Characterisation of the mob locus of Rhodobacter sphaeroides WS8: mobA is the only gene required for molybdopterin guanine dinucleotide synthesis

The mob genes of several bacteria have been implicated in the conversion of molybdopterin to molybdopterin guanine dinucleotide. The mob locus of Rhodobacter sphaeroides WS8 comprises three genes, mobABC. Chromosomal in-frame deletions in each of the mob genes have been constructed. The mobA mutant strain has inactive DMSO reductase and periplasmic nitrate reductase activities (both molybdopterin guanine dinucleotide-requiring enzymes), but the activity of xanthine dehydrogenase, a molybdopterin enzyme, is unaffected. The inability of a mobA mutant to synthesise molybdopterin guanine dinucleotide is confirmed by analysis of cell extracts of the mobA strain for molybdenum cofactor forms following iodine oxidation. Mutations in mobB and mobC are not impaired for molybdoenzyme activities and accumulate wild-type levels of molybdopterin and molybdopterin guanine dinucleotide, indicating they are not compromised in molybdenum cofactor synthesis. In the mobA mutant strain, the inactive DMSO reductase is found in the periplasm, suggesting that molybdenum cofactor insertion is not necessarily a pre-requisite for export.     

Molybdenum cofactor biosynthesis in Escherichia coli. Requirement of the chlB gene product for the formation of molybdopterin guanine dinucleotide.

The chlorate-resistant mutants of Escherichia coli are affected in the biosynthesis of the molybdenum cofactor and show pleiotropic loss of the activities of those enzymes which require the cofactor. The molybdenum cofactor in all molybdoenzymes other than nitrogenase is a complex of the metal with a unique pterin termed molybdopterin. The molybdenum cofactor in a number of E. coli enzymes has been shown to contain GMP in addition to the metal-molybdopterin complex, with the GMP appended in pyrophosphate linkage to the terminal phosphate ester on the molybdopterin side chain. In this paper, we have examined the biochemistry of the chlB mutant and show that the gene product of the chlB locus is essential for the addition of the GMP moiety to form molybdopterin guanine dinucleotide, a step which occurs late in the cofactor biosynthetic pathway in E. coli. Sensitive techniques were developed for the identification of fluorescent derivatives of molybdopterin and of molybdopterin guanine dinucleotide in extracts of E. coli cells. Wild type cells were shown to contain both molybdopterin and molybdopterin guanine dinucleotide, while cells of chlB mutants were found to contain elevated levels of molybdopterin but no detectable molybdopterin guanine dinucleotide.     

A pAO1-encoded molybdopterin cofactor gene (moaA) ofArthrobacter nicotinovorans: characterization and site-directed mutagenesis of the encoded protein

A gene homologous tomoaA, the gene responsible for the expression of a protein involved in an early step in the synthesis of the molybdopterin cofactor ofEscherichia coli, was found to be located 2.7-kb upstream of the nicotine dehydrogenase (ndh) operon on the catabolic plasmid pAO1 ofArthrobacter nicotinovorans. The MoaA protein, containing 354 amino acids, migrated on an SDS-polyacrylamide gel with an apparent molecular weight of 40,000, in good agreement with the predicted molecular weight of 38,880. The pAO1-encodedmoaA gene fromA. nicotinovorans was expressed inE. coli as an active protein that functionally complementedmoaA mutants. Its reduced amino acid sequence shows 43% identity to theE. coli MoaA, 44% to the NarAB gene product fromBacillus subtilis, and 42% to the gene product of two contiguous ORFs fromMethanobacterium formicicum. N-terminal sequences, including the motif CxxxCxYC, are conserved among the MoaA and NarAB proteins. This motif is also present in proteins involved in PQQ cofactor synthesis in almost all the NifB proteins reported so far and in thefixZ gene product fromRhizobium leguminosarum. Mutagenesis of any of these three conserved cysteine residues to serine abolished the biological activity of MoaA, while substitution of the tyrosine by either serine, phenylalanine, or alanine did not alter the capacity of the protein to complement themoaA mutation inE. coli. A second Cys-rich domain with the motif FCxxC(13x)C is found close to the C-terminus of MoaA and NarAB proteins. These two Cys-rich sequences may be involved in the coordination of a metal ions. The pAO1 copy ofmoaA may not be unique in theA. nicotinovorans genome since the molybdopterin cofactor oxidation products were detected in cell extracts from a plasmidless strain.     

A Functional mobA Gene for Molybdopterin Cytosine Dinucleotide Cofactor Biosynthesis Is Required for Activity and Holoenzyme Assembly of the Heterotrimeric Nicotine Dehydrogenases of Arthrobacter nicotinovorans

Two Arthrobacter nicotinovorans molybdenum enzymes hydroxylate the pyridine ring of nicotine. Molybdopterin cytosine dinucleotide (MCD) was determined to be a cofactor of these enzymes. A mobA gene responsible for the formation of MCD could be identified and its function shown to be required for assembly of the heterotrimeric molybdenum enzymes.     

Molybdopterin dinucleotide biosynthesis in Escherichia coli: identification of amino acid residues of molybdopterin dinucleotide transferases that determine specificity for binding of guanine or cytosine nucleotides

The molybdenum cofactor is modified by the addition of GMP or CMP to the C4' phosphate of molybdopterin forming the molybdopterin guanine dinucleotide or molybdopterin cytosine dinucleotide cofactor, respectively. The two reactions are catalyzed by specific enzymes as follows: the GTP:molybdopterin guanylyltransferase MobA and the CTP:molybdopterin cytidylyltransferase MocA. Both enzymes show 22% amino acid sequence identity and are specific for their respective nucleotides. Crystal structure analysis of MobA revealed two conserved motifs in the N-terminal domain of the protein involved in binding of the guanine base. Based on these motifs, we performed site-directed mutagenesis studies to exchange the amino acids to the sequence found in the paralogue MocA. Using a fully defined in vitro system, we showed that the exchange of five amino acids was enough to obtain activity with both GTP and CTP in either MocA or MobA. Exchange of the complete N-terminal domain of each protein resulted in the total inversion of nucleotide specificity activity, showing that the N-terminal domain determines nucleotide recognition and binding. Analysis of protein-protein interactions showed that the C-terminal domain of either MocA or MobA determines the specific binding to the respective acceptor protein.     

Structural analysis and molybdenum-dependent expression of the pAO1-encoded nicotine dehydrogenase genes of Arthrobacter nicotinovorans

The genes of nicotine dehydrogenase (NDH) were identified, cloned and sequenced from the catabolic plasmid pA01 of Arthrobacter nicotinovorans. In immediate proximity to this gene cluster is the beginning of the 6-hydroxy-L-niotine oxidase (6-HLNO) gene. NDH is composed of three subunits (A, B and C) of M_r 30011, 14924 and 87677. It belongs to a family of bacterial hydroxylases with a similar subunit structure; they have molybdopterin dinucleotide, FAD and Fe-S clusters as cofactors. Here the first complete primary structure of a bacterial hydroxylase is provided. Sequence alignments of each of the NDH subunits show similarities to the sequences of eukaryotic xanthine dehydrogenase (XDH) but not to other known molybdenum-containing bacterial enzymes. Based on alignment with XDH it is inferred that the smallest subunit (NDHB) carries an iron-sulphur cluster, that the middle-sized subunit (NDHA) binds FAD, and that the largest NDH subunit (NDHC) corresponds to the molybdopterin-binding domain of XDH. Expression of both the ndh and the 6-hlno genes required the presence of nicotine and molybdenum in the culture medium. Tungsten inhibited enzyme activity but not the synthesis of the enzyme protein. The enzyme was found in A. nicotinovorans cells in a soluble form and in a membrane-associated form. In the presence of tungsten the fraction of membrane-associated NDH increased.     

Molybdopterin guanine dinucleotide cofactor in Synechococcus sp. nitrate reductase: identification of mobA and isolation of a putative moeB gene

Cathepsin B and other lysosomal cysteine proteinases are synthesized as inactive zymogens, which are converted to their mature forms by other proteases or by autocatalytic processing. Procathepsin B autoactivation was shown in vitro at pH 4.5 to be a bimolecular process with K(s) and k(cat) values of 2.1+/-0.9 microM and 0.12+/-0.02 s(-1)6.0. However, in the presence of 0.5 microg/ml of dextran sulfate, relatively rapid processing is observed even at pH 6.5 (t(1/2) approximately 90 min), suggesting that glycosaminoglycans are involved in in vivo processing of lysosomal cysteine proteases.     

A novel gamma-N-methylaminobutyrate demethylating oxidase involved in catabolism of the tobacco alkaloid nicotine by Arthrobacter nicotinovorans pAO1.

Nicotine catabolism, linked in Arthrobacter nicotinovorans to the presence of the megaplasmid pAO1, leads to the formation of gamma-N-methylaminobutyrate from the pyrrolidine ring of the alkaloid. Until now the metabolic fate of gamma-N-methylaminobutyrate has been unknown. pAO1 carries a cluster of ORFs with similarity to sarcosine and dimethylglycine dehydrogenases and oxidases, to the bifunctional enzyme methylenetetrahydrofolate dehydrogenase/cyclohydrolase and to formyltetrahydrofolate deformylase. We cloned and expressed the gene carrying the sarcosine dehydrogenase-like ORF and showed, by enzyme activity, spectrophotometric methods and identification of the reaction product as gamma-aminobutyrate, that the predicted 89 395 Da flavoprotein is a demethylating gamma-N-methylaminobutyrate oxidase. Site-directed mutagenesis identified His67 as the site of covalent attachment of FAD and confirmed Trp66 as essential for FAD binding, for enzyme activity and for the spectral properties of the wild-type enzyme. A Km of 140 microm and a kcat of 800 s(-1) was determined when gamma-N-methylaminobutyrate was used as the substrate. Sarcosine was also turned over by the enzyme, but at a rate 200-fold slower than gamma-N-methylaminobutyrate. This novel enzyme activity revealed that the first step in channelling the gamma-N-methylaminobutyrate generated from nicotine into the cell metabolism proceeds by its oxidative demethylation.     

The tightly bound calcium of MauG is required for tryptophan tryptophylquinone cofactor biosynthesis

The diheme enzyme MauG catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. The crystal structure of the MauG-preMADH complex revealed the presence of a Ca(2+) in proximity to the two hemes [Jensen, L. M. R., Sanishvili, R., Davidson, V. L., and Wilmot, C. M. (2010) Science 327, 1392-1394]. This Ca(2+) did not readily dissociate; however, after extensive treatment with EGTA or EDTA MauG was no longer able to catalyze TTQ biosynthesis and exhibited altered absorption and resonance Raman spectra. The changes in spectral features are consistent with Ca(2+)-dependent changes in heme spin state and conformation. Addition of H(2)O(2) to the Ca(2+)-depleted MauG did not yield spectral changes characteristic of formation of the bis-Fe(IV) state which is stabilized in native MauG. After addition of Ca(2+) to the Ca(2+)-depleted MauG, full TTQ biosynthesis activity and reactivity toward H(2)O(2) were restored, and the spectral properties returned to those of native MauG. Kinetic and equilibrium studies of Ca(2+) binding to Ca(2+)-depleted MauG indicated a two-step mechanism. Ca(2+) initially reversibly binds to Ca(2+)-depleted MauG (K(d) = 22.4 μM) and is followed by a relatively slow (k = 1.4 × 10(-3) s(-1)) but highly favorable (K(eq) = 4.2) conformational change, yielding an equilibrium dissociation constant K(d,eq) value of 5.3 μM. The circular dichroism spectra of native and Ca(2+)-depleted MauG were essentially the same, consistent with Ca(2+)-induced conformational changes involving domain or loop movements rather than general unfolding or alteration of secondary structure. These results are discussed in the context of the structures of MauG and heme-containing peroxidases.     

Zasp is required for the assembly of functional integrin adhesion sites

The integrin family of heterodimeric transmembrane receptors mediates cell–matrix adhesion. Integrins often localize in highly organized structures, such as focal adhesions in tissue culture and myotendinous junctions in muscles. Our RNA interference screen for genes that prevent integrin-dependent cell spreading identifies Z band alternatively spliced PDZ-motif protein (zasp), encoding the only known Drosophila melanogaster Alp/Enigma PDZ-LIM domain protein. Zasp localizes to integrin adhesion sites and its depletion disrupts integrin adhesion sites. In tissues, Zasp colocalizes with βPS integrin in myotendinous junctions and with α-actinin in muscle Z lines. Zasp also physically interacts with α-actinin. Fly larvae lacking Zasp do not form Z lines and fail to recruit α-actinin to the Z line. At the myotendinous junction, muscles detach in zasp mutants with the onset of contractility. Finally, Zasp interacts genetically with integrins, showing that it regulates integrin function. Our observations point to an important function for Zasp in the assembly of integrin adhesion sites both in cell culture and in tissues.     

Crystal structure and catalytic mechanism of the MbnBC holoenzyme required for methanobactin biosynthesis

Methanobactins(Mbns)are a family of copper-binding peptides involved in copper uptake by methanotrophs,and are potential therapeutic agents for treating disease...     

NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli

The formation of active membrane-bound nitrate reductase A in Escherichia coli requires the presence of three subunits, NarG, NarH and NarI, as well as a fourth protein, NarJ, that is not part of the active nitrate reductase. In narJ strains, both NarG and NarH subunits are associated in an unstable and inactive NarGH complex. A significant activation of this complex was observed in vitro after adding purified NarJ-6His polypeptide to the cell supernatant of a narJ strain. Once the apo-enzyme NarGHI of a narJ mutant has become anchored to the membrane via the NarI subunit, it cannot be reactivated by NarJ in vitro . NarJ protein specifically recognizes the catalytic NarG subunit. Fluorescence, electron paramagnetic resonance (EPR) spectroscopy and molybdenum quantification based on inductively coupled plasma emission spectroscopy (ICPES) clearly indicate that, in the absence of NarJ, no molybdenum cofactor is present in the NarGH complex. We propose that NarJ is a specific chaperone that binds to NarG and may thus keep it in an appropriate competent-open conformation for the molybdenum cofactor insertion to occur, resulting in a catalytically active enzyme. Upon insertion of the molybdenum cofactor into the apo-nitrate reductase, NarJ is then dissociated from the activated enzyme.     

A disulfide bond is required for functional assembly of NCX1 from complementary fragments

The cardiac Na(+)-Ca(2+) exchanger consists of a single polypeptide with two transmembrane segment (TMS) clusters separated by a large intracellular loop between TMS5 and TMS6 (Nicoll et al. (1999) J. Biol. Chem. 274, 910-917; Iwamoto et al. (1999) FEBS Lett. 446, 264-268). A "split" exchanger can be expressed by dividing the exchanger cDNA into two fragments so that the NH(2)- and CO(2)H-terminal portions of the protein are expressed as separate polypeptides in HEK293 cells. Expression of partial exchanger molecules did not result in detectable exchanger activity. Cells coexpressing both portions of the exchanger, however, displayed between 30 and 50% of the activity of the intact wild-type exchanger. The full-length exchanger contains a disulfide bond between residues 14 or 20 and 792. We examined the role of this disulfide bond in the split exchanger by mutagenesis and expression studies. Our results indicate that the function of the exchanger requires both TMS clusters and that the C(14 or 20)/C792 disulfide bond is essential for expression of active exchangers from half molecules.     

A domain of RecC required for assembly of the regulatory RecD subunit into the Escherichia coli RecBCD holoenzyme

The heterotrimeric RecBCD enzyme of Escherichia coli is required for the major pathway of double-strand DNA break repair and genetic exchange. Assembled as a heterotrimer, the enzyme has potent nuclease and helicase activity. Analysis of recC nonsense and deletion mutations revealed that the C terminus of RecC is required for assembly of the RecD subunit into RecBCD holoenzyme but not for recombination proficiency; the phenotype of these mutations mimics that of recD deletion mutations. Partial proteolysis of purified RecC polypeptide yielded a C-terminal fragment that corresponds to the RecD-interaction domain. RecD is essential for nuclease activity, regulation by the recombination hotspot Chi, and high affinity for DNA ends. The RecC-RecD interface thus appears critical for the regulation of RecBCD enzyme via the assembly and, we propose, disassembly or conformational change of the RecD subunit.     

Sequence of the 165-kilobase catabolic plasmid pAO1 from Arthrobacter nicotinovorans and identification of a pAO1-dependent nicotine uptake system

The 165-kb catabolic plasmid pAO1 enables the gram-positive soil bacterium Arthrobacter nicotinovorans to grow on the tobacco alkaloid L-nicotine. The 165,137-nucleotide sequence, with an overall G+C content of 59.7%, revealed, besides genes and open reading frames (ORFs) for nicotine degradation, a complete set of ORFs for enzymes essential for the biosynthesis of the molybdenum dinucleotide cofactor, as well as ORFs related to uptake and utilization of carbohydrates, sarcosine, and amino acids. Of the 165 ORFs, approximately 50% were related to metabolic functions. pAO1 conferred to A. nicotinovorans the ability to take up L-[(14)C]nicotine from the medium, with an K(m) of 5.6 +/- 2.2 micro M. ORFs of putative nicotine transporters formed a cluster with the gene of the D-nicotine-specific 6-hydroxy-D-nicotine oxidase. ORFs related to replication, chromosome partitioning, and natural transformation functions (dprA) were identified on pAO1. Few ORFs showed similarity to known conjugation-promoting proteins, but pAO1 could be transferred by conjugation to a pAO1-negative strain at a rate of 10(-2) to 10(-3) per donor. ORFs with no known function represented approximately 35% of the pAO1 sequence. The positions of insertion sequence elements and composite transposons, corroborated by the G+C content of the pAO1 sequence, suggest a modular composition of the plasmid.     

A gene required for very short patch repair in Escherichia coli is adjacent to the DNA cytosine methylase gene.

Deamination of 5-methylcytosine in DNA results in T/G mismatches. If unrepaired, these mismatches can lead to C-to-T transition mutations. The very short patch (VSP) repair process in Escherichia coli counteracts the mutagenic process by repairing the mismatches in favor of the G-containing strand. Previously we have shown that a plasmid containing an 11-kilobase fragment from the E. coli chromosome can complement a chromosomal mutation defective in both cytosine methylation and VSP repair. We have now mapped the regions essential for the two phenotypes. In the process, we have constructed plasmids that complement the chromosomal mutation for methylation, but not for repair, and vice versa. The genes responsible for these phenotypes have been identified by DNA sequence analysis. The gene essential for cytosine methylation, dcm, is predicted to code for a 473-amino-acid protein and is not required for VSP repair. It is similar to other DNA cytosine methylases and shares extensive sequence similarity with its isoschizomer, EcoRII methylase. The segment of DNA essential for VSP repair contains a gene that should code for a 156-amino-acid protein. This gene, named vsr, is not essential for DNA methylation. Remarkably, the 5' end of this gene appears to overlap the 3' end of dcm. The two genes appear to be transcribed from a common promoter but are in different translational registers. This gene arrangement may assure that Vsr is produced along with Dcm and may minimize the mutagenic effects of cytosine methylation.     

Characterization of Escherichia coli MoeB and its involvement in the activation of molybdopterin synthase for the biosynthesis of the molybdenum cofactor.

Amino acid sequence comparisons of Escherichia coli MoeB suggested that the MoeB-dependent formation of a C-terminal thiocarboxylate on the MoaD subunit of molybdopterin synthase might resemble the ubiquitin-activating step in the ubiquitin-targeted degradation of proteins in eukaryotes. To determine the exact role of MoeB in molybdopterin biosynthesis, the protein was purified after homologous overexpression. Using purified proteins, we have demonstrated the ATP-dependent formation of a complex of MoeB and MoaD adenylate that is stable to gel filtration. Mass spectrometry of the complex revealed a peak of a molecular mass of 9,073 Da, the expected mass of MoaD adenylate. However, unlike the ubiquitin activation reaction, the formation of a thioester intermediate between MoeB and MoaD could not be observed. There was also no evidence for a MoeB-bound sulfur during the sulfuration of MoaD. Amino acid substitutions were generated in every cysteine residue in MoeB. All of these exhibited activity comparable to the wild type, with the exception of mutations in cysteine residues located in putative Zn-binding motifs. For these cysteines, loss of activity correlated with loss of metal binding.     

The maize Viviparous10/Viviparous13 locus encodes the Cnx1 gene required for molybdenum cofactor biosynthesis

Abscisic acid (ABA), auxin and nitrate are important signaling molecules that affect plant growth responses to the environment. The synthesis or metabolism of these compounds depends on the molybdenum cofactor (MoCo). We show that maize (Zea mays) viviparous10 (vp10) mutants have strong precocious germination and seedling lethal phenotypes that cannot be rescued with tissue culture. We devised a novel PCR-based method to clone a transposon-tagged allele of vp10, and show that Vp10 encodes the ortholog of Cnx1, which catalyzes the final common step of MoCo synthesis. The seedling phenotype of vp10 mutants is consistent with disruptions in ABA and auxin biosynthesis, as well as a disruption in nitrate metabolism. Levels of ABA and auxin are reduced in vp10 mutants, and vp10 seedlings lack MoCo-dependent enzyme activities that are repairable with exogenous molybdenum. vp10 and an Arabidopsis cnx1 mutant, chlorate6 (chl6), have similar defects in aldehyde oxidase (AO) enzyme activity, which is required for ABA synthesis. Surprisingly, chl6 mutants do not show defects in abiotic stress responses. These observations confirm an orthologous function for Cnx1 and Vp10, as well as defining a characteristic viviparous phenotype to identify other maize cnx mutants. Finally, the vp10 mutant phenotype suggests that cnx mutants can have auxin- as well as ABA-biosynthesis defects, while the chl6 mutant phenotype suggests that low levels of AO activity are sufficient for normal abiotic stress responses.     

Molecular cloning and functional expression of the rfaE gene required for lipopolysaccharide biosynthesis in Salmonella typhimurium

The rfaE (WaaE) gene of Salmonella typhimurium is known to be located at 76min on the genetic map outside of the rfa gene cluster encoding core oligosaccharide biosynthesis of lipopolysaccharide(LPS). The rfaE mutant synthesizes heptose-deficient LPS; its LPS consists of only lipid A and 3-deoxy-D-manno-octulosonic acid (KDO), and the rfaE gene is believed to be involved in the formation of ADP-L-glycero-D-manno-heptose. Mutants, which make incomplete LPS, are known as rough mutants. Salmonella typhimurium deep-rough mutants affected in the heptose region of the inner core often show reduced growth rate, sensitivity to high temperature and hypersensitivity to hydrophobic antibiotics. We have cloned the rfaE gene of S. typhimurium. The chromosomal region carrying this gene was isolated by screening a genomic library of S. typhimurium using the complementation of S. typhimurium rfaE mutant. The 2.6-Kb insert in the plasmid pHEPs appears to carry a functional rfaE gene. SL1102 (rfaE543) makes heptose-deficient LPS and has a deep rough phenotype, but pHEPs complement the rfaE543 mutation to give the smooth phenotype. The sensitivity of SL1102 to bacteriophages (P22.c2, Felix-O, Br60) which use LPS as their receptor for adsorption is changed to that of wild-type strain. The permeability barrier of SL1102 to hydrophobic antibiotics (novobiocin) is restored to that of wild-type. LPS produced by SL1102 (rfaE543) carrying pHEPs makes LPS indistinguishable from that of smooth strains. The rfaE gene encoded a polypeptide of 477 amino acid residues highly homologous to the S. enterica rfaE protein (98% identity), E. coli (93% identity), Yersenia pestis (85% identity), Haemophilus influenzae (70% identity) and Helicobacter pyroli (41% identity) with a molecular weight 53 kDa.