YcbX and yiiM, two novel determinants for resistance of Escherichia coli to N-hydroxylated base analogues

We have shown previously that lack of molybdenum cofactor (MoCo) in Escherichia coli leads to hypersensitivity to the mutagenic and toxic effects of N -hydroxylated base analogues, such as 6- N -hydroxylaminopurine (HAP). However, the nature of the MoCo-dependent mechanism is unknown, as inactivation of all known and putative E. coli molybdoenzymes does not produce any sensitivity. Presently, we report on the isolation and characterization of two novel HAP-hypersensitive mutants carrying defects in the ycbX or yiiM open reading frames. Genetic analysis suggests that the two genes operate within the MoCo-dependent pathway. In the absence of the ycbX - and yiiM -dependent pathways, biotin sulfoxide reductase plays also a role in the detoxification pathway. YcbX and YiiM are hypothetical members of the MOSC protein superfamily, which contain the C-terminal domain (MOSC) of the eukaryotic MoCo sulphurases. However, deletion of ycbX or yiiM did not affect the activity of human xanthine dehydrogenase expressed in E. coli , suggesting that the role of YcbX and YiiM proteins is not related to MoCo sulphuration. Instead, YcbX and YiiM may represent novel MoCo-dependent enzymatic activities. We also demonstrate that the MoCo/YcbX/YiiM-dependent detoxification of HAP proceeds by reduction to adenine.     

Role for CysJ Flavin Reductase in Molybdenum Cofactor-Dependent Resistance of Escherichia coli to 6-N-Hydroxylaminopurine

We have previously described a novel Escherichia coli detoxification system for the removal of toxic and mutagenic N-hydroxylated nucleobases and related compounds that requires the molybdenum cofactor. Two subpathways (ycbX and yiiM) were identified, each employing a novel molybdo activity capable of inactivating N-hydroxylated compounds by reduction to the corresponding amine. In the present study, we identify the cysJ gene product as one additional component of this system. While the CysJ protein has been identified as the NADPH:flavin oxidoreductase component of the CysJI sulfite reductase complex (CysJ₈I₄), we show that the role of CysJ in base analog detoxification is unique and independent of CysI and sulfite reductase. We further show that CysJ functions as a specific partner of the YcbX molybdoenzyme. We postulate that the function of CysJ in this pathway is to provide, via its NADPH:flavin reductase activity, the reducing equivalents needed for the detoxification reaction at the YcbX molybdocenter. In support of the proposed interaction of the CysJ and YcbX proteins, we show that an apparent CysJ-YcbX “hybrid” protein from two Vibrio species is capable of compensating for a double cysJ ycbX defect in E. coli.Mutagenic base analogs are chemically modified nucleobases that can be incorporated in the cellular metabolism through purine or pyrimidine salvage pathways. Once converted to the deoxynucleoside triphosphate (dNTP) level, they may participate in DNA replication in an error-prone manner because of their ambivalent base-pairing capacity (11). Such synthetic base analogs are often used as a sensitive tool for studying DNA replication fidelity, DNA repair, or the metabolism of nucleic acid precursors. Mutagenic base analogs such as 8-oxoguanine or 3-methyladenine can also be formed in vivo as a consequence of normal cellular metabolism or produced by chemical and physical factors, such as alkylating agents or ionizing radiation.An important group of mutagenic and cytotoxic analogs are the N-hydroxylated nucleobases (or ribosides) such as 6-N-hydroxylaminopurine (HAP), 2-amino-HAP, or N⁴-hydroxycytidine (15). Specifically, HAP was found to be a very strong mutagen in bacteria and fungi, as well as mammalian cells (2, 20, 27). Some data have suggested that HAP may also be formed in vivo under oxidative stress (30) or as a by-product of certain purine salvage/interconversion pathways (5, 22).The genetic control of HAP-induced mutagenesis has been studied in some detail in the yeast Saccharomyces cerevisiae and in the bacterium Escherichia coli. In S. cerevisiae, resistance to HAP depends primarily on genes involved in adjusting and regulating the DNA or RNA precursor pools (HAM1 [ITP/XTPase], AAH1 [adenine aminohydrolase], and ADE genes involved in de novo AMP biosynthesis) (34).In E. coli, the major pathway that protects cells against HAP and related N-hydroxylated compounds is controlled by the moa, moe, and mog genes, which are required for biosynthesis of molybdenum cofactor (MoCo) (18, 19). MoCo is an essential cofactor for a varied group of oxidoreductases that are widely distributed from bacteria to humans. Chemically, MoCo is a pterin derivative (molybdopterin) that coordinates a molybdenum atom that serves as a catalytic redox center (for reviews, see references 23, 28, and 29). Based on catalytic details and sequence homology, molybdopterin-containing enzymes have been divided in four families: the xanthine oxidase family, the sulfite oxidase family, the dimethyl sulfoxide (DMSO) reductase family, and the aldehyde ferredoxin oxidoreductase family (14, 16). However, our previous studies on the MoCo-dependent resistance to HAP showed that none of the known or putative E. coli members of these families are responsible for the major HAP resistance mechanism (19). Instead, we discovered that HAP resistance is dependent on two newly described proteins, YcbX and YiiM, that are characterized by a so-called MOSC domain (molybdenum cofactor sulfurase C-terminal domain) (1, 17). This domain was first described as part of eukaryotic MoCo sulfurases (MOSs) (1), and it most likely represents a novel class of MoCo-binding domain, as indicated by studies on two mammalian MOSC-containing proteins (mARC1 and mARC2) discovered in mitochondria (12, 13).Our studies in E. coli showed that cell-free bacterial extracts were capable of converting HAP to adenine by an N-reductive reaction (17). Importantly, this conversion was entirely dependent on the presence of MoCo and the YcbX or YiiM proteins (17). Consequently, we suggested that this reduction of HAP to adenine forms the basis of the in vivo MoCo-dependent detoxification in E. coli (17). Interestingly, the mammalian MOSC-containing proteins mARC1 and mARC2 were shown to mediate the reduction of the N-hydroxylated prodrug benzamidoxime to its active amino form benzamidine (12, 13). Thus, the reduction of N-hydroxylated compounds may be a defining feature for the broadly distributed MOSC proteins (1).Our previous analyses also revealed that the E. coli ycbX and yiiM genes define two independent subpathways within the MoCo-dependent system (17). This is illustrated in the overall scheme shown in Fig. ​Fig.1.1. MoCo is synthesized in a series of steps from GTP by-products of the moa, moe, and mog operons. MoCo is then used as a cofactor for the YcbX and YiiM proteins, which reduce the N-hydroxylated compound to the corresponding amino form. The ycbX and yiiM pathways are genetically distinct as determined by epistasis experiments (17). They also differ by their substrate specificity patterns: YcbX protects most strongly against HAP, whereas YiiM has its largest effects toward hydroxylamine (NH₂OH) (17).Open in a separate windowFIG. 1.Genetic framework for the major molybdenum cofactor (MoCo)-dependent pathways of detoxification of N-hydroxylated base analogs in E. coli (17). moaA to mogA indicate the series of genes required for MoCo biosynthesis (19, 28), while ycbX and yiiM represent the two independent subpathways identified within the MoCo-dependent pathway (17). Specifically, ycbX and yiiM produce apoenzymes that react with MoCo to form the active YcbX and YiiM proteins. The diagram also indicates the differential specificity of the two subpathways toward the model N-hydroxylated compounds used in our studies: 6-N-hydroxylaminopurine (HAP), 2-amino-HAP (AHAP), and hydroxylamine (NH₂OH). For simplicity, the diagram does not distinguish between the MPT and MGD forms of MoCo (19). As shown elsewhere (19), YcbX and YiiM likely employ the MPT form. One additional, minor pathway for HAP detoxification dependent on biotin sulfoxide reductase (an MGD-requiring enzyme) is observable only in the double ycbX yiiM-deficient background and is likewise not shown here (see reference 17 for details).Prior to the establishment of this scheme of YcbX and YiiM as molybdoproteins, we had entertained certain alternative possibilities for the precise function of the ycbX and yiiM open reading frames (ORFs), including a possible role in MoCo sulfuration (which is a required modification of MoCo in certain molybdoenzymes, such as xanthine oxidase) (23, 29). This sulfuration model was ultimately eliminated (17), but certain experiments related to this hypothesis yielded interesting further clues regarding the detailed mechanisms of HAP resistance. These observations included an unexpected HAP-sensitive phenotype for cysJ mutants as well as a noted sensitization of wild-type strains to HAP by l-cysteine. In the present work, we describe these experiments and show the cysJ gene to be an essential component of the ycbX branch of HAP resistance. In a related mechanism, the observed sensitization of wild-type strains by l-cysteine results from the suppression, by l-cysteine, of the cys regulon. Overall, our experiments suggest that CysJ is a specific protein partner of YcbX and that CysJ mediates the N-reductive reaction through its NADPH:flavin oxidoreductase activity. This activity provides reducing equivalents to its partner YcbX, which ultimately performs the reduction of HAP to nontoxic adenine at its molybdocenter.     

Evidence for MoeA-dependent formation of the molybdenum cofactor from molybdate and molybdopterin in<Emphasis Type="Italic"> Escherichia coli</Emphasis>

The function of the MoeA protein in the biosynthesis of the molybdenum cofactor (MoCo) was analyzed in vitro, using purified His(6)-MoeA from Escherichia coli, molybdopterin (MPT) isolated from buttermilk xanthine oxidase and molybdate. The formation of MoCo was monitored by the reconstitution of nitrate reductase activity in extracts of the Neurospora crassa nit-1 mutant. Formation of MoCo from MPT and molybdate required MoeA and L-cysteine or glutathione. The reaction proceeded at micromolar molybdate levels and was time- and MoeA concentration-dependent. A physical interaction between MoeA and MPT was demonstrated by HPLC analysis of MoeA-bound MPT.     

Direct transfer of molybdopterin cofactor to aponitrate reductase from a carrier protein in Chlamydomonas reinhardtii.

A Chlamydomonas reinhardtii molybdenum cofactor (MoCo)-carrier protein (CP), capable of reconstituting nitrate reductase activity with apoprotein from the Neurospora crassa mutant nit-1, was subjected to experiments of diffusion through a dialysis membrane and gel filtration. CP bonded firmly MoCo and did not release it efficiently unless aponitrate reductase was present in the incubation mixture. Stability of MoCo bound to CP against air and heat was very similar to that of free-MoCo released from milk xanthine oxidase. Our data strongly suggest that MoCo is directly transferred from CP to aponitrate reductase to form an active enzyme.     

Identification by mutational analysis of four critical residues in the molybdenum cofactor domain of eukaryotic nitrate reductase

The nucleotide sequence of the nitrate reductase (NR) molybdenum cofactor (MoCo) domain was determined in four Nicotiana plumbaginifolia mutants affected in the NR apoenzyme gene. In each case, missense mutations were found in the MoCo domain which affected amino acids that were conserved not only among eukaryotic NRs but also in animal sulfite oxidase sequences. Moreover an abnormal NR molecular mass was observed in three mutants, suggesting that the integrity of the MoCo domain is essential for a proper assembly of holo-NR. These data allowed to pinpoint critical residues in the NR MoCo domain necessary for the enzyme activity but also important for its quaternary structure.     

Regulation of molybdenum cofactor species in the green alga Chlamydomonas reinhardtii

Molybdenum cofactor (MoCo) of molybdoenzymes is constitutively produced in cells of the green alga Chlamydomonas reinhardtii grown in ammonium media, under which conditions certain molybdoenzymes are not synthesized. In soluble form, MoCo was found to be present in several forms: (i) as a low Mr free species; (ii) bound to a MoCo-carrier protein of about 50 kDa that could release MoCo to directly reconstitute in vitro nitrate reductase activity in the nit-1 mutant of Neurospora crassa, but not to Thiol-Sepharose which, in contrast, bonded free MoCo; and (iii) bound to other proteins, putatively constitutive molybdoenzymes, which only released MoCo after a denaturing treatment. The amount of total MoCo (free, carrier-bound and heat releasable forms) was dependent on the growth phase of cell cultures. Constitutive levels of total MoCo in ammonium-grown cells markedly increased when cells were transferred to media lacking ammonium (nitrate, urea or nitrogen-free media). This increase did not require de novo protein synthesis and was stimulated by light. Levels of both total MoCo and free plus carrier-bound MoCo seemed to be unrelated to either nitrate reductase synthesis or functioning of nit-1 and nit-2 genes responsible for nitrate reductase structure and regulation, respectively. Results suggest that MoCo is continuously synthesized in C. reinhardtii and that its levels are regulated by ammonium in a way independent of nitrate reductase synthesis.     

A mutation in the gene for the neurotransmitter receptor-clustering protein gephyrin causes a novel form of molybdenum cofactor deficiency

Gephyrin was originally identified as a membrane-associated protein that is essential for the postsynaptic localization of receptors for the neurotransmitters glycine and GABA(A). A sequence comparison revealed homologies between gephyrin and proteins necessary for the biosynthesis of the universal molybdenum cofactor (MoCo). Because gephyrin expression can rescue a MoCo-deficient mutation in bacteria, plants, and a murine cell line, it became clear that gephyrin also plays a role in MoCo biosynthesis. Human MoCo deficiency is a fatal disease resulting in severe neurological damage and death in early childhood. Most patients harbor MOCS1 mutations, which prohibit formation of a precursor, or carry MOCS2 mutations, which abrogate precursor conversion to molybdopterin. The present report describes the identification of a gephyrin gene (GEPH) deletion in a patient with symptoms typical of MoCo deficiency. Biochemical studies of the patient's fibroblasts demonstrate that gephyrin catalyzes the insertion of molybdenum into molybdopterin and suggest that this novel form of MoCo deficiency might be curable by molybdate supplementation.     

Mutations of the silkworm molybdenum cofactor sulfurase gene,og, cause translucent larval skin

Normal silkworms (Bombyx mori) have opaque larval skin due to uric acid accumulation in the epidermis while a mutant, og, is translucent owing to a deficiency in xanthine dehydrogenase (XDH), which synthesizes uric acid. Molybdenum cofactor (MoCo) sulfurase is responsible for XDH activation in various organisms. A silkworm MoCo sulfurase gene was cloned and found to be on the og locus, whose mutant alleles, og(k) and og(t), show premature stop codons, proving that og is the MoCo sulfurase gene. It was observed that a miniature inverted-repeat transposable element (MITE), named Organdy, when inserted in an og(t) mutant allele exon, causes unstable splicing of a downstream intron leading to incomplete open reading frames.     

Quantitation of molybdopterin oxidation product in wild-type and molybdenum cofactor deficient mutants of Chlamydomonas reinhardtii.

A simple and reliable procedure of oxidation of molybdenum cofactor (MoCo) from molybdoenzymes by autoclaving samples at 120 degrees C for 20 min yielded a single predominant fluorescent species that could be quantitatively determined by reverse phase high performance liquid chromatography. This method allowed detection and quantitation of molybdopterin in cell-free extracts of the green alga Chlamydomonas reinhardtii. The MoCo oxidation product from C. reinhardtii has the same chromatographic and spectral properties as that of milk xanthine oxidase and chicken liver sulfite oxidase. The oxidized species was also detected in molybdenum cofactor mutants of Chlamydomonas reinhardtii defective at the nit-3, nit-4, nit-5/nit-6 and nit-7 loci, which strongly suggests that active molybdenum cofactor itself is not directly involved in the control of its own biosynthetic pathway.     

Molybdenum cofactor deficiency causes translucent integument,male-biased lethality,and flaccid paralysis in the silkworm Bombyx mori

Uric acid accumulates in the epidermis of Bombyx mori larvae and renders the larval integument opaque and white. Yamamoto translucent (oya) is a novel spontaneous mutant with a translucent larval integument and unique phenotypic characteristics, such as male-biased lethality and flaccid larval paralysis. Xanthine dehydrogenase (XDH) that requires a molybdenum cofactor (MoCo) for its activity is a key enzyme for uric acid synthesis. It has been observed that injection of a bovine xanthine oxidase, which corresponds functionally to XDH and contains its own MoCo activity, changes the integuments of oya mutants from translucent to opaque and white. This finding suggests that XDH/MoCo activity might be defective in oya mutants. Our linkage analysis identified an association between the oya locus and chromosome 23. Because XDH is not linked to chromosome 23 in B. mori, MoCo appears to be defective in oya mutants. In eukaryotes, MoCo is synthesized by a conserved biosynthesis pathway governed by four loci (MOCS1, MOCS2, MOCS3, and GEPH). Through a candidate gene approach followed by sequence analysis, a 6-bp deletion was detected in an exon of the B. mori molybdenum cofactor synthesis-step 1 gene (BmMOCS1) in the oya strain. Moreover, recombination was not observed between the oya and BmMOCS1 loci. These results indicate that the BmMOCS1 locus is responsible for the oya locus. Finally, we discuss the potential cause of male-biased lethality and flaccid paralysis observed in the oya mutants.     

Mechanism of gallic acid biosynthesis in bacteria (Escherichia coli) and walnut (Juglans regia)

Gallic acid (GA), a key intermediate in the synthesis of plant hydrolysable tannins, is also a primary anti-inflammatory, cardio-protective agent found in wine, tea, and cocoa. In this publication, we reveal the identity of a gene and encoded protein essential for GA synthesis. Although it has long been recognized that plants, bacteria, and fungi synthesize and accumulate GA, the pathway leading to its synthesis was largely unknown. Here we provide evidence that shikimate dehydrogenase (SDH), a shikimate pathway enzyme essential for aromatic amino acid synthesis, is also required for GA production. Escherichia coli (E. coli) aroE mutants lacking a functional SDH can be complemented with the plant enzyme such that they grew on media lacking aromatic amino acids and produced GA in vitro. Transgenic Nicotiana tabacum lines expressing a Juglans regia SDH exhibited a 500% increase in GA accumulation. The J. regia and E. coli SDH was purified via overexpression in E. coli and used to measure substrate and cofactor kinetics, following reduction of NADP(+) to NADPH. Reversed-phase liquid chromatography coupled to electrospray mass spectrometry (RP-LC/ESI-MS) was used to quantify and validate GA production through dehydrogenation of 3-dehydroshikimate (3-DHS) by purified E. coli and J. regia SDH when shikimic acid (SA) or 3-DHS were used as substrates and NADP(+) as cofactor. Finally, we show that purified E. coli and J. regia SDH produced GA in vitro.     

Tissue-specific regulation of rice molybdenum cofactor sulfurase gene in response to salt stress and ABA

Molybdenum-containing aldehyde oxidase is a key enzyme for catalyzing the final step of abscisic acid (ABA) biosynthesis in plants. Sulfuration of the molybdenum cofactor (MoCo) is an essential step for activating aldehyde oxidase. The molybdenum cofactor sulfurase (MCSU) that transfers the sulfur ligand to aldehyde oxidase-bound MoCo is thus considered an important factor in regulating the ABA levels in plant tissues. In this study, we identified the rice MCSU cDNA (OsMCSU), which is the first MCSU gene cloned in monocot species. According to the functional domain analysis of the predicted amino acid sequence, the OsMCSU protein contains a Nifs domain at its N-terminus and a MOSC domain at the C-terminus. Expression of the OsMCSU gene was up-regulated by salt stress in root tissues of rice seedlings, but this effect was not observed in leaf tissues. In roots, regulations of OsMCSU expressions could be mediated by both ABA-dependent and ABA-independent signaling pathways under salt stress condition.     

Hypersensitivity of Escherichia coli Delta(uvrB-bio) mutants to 6-hydroxylaminopurine and other base analogs is due to a defect in molybdenum cofactor biosynthesis

We have shown previously that Escherichia coli and Salmonella enterica serovar Typhimurium strains carrying a deletion of the uvrB-bio region are hypersensitive to the mutagenic and toxic action of 6-hydroxylaminopurine (HAP) and related base analogs. This sensitivity is not due to the uvrB excision repair defect associated with this deletion because a uvrB point mutation or a uvrA deficiency does not cause hypersensitivity. In the present work, we have investigated which gene(s) within the deleted region may be responsible for this effect. Using independent approaches, we isolated both a point mutation and a transposon insertion in the moeA gene, which is located in the region covered by the deletion, that conferred HAP sensitivity equal to that conferred by the uvrB-bio deletion. The moeAB operon provides one of a large number of genes responsible for biosynthesis of the molybdenum cofactor. Defects in other genes in the same pathway, such as moa or mod, also lead to the same HAP-hypersensitive phenotype. We propose that the molybdenum cofactor is required as a cofactor for an as yet unidentified enzyme (or enzymes) that acts to inactivate HAP and other related compounds.     

Acyl carrier protein is a cellular target for the antibacterial action of the pantothenamide class of pantothenate antimetabolites

Pantothenate is the precursor of the essential cofactor coenzyme A (CoA). Pantothenate kinase (CoaA) catalyzes the first and regulatory step in the CoA biosynthetic pathway. The pantothenate analogs N-pentylpantothenamide and N-heptylpantothenamide possess antibiotic activity against Escherichia coli. Both compounds are substrates for E. coli CoaA and competitively inhibit the phosphorylation of pantothenate. The phosphorylated pantothenamides are further converted to CoA analogs, which were previously predicted to act as inhibitors of CoA-dependent enzymes. Here we show that the mechanism for the toxicity of the pantothenamides is due to the inhibition of fatty acid biosynthesis through the formation and accumulation of the inactive acyl carrier protein (ACP), which was easily observed as a faster migrating protein using conformationally sensitive gel electrophoresis. E. coli treated with the pantothenamides lost the ability to incorporate [1-(14)C]acetate to its membrane lipids, indicative of the inhibition of fatty acid synthesis. Cellular CoA was maintained at the level sufficient for bacterial protein synthesis. Electrospray ionization time-of-flight mass spectrometry confirmed that the inactive ACP was the product of the transfer of the inactive phosphopantothenamide moiety of the CoA analog to apo-ACP, forming the ACP analog that lacks the sulfhydryl group for the attachment of acyl chains for fatty acid synthesis. Inactive ACP accumulated in pantothenamide-treated cells because of the active hydrolysis of regular ACP and the slow turnover of the inactive prosthetic group. Thus, the pantothenamides are pro-antibiotics that inhibit fatty acid synthesis and bacterial growth because of the covalent modification of ACP.     

The Arabidopsis LOS5/ABA3 locus encodes a molybdenum cofactor sulfurase and modulates cold stress- and osmotic stress-responsive gene expression

To understand low temperature and osmotic stress signaling in plants, we isolated and characterized two allelic Arabidopsis mutants, los5-1 and los5-2, which are impaired in gene induction by cold and osmotic stresses. Expression of RD29A-LUC (the firefly luciferase reporter gene under the control of the stress-responsive RD29A promoter) in response to cold and salt/drought is reduced in the los5 mutants, but the response to abscisic acid (ABA) remains unaltered. RNA gel blot analysis indicates that the los5 mutation reduces the induction of several stress-responsive genes by cold and severely diminishes or even completely blocks the induction of RD29A, COR15, COR47, RD22, and P5CS by osmotic stresses. los5 mutant plants are compromised in their tolerance to freezing, salt, or drought stress. los5 plants are ABA deficient, as indicated by increased transpirational water loss and reduced accumulation of ABA under drought stress in the mutant. A comparison with another ABA-deficient mutant, aba1, reveals that the impaired low-temperature gene regulation is specific to the los5 mutation. Genetic tests suggest that los5 is allelic to aba3. Map-based cloning reveals that LOS5/ABA3 encodes a molybdenum cofactor (MoCo) sulfurase. MoCo sulfurase catalyzes the generation of the sulfurylated form of MoCo, a cofactor required by aldehyde oxidase that functions in the last step of ABA biosynthesis in plants. The LOS5/ABA3 gene is expressed ubiquitously in different plant parts, and the expression level increases in response to drought, salt, or ABA treatment. Our results show that LOS5/ABA3 is a key regulator of ABA biosynthesis, stress-responsive gene expression, and stress tolerance.     

Structure--activity study of the antibacterial peptide fallaxin

Fallaxin is a 25-mer antibacterial peptide amide, which was recently isolated from the West Indian mountain chicken frog Leptodactylus fallax. Fallaxin has been shown to inhibit the growth of several Gram-negative bacteria including Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Here, we report a structure-activity study of fallaxin based on 65 analogs, including a complete alanine scan and a full set of N- and C-terminal truncated analogs. The fallaxin analogs were tested for hemolytic activity and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate resistant S. aureus, (VISA), methicillin-susceptible S. aureus (MSSA), E. coli, K. pneumoniae, and P. aeruginosa. We identified several analogs, which showed improved antibacterial activity compared to fallaxin. Our best candidate was FA12, which displayed MIC values of 3.12, 25, 25, and 50 muM against E. coli, K. pneumoniae, MSSA, and VISA, respectively. Furthermore, we correlated the antibacterial activity with various structural parameters such as charge, hydrophobicity H, mean hydrophobic moment mu(H), and alpha-helicity. We were able to group the active and inactive analogs according to mean hydrophobicity H and mean hydrophobic moment mu(H). Far-UV CD-spectroscopy experiments on fallaxin and several analogs in buffer, in TFE, and in membrane mimetic environments (small unilamellar vesicles) indicated that a coiled-coil conformation could be an important structural trait for antibacterial activity. This study provides data that support fallaxin analogs as promising lead structures in the development of new antibacterial agents.     

Functional analysis of molybdopterin biosynthesis in mycobacteria identifies a fused molybdopterin synthase in Mycobacterium tuberculosis

Most mycobacterial species possess a full complement of genes for the biosynthesis of molybdenum cofactor (MoCo). However, a distinguishing feature of members of the Mycobacterium tuberculosis complex is their possession of multiple homologs associated with the first two steps of the MoCo biosynthetic pathway. A mutant of M. tuberculosis lacking the moaA1-moaD1 gene cluster and a derivative in which moaD2 was also deleted were significantly impaired for growth in media containing nitrate as a sole nitrogen source, indicating a reduced availability of MoCo to support the assimilatory function of the MoCo-dependent nitrate reductase, NarGHI. However, the double mutant displayed residual respiratory nitrate reductase activity, suggesting that it retains the capacity to produce MoCo. The M. tuberculosis moaD and moaE homologs were further analyzed by expressing these genes in mutant strains of M. smegmatis that lacked one or both of the sole molybdopterin (MPT) synthase-encoding genes, moaD2 and moaE2, and were unable to grow on nitrate, presumably as a result of the loss of MoCo-dependent nitrate assimilatory activity. Expression of M. tuberculosis moaD2 in the M. smegmatis moaD2 mutant and of M. tuberculosis moaE1 or moaE2 in the M. smegmatis moaE2 mutant restored nitrate assimilation, confirming the functionality of these genes in MPT synthesis. Expression of M. tuberculosis moaX also restored MoCo biosynthesis in M. smegmatis mutants lacking moaD2, moaE2, or both, thus identifying MoaX as a fused MPT synthase. By implicating multiple synthase-encoding homologs in MoCo biosynthesis, these results suggest that important cellular functions may be served by their expansion in M. tuberculosis.