruction of the tubulin heterodimer and of microtubules. Arl2 specifically prevents destruction of tubulin and microtubules by cofactor D, but not by cofactor E. We generated mutant forms of Arl2 based on the known properties of classical Ras-family mutations. Experiments using these altered forms of Arl2 in vitro and in vivo demonstrate that it is GDP-bound Arl2 that interacts with cofactor D, thereby averting tubulin and microtubule destruction. These data establish a role for Arl2 in modulating the interaction of tubulin-folding cofactors with native tubulin in vivo.     

Chaperone action of a versatile small heat shock protein from Methanococcoides burtonii, a cold adapted archaeon

The Methanococcoides burtonii small heat shock protein (Mb-sHsp) is an alphaB-crystallin homolog that delivers protein stabilizing and protective functions to model enzymes, presumably reflecting its role as a molecular chaperone in vivo. Although the gene encoding Mb-shsp was cloned from a cold-adapted microorganism, the Mb-sHsp is an efficient protein chaperone at temperatures far above the optimum growth temperature of M. burtonii. We show that Mb-sHsp can prevent aggregation in E. coli cell free extracts at 60 degrees C for 4 h and can stabilize bovine liver glutamate dehydrogenase for 3 h at 50 degrees C. Surface plasmon resonance was used to determine the binding affinity of Mb-sHsp for denatured proteins. Mb-sHsp bound tightly to denatured lysozyme but not to the native form. When Mb-Cpn and Mg(2+)-ATP were added to the reaction, bound lysozyme was released from Mb-sHsp establishing that Mb-Cpn is able to off-load folding intermediates from Mb-sHsp. In addition, Mb-sHsp and Mb-Cpn also function cooperatively to protect an enzyme substrate. Through characterization of these M. burtonii chaperones, we were able to reconstitute a key heat shock regulated protein folding function of this cold adapted organism in vitro.     

共表达HAC1和分子伴侣基因对甘露聚糖酶在毕赤酵母中表达的影响

为了提高甘露聚糖酶ManA在毕赤酵母中分泌表达的酶活,选择毕赤酵母内质网未折叠蛋白反应(Unfolded protein response,UPR)激活调控因子HAC1与5种毕赤酵母蛋白折叠相关的分子伴侣ERO1、PDI、PDI1、CPR5、BiP,通过构建pPICZA-HAC1等6种胞内表达重组质粒,分别电转化至分泌表达ManA的毕赤酵母重组菌中胞内共表达,并分析其重组菌摇瓶发酵时ManA表达的影响。结果发现在摇瓶发酵水平,胞内共表达HAC1、ERO1、PDI的重组菌发酵上清液中的ManA酶活力分别提高了26%、15%、20%,其重组菌发酵上清液的酶活力分别达到1 014 U/mL、925 U/mL、965 U/mL。通过对各重组菌上清液酶活力、胞内滞留酶活力、上清液蛋白浓度数据进行分析,进一步选择将HAC1、ERO1、PDI进行两基因或三基因组合,并分别在分泌表达ManA的重组菌胞内共表达,但各共表达重组菌发酵上清液的酶活力都没有进一步的提升。单独共表达HAC1或者分子伴侣ERO1、PDI可以辅助ManA的正确折叠,提高其蛋白表达。     

Molybdenum cofactor negative mutants of Escherichia coli use citrate anaerobically

Anaerobically, Escherichia coli cannot grow using either glycerol or citrate as sole carbon and energy source. However, it has been reported that a mixture of glycerol and citrate will support growth. We have found that wild-type strains of E. coli K-12 do not grow on glycerol plus citrate anaerobically. However, growth eventually occurs due to the frequent appearance of mutants. We found that such Cit+ mutants were defective in anaerobic respiration with nitrate or trimethylamine-N-oxide and were chlorate resistant (i.e. molybdenum cofactor deficient). Conversely, well characterized mutants in any of chlA, B, D, E, G and N were also able to use citrate anaerobically. No anaerobic growth differences between wild type and chl mutants were observed either with fermentable sugars or with glycerol plus fumarate or glycerol plus tartrate. Citrate lyase was induced anaerobically by citrate and repressed by glucose in both wild type strains and chl mutants. Furthermore, levels of citrate lyase, fumarate reductase, malate dehydrogenase, fumarase and alcohol dehydrogenase were similar in both types of strains under anaerobic conditions. It is conceivable that a functioning molybdenum cofactor prevents use of citrate by keeping citrate lyase in the inactive form.     

Survival of the Aerobic Denitrifier Pseudomonas stutzeri Strain TR2 during Co-Culture with Activated Sludge under Denitrifying Conditions

The aerobic denitrifier Pseudomonas stutzeri TR2 (strain TR2) has the potential to reduce nitrous oxide emissions during the wastewater treatment process. In this application, it is important to find the best competitive survival conditions for strain TR2 in complex ecosystems. To that end, we examined co-cultures of strain TR2 with activated sludge via five passage cultures in a medium derived from treated piggery wastewater that contained a high concentration of ammonium. The results are as follows: (i) The medium supported the proliferation of strain TR2 (P. stutzeri strains) under denitrifying conditions. (ii) Nitrite was a better denitrification substrate than nitrate for TR2 survival. (iii) Strain TR2 also demonstrated strong survival even under aerobic conditions. This suggests that strain TR2 is effectively augmented to the wastewater treatment process, aiding in ammonium-nitrogen removal and reducing nitrous oxide production with a partial nitrification technique in which nitrite accumulates.     

Nitrogen limitation of heterotrophic biofilms in boreal streams

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Rescue of molybdenum cofactor biosynthesis in gephyrin-deficient mice by a Cnx1 transgene

Gephyrin is a bifunctional protein which is essential for both synaptic clustering of inhibitory neurotransmitter receptors in the central nervous system and the biosynthesis of the molybdenum cofactor (MoCo) in peripheral tissues. Mice deficient in gephyrin die early postnatally and display a loss of glycine receptors (GlyRs) and many GABA(A) receptor (GABA(A)R) subtypes from postsynaptic sites. In addition, the activities of the MoCo-dependent enzymes xanthine dehydrogenase and sulfite oxidase are reduced to background levels in the liver and intestine of these animals. To genetically separate the different consequences of gephyrin deficiency, we expressed a transgene of the plant homolog Cnx1, known to rescue mammalian MoCo deficiency, on the background of gephyrin knockout mice. Cnx1 partially restored sulfite oxidase activity in the liver of the transgenic animals, whereas early lethality and the loss of GlyR clustering were unaltered. Our data suggest that the loss of neurotransmitter receptor clustering at inhibitory synapses causes the early lethality of gephyrin deficient mice.     

The active site of the molybdenum cofactor biosynthetic protein domain Cnx1G

The final step of molybdenum cofactor biosynthesis in plants is catalyzed by the two-domain protein Cnx1. The G domain of Cnx1 (Cnx1G) binds molybdopterin with high affinity and transfers molybdenum to molybdopterin. Here, we describe the functional and structural characterization of structure-based Cnx1G mutants. For molybdopterin binding residues Thr542 and Ser573 were found to be important because different mutations of those residues resulted in 7- to 26-fold higher k(D) values for molybdopterin binding. Furthermore, we showed that the terminal phosphate of molybdopterin is directly involved in protein-pterin interactions as dephosphorylated molybdopterin binds with one magnitude of order lower affinity to the wild-type protein. Molybdopterin binding was not affected in mutants defective in Ser476, Asp486, or Asp515. However, molybdenum insertion was completely abolished, indicating their important role for catalysis. Based on these results we propose the binding of molybdopterin to a large depression in the structure of Cnx1G formed by beta5, alpha5, beta6, and alpha6, whereas the negatively charged depression formed by the loop between beta3 and alpha4, the N-terminal end of alpha2, the 3(10) helix, and the region between beta6 and alpha6 is involved in catalysis.     

Effective cotranslational folding of firefly luciferase without chaperones of the Hsp70 family

Molecular chaperones of the Hsp70 family (bacterial DnaK, DnaJ, and GrpE) were shown to be strictly required for refolding of firefly luciferase from a denatured state and thus for effective restoration of its activity. At the same time the luciferase was found to be synthesized in an Escherichia coli cell-free translation system in a highly active state in the extract with no chaperone activity. The addition of the chaperones to the extract during translation did not raise the activity of the enzyme. The abrupt arrest of translation by the addition of a translational inhibitor led to immediate cessation of the enzyme activity accumulation, indicating the cotranslational character of luciferase folding. The results presented suggest that the chaperones of the Hsp70 family are not required for effective cotranslational folding of firefly luciferase.     

The influence of growth conditions on the synthesis of molybdenum cofactor in Proteus mirabilis

Cell-free extracts of Proteus mirabilis were able to reconstitute NADPH-dependent assimilatory nitrate reductase in crude extracts of the Neurospora crassa mutant strain nit-1, lacking molybdenum cofactor. Molybdenum cofactor was formed in the cytoplasm of the bacterium even in the presence of oxygen during growth though under these conditions no molybdo enzymes are formed. As a consequence no cofactor could be released by acid treatment from membranes of cells grown aerobically. The amount of cofactor released from membranes of cells grown anaerobically under various conditions was proportional to the amount of molybdo enzymes formed. During growth in the presence of tungstate a cofactor, which lacks molybdenum, was found in the cytoplasm. For detection of this so-called demolybdo cofactor the presence of molybdate during reconstitution was essential. Moreover, the cytoplasmic cofactor pool in cells grown in the presence of tungstate appeared to be two to three times higher than in cells grown under similar conditions without tungstate. After anaerobic growth in the presence of tungstate, the inactive demolybdo reductases were shown to contain partly no cofactor and partly a demolybdo cofactor. The P. mirabilis chlorate resistant mutant S 556 did not contain molybdenum cofactor. In two other chl-mutants the cofactor activity was the same as in the wild type.     

PCR-DGGE analysis of bacterial community dynamics in kava beverages during refrigeration

Aims: Kava beverages are highly perishable even under refrigerated conditions. This study aimed to investigate the bacterial community dynamics in kava beverages during refrigeration. Methods and Results: Four freshly made kava beverages were obtained from kava bars and stored at 4°C. On days 0, 3 and 6, the aerobic plate count (APC), lactic acid bacteria (LAB) count and yeast and mould count (YMC) of the samples were determined. Meanwhile, bacterial DNA was extracted from each sample and subjected to the polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE). Moreover, species‐specific PCR assays were employed to identify predominant Pseudomonas spp. involved in kava spoilage. Over the storage period, the APC, LAB count and YMC of the four kava beverages all increased, whereas their pH values decreased. The DGGE profile revealed diverse bacterial populations in the samples. LAB, such as Weissella soli, Lactobacillus spp. and Lactococcus lactis, were found in the kava beverages. Species‐specific PCR assays detected Pseudomonas putida and Pseudomonas fluorescens in the samples; Ps. fluorescens became dominant during refrigeration. Conclusions: LAB and Pseudomonas may play a significant role in the spoilage of kava beverages. Significance and Impact of the Study: This study provides important information that may be used to extend the shelf life of kava beverages.