Cloning and Characterization of AtRGP1 : A Reversibly Autoglycosylated Arabidopsis Protein Implicated in Cell Wall Biosynthesis

A reversibly glycosylated polypeptide from pea (Pisum sativum) is thought to have a role in the biosynthesis of hemicellulosic polysaccharides. We have investigated this hypothesis by isolating a cDNA clone encoding a homolog of Arabidopsis thaliana, Reversibly Glycosylated Polypeptide-1 (AtRGP1), and preparing antibodies against the protein encoded by this gene. Polyclonal antibodies detect homologs in both dicot and monocot species. The patterns of expression and intracellular localization of the protein were examined. AtRGP1 protein and RNA concentration are highest in roots and suspension-cultured cells. Localization of the protein shows it to be mostly soluble but also peripherally associated with membranes. We confirmed that AtRGP1 produced in Escherichia coli could be reversibly glycosylated using UDP-glucose and UDP-galactose as substrates. Possible sites for UDP-sugar binding and glycosylation are discussed. Our results are consistent with a role for this reversibly glycosylated polypeptide in cell wall biosynthesis, although its precise role is still unknown.The primary cell wall of dicot plants is laid down by young cells prior to the cessation of elongation and secondary wall deposition. Making up to 90% of the cell''s dry weight, the extracellular matrix is important for many processes, including morphogenesis, growth, disease resistance, recognition, signaling, digestibility, nutrition, and decay. The composition of the cell wall has been extensively described (Bacic et al., 1988; Levy and Staehelin, 1992; Zablackis et al., 1995), and yet many questions remain unanswered regarding the synthesis and interaction of these components to provide cells with a functional wall (Carpita and Gibeaut, 1993; Carpita et al., 1996).Heteropolysaccharide biosynthesis can be divided into four steps: (a) chain or backbone initiation, (b) elongation, (c) side-chain addition, and (d) termination and extracellular deposition (Waldron and Brett, 1985). The similarity between various polysaccharide backbones leads to the prediction that the synthesizing machinery would be conserved between them. For example, the backbone of xyloglucan polymers, β-1,4 glucan, can be synthesized independently of or concurrently with side-chain addition (Campbell et al., 1988; White et al., 1993), and this polymer and the chains that make up cellulose are identical. The later addition of side chains to xyloglucan are catalyzed by specific transferases (Kleene and Berger, 1993) such as xylosyltransferase (Campbell et al., 1988), galactosyltransferase, and fucosyltransferase (Faïk et al., 1997), all of which are localized to the Golgi compartment (Brummell et al., 1990; Driouich et al., 1993; Staehelin and Moore, 1995).The enzymes involved in wall biosynthesis have been recalcitrant to isolation (Carpita et al., 1996; Albersheim et al., 1997). Only recently has the first gene encoding putative cellulose biosynthetic enzymes, celA, been isolated from cotton (Gossypium hirsutum) and rice (Oryza sativa; Pear et al., 1996).During studies of polysaccharide synthesis in pea (Pisum sativum) Golgi membranes, Dhugga et al. (1991) identified a 41-kD protein doublet that they suggested was involved in polysaccharide synthesis. The authors showed that this protein could be glycosylated by radiolabeled UDP-Glc but that this labeling could be reversibly competed with by unlabeled UDP-Glc, UDP-Xyl, and UDP-Gal, the sugars that make up xyloglucan (Hayashi, 1989). The 41-kD protein was named PsRGP1 (P. sativum Reversibly Glycosylated Polypeptide-1; Dhugga et al., 1997). Furthermore, the conditions that stimulate or inhibit Golgi-localized β-glucan synthase activity are the same conditions that stimulate or inhibit the glycosylation of PsRGP1 (Dhugga et al., 1991). To address the role of this protein in polysaccharide synthesis, the authors purified the polypeptides and obtained the sequences from tryptic peptides (Dhugga and Ray, 1994). Antibodies raised against PsRGP1 showed that it is soluble and localized to the plasma membrane (Dhugga et al., 1991) and Golgi compartment (Dhugga et al., 1997). In addition to its Golgi localization, the steady-state glycosylation of PsRGP1 is approximately 10:7:3 (UDP-Glc:-Xyl:-Gal), which is similar to the typical sugar composition of xyloglucan (1.0:0.75:0.25; Dhugga et al., 1997).We were interested in studying various aspects of cell wall metabolism, including the synthesis of polysaccharides and their delivery to the cell wall. Studies in pea have shown that a 41-kD protein may be involved in cell wall polysaccharide synthesis, possibly that of xyloglucan (Dhugga et al., 1997). Here we report the characterization of AtRGP1 (Arabidopsis thaliana Reversibly Glycosylated Polypeptide-1), a soluble protein that can also be found weakly associated with membrane fractions, most likely the Golgi fraction. The reversible nature of the glycosylation of this Arabidopsis homolog by the substrates used to make polysaccharides (nucleotide sugars) suggests a possible role for AtRGP1 in polysaccharide biosynthesis.     

The GA2 Locus of Arabidopsis thaliana Encodes ent-Kaurene Synthase of Gibberellin Biosynthesis

The ga2 mutant of Arabidopsis thaliana is a gibberellin-deficient dwarf. Previous biochemical studies have suggested that the ga2 mutant is impaired in the conversion of copalyl diphosphate to ent-kaurene, which is catalyzed by ent-kaurene synthase (KS). Overexpression of the previously isolated KS cDNA from pumpkin (Cucurbita maxima) (CmKS) in the ga2 mutant was able to complement the mutant phenotype. A genomic clone coding for KS, AtKS, was isolated from A. thaliana using CmKS cDNA as a heterologous probe. The corresponding A. thaliana cDNA was isolated and expressed in Escherichia coli as a fusion protein. The fusion protein showed enzymatic activity that converted [³H]copalyl diphosphate to [³H]ent-kaurene. The recombinant AtKS protein derived from the ga2–1 mutant is truncated by 14 kD at the C-terminal end and does not contain significant KS activity in vitro. Sequence analysis revealed that a C-2099 to T base substitution, which converts Gln-678 codon to a stop codon, is present in the AtKS cDNA from the ga2–1 mutant. Taken together, our results show that the GA2 locus encodes KS.     

An in Vitro System from Maize Seedlings for Tryptophan-Independent Indole-3-Acetic Acid Biosynthesis

The enzymatic synthesis of indole-3-acetic acid (IAA) from indole by an in vitro preparation from maize (Zea mays L.) that does not use tryptophan (Trp) as an intermediate is described. Light-grown seedlings of normal maize and the maize mutant orange pericarp were shown to contain the necessary enzymes to convert [¹⁴C]indole to IAA. The reaction was not inhibited by unlabeled Trp and neither [¹⁴C]Trp nor [¹⁴C]serine substituted for [¹⁴C]indole in this in vitro system. The reaction had a pH optimum greater than 8.0, required a reducing environment, and had an oxidation potential near that of ascorbate. The results obtained with this in vitro enzyme preparation provide strong, additional evidence for the presence of a Trp-independent IAA biosynthesis pathway in plants.     

Cloning of Nicotianamine Synthase Genes, Novel Genes Involved in the Biosynthesis of Phytosiderophores

Nicotianamine synthase (NAS), the key enzyme in the biosynthetic pathway for the mugineic acid family of phytosiderophores, catalyzes the trimerization of S-adenosylmethionine to form one molecule of nicotianamine. We purified NAS protein and isolated the genes nas1, nas2, nas3, nas4, nas5-1, nas5-2, and nas6, which encode NAS and NAS-like proteins from Fe-deficient barley (Hordeum vulgare L. cv Ehimehadaka no. 1) roots. Escherichia coli expressing nas1 showed NAS activity, confirming that this gene encodes a functional NAS. Expression of nas genes as determined by northern-blot analysis was induced by Fe deficiency and was root specific. The NAS genes form a multigene family in the barley and rice genomes.     

Mycobacterium tuberculosis Glucosyl-3-Phosphoglycerate Synthase: Structure of a Key Enzyme in Methylglucose Lipopolysaccharide Biosynthesis

Tuberculosis constitutes today a serious threat to human health worldwide, aggravated by the increasing number of identified multi-resistant strains of Mycobacterium tuberculosis, its causative agent, as well as by the lack of development of novel mycobactericidal compounds for the last few decades. The increased resilience of this pathogen is due, to a great extent, to its complex, polysaccharide-rich, and unusually impermeable cell wall. The synthesis of this essential structure is still poorly understood despite the fact that enzymes involved in glycosidic bond synthesis represent more than 1% of all M. tuberculosis ORFs identified to date. One of them is GpgS, a retaining glycosyltransferase (GT) with low sequence homology to any other GTs of known structure, which has been identified in two species of mycobacteria and shown to be essential for the survival of M. tuberculosis. To further understand the biochemical properties of M. tuberculosis GpgS, we determined the three-dimensional structure of the apo enzyme, as well as of its ternary complex with UDP and 3-phosphoglycerate, by X-ray crystallography, to a resolution of 2.5 and 2.7 Å, respectively. GpgS, the first enzyme from the newly established GT-81 family to be structurally characterized, displays a dimeric architecture with an overall fold similar to that of other GT-A-type glycosyltransferases. These three-dimensional structures provide a molecular explanation for the enzyme''s preference for UDP-containing donor substrates, as well as for its glucose versus mannose discrimination, and uncover the structural determinants for acceptor substrate selectivity. Glycosyltransferases constitute a growing family of enzymes for which structural and mechanistic data urges. The three-dimensional structures of M. tuberculosis GpgS now determined provide such data for a novel enzyme family, clearly establishing the molecular determinants for substrate recognition and catalysis, while providing an experimental scaffold for the structure-based rational design of specific inhibitors, which lay the foundation for the development of novel anti-tuberculosis therapies.     

拟南芥psy基因cDNA的克隆及其植物表达载体的构建

为了获得胚乳组织特异性表达八氢番茄红素的转基因小麦,以拟南芥幼叶RNA为模板,由特异型引物通过RT-PCR一步法得到大小约为1.3kb的基因片段,将此片段连接在克隆载体pMD18-T进行测序,结果表明,该基因片段为八氢番茄红素合成酶基因(psy)cDNA片段。将psy基因片段正向插入植物表达载体pLRPT中高分子量麦谷蛋白亚基基因1Dx5启动子与nos终止子之间,pLRPT载体无1Dx5基因开放阅读框,运用菌落PCR对重组子进行筛选与鉴定,说明拟南芥psy基因已正确插入pL-RPT,成功构建了植物表达载体pLRPTPSY。     

Purification and cDNA Cloning of Isochorismate Synthase from Elicited Cell Cultures of Catharanthus roseus

Isochorismate is an important metabolite formed at the end of the shikimate pathway, which is involved in the synthesis of both primary and secondary metabolites. It is synthesized from chorismate in a reaction catalyzed by the enzyme isochorismate synthase (ICS; EC 5.4.99.6). We have purified ICS to homogeneity from elicited Catharanthus roseus cell cultures. Two isoforms with an apparent molecular mass of 64 kD were purified and characterized. The K_m values for chorismate were 558 and 319 μm for isoforms I and II, respectively. The isoforms were not inhibited by aromatic amino acids and required Mg²⁺ for enzyme activity. Polymerase chain reaction on a cDNA library from elicited C. roseus cells with a degenerated primer based on the sequence of an internal peptide from isoform II resulted in an amplification product that was used to screen the cDNA library. This led to the first isolation, to our knowledge, of a plant ICS cDNA. The cDNA encodes a protein of 64 kD with an N-terminal chloroplast-targeting signal. The deduced amino acid sequence shares homology with bacterial ICS and also with anthranilate synthases from plants. Southern analysis indicates the existence of only one ICS gene in C. roseus.The shikimate pathway is a major pathway in primary and secondary plant metabolism (Herrmann, 1995). It provides chorismate for the synthesis of the aromatic amino acids Phe, Tyr, and Trp, which are used in protein biosynthesis, but also serves as a precursor for a wide variety of aromatic substances (Herrmann, 1995; Weaver and Hermann, 1997; Fig. ​Fig.1a).1a). Chorismate is also the starting point of a biosynthetic pathway leading to phylloquinones (vitamin K₁) and anthraquinones (Poulsen and Verpoorte, 1991). The first committed step in this pathway is the conversion of chorismate into isochorismate, which is catalyzed by ICS (Poulsen and Verpoorte, 1991; Fig. ​Fig.1b).1b). Its substrate, chorismate, plays a pivotal role in the synthesis of shikimate-pathway-derived compounds, and its distribution over the various pathways is expected to be tightly regulated. Elicited cell cultures of Catharanthus roseus provide an example of the partitioning of chorismate. Concurrently, these cultures produce both Trp-derived indole alkaloids and DHBA (Moreno et al., 1994). In bacteria DHBA is synthesized from isochorismate (Young et al., 1969). Elicitation of C. roseus cell cultures with a fungal extract induces not only several enzymes of the indole alkaloid biosynthetic pathway (Pasquali et al., 1992) but also ICS (Moreno et al., 1994). Information concerning the expression and biochemical characteristics of the enzymes that compete for available chorismate (ICS, CM, and AS) may help us to understand the regulation of the distribution of this precursor over the various pathways. Such information is already available for CM (Eberhard et al., 1996) and AS (Poulsen et al., 1993; Bohlmann et al., 1995) but not for ICS. Figure 1a, Position of ICS in the plant metabolism. SA, Salicylic acid, OSB, o-succinylbenzoic acid. b, Reaction catalyzed by ICS.Isochorismate plays an important role in bacterial and plant metabolism as a precursor of o-succinylbenzoic acid, an intermediate in the biosynthesis of menaquinones (vitamin K₂) (Weische and Leistner, 1985) and phylloquinones (vitamin K₁; Poulsen and Verpoorte, 1991). In bacteria isochorismate is also a precursor of siderophores such as DHBA (Young et al., 1969), enterobactin (Walsh et al., 1990), amonabactin (Barghouthi et al., 1991), and salicylic acid (Serino et al., 1995). Although evidence from tobacco would indicate that salicylic acid in plants is derived from Phe via benzoic acid (Yalpani et al., 1993; Lee et al., 1995; Coquoz et al., 1998), it cannot be excluded that it is also synthesized from isochorismate. In the secondary metabolism of higher plants, isochorismate is a precursor for the biosynthesis of anthraquinones (Inoue et al., 1984; Sieweke and Leistner, 1992), naphthoquinones (Müller and Leistner, 1978), catalpalactone (Inouye et al., 1975), and certain alkaloids in orchids (Leete and Bodem, 1976).ICS was first extracted and partially purified from crude extracts of Aerobacter aerogenes (Young and Gibson, 1969). Later, ICS activity was detected in protein extracts of cell cultures from plants of the Rubiaceae, Celastraceae, and Apocynaceae families (Ledüc et al., 1991; Poulsen et al., 1991; Poulsen and Verpoorte, 1992). Genes encoding ICS have been cloned from bacteria such as Escherichia coli (Ozenberger et al., 1989), Pseudomonas aeruginosa (Serino et al., 1995), Aeromonas hydrophila (Barghouthi et al., 1991), Flavobacterium K_3–15 (Schaaf et al., 1993), Hemophilus influenzae (Fleischmann et al., 1995), and Bacillus subtilis (Rowland and Taber, 1996). Both E. coli and B. subtilis have two distinct ICS genes; one is involved in siderophore biosynthesis and the other is involved in menaquinone production (Daruwala et al., 1996, 1997; Müller et al., 1996; Rowland and Taber, 1996). The biochemical properties of the two ICS enzymes from E. coli are different (Daruwala et al., 1997; Liu et al., 1990). Sequence analysis has revealed that the bacterial ICS enzymes share homology with the chorismate-utilizing enzymes AS and p-aminobenzoate synthase, suggesting that they share a common evolutionary origin (Ozenberger et al., 1989).Much biochemical and molecular data concerning the shikimate pathway in plants have accumulated in recent years (Schmid and Amrhein, 1995; Weaver and Hermann, 1997), but relatively little work has been done on ICS from higher plants. The enzyme has been partially purified from Galium mollugo cell cultures (Ledüc et al., 1991, 1997), but purification of the ICS protein to homogeneity has remained elusive, probably because of instability of the enzyme.Our interests focus on the role of ICS in the regulation of chorismate partitioning over the various pathways. Furthermore, we studied ICS in C. roseus to gain insight into the biosynthesis of DHBA in higher plants (Moreno et al., 1994). In this paper we report the first purification, to our knowledge, of ICS to homogeneity from a plant source and the cloning of the corresponding cDNA.     

Microsomal Electron Transfer in Higher Plants: Cloning and Heterologous Expression of NADH-Cytochrome b5 Reductase from Arabidopsis

AtCBR, a cDNA encoding NADH-cytochrome (Cyt) b₅ reductase, and AtB5-A and AtB5-B, two cDNAs encoding Cyt b₅, were isolated from Arabidopsis. The primary structure deduced from the AtCBR cDNA was 40% identical to those of the NADH-Cyt b₅ reductases of yeast and mammals. A recombinant AtCBR protein prepared using a baculovirus system exhibited typical spectral properties of NADH-Cyt b₅ reductase and was used to study its electron-transfer activity. The recombinant NADH-Cyt b₅ reductase was functionally active and displayed strict specificity to NADH for the reduction of a recombinant Cyt b₅ (AtB5-A), whereas no Cyt b₅ reduction was observed when NADPH was used as the electron donor. Conversely, a recombinant NADPH-Cyt P450 reductase of Arabidopsis was able to reduce Cyt b₅ with NADPH but not with NADH. To our knowledge, this is the first evidence in higher plants that both NADH-Cyt b₅ reductase and NADPH-Cyt P450 reductase can reduce Cyt b₅ and have clear specificities in terms of the electron donor, NADH or NADPH, respectively. This substrate specificity of the two reductases is discussed in relation to the NADH- and NADPH-dependent activities of microsomal fatty acid desaturases.     

Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguinis

Streptococcus sanguinis is a cause of infective endocarditis and has been shown to require a manganese transporter called SsaB for virulence and O₂ tolerance. Like certain other pathogens, S. sanguinis possesses aerobic class Ib (NrdEF) and anaerobic class III (NrdDG) ribonucleotide reductases (RNRs) that perform the essential function of reducing ribonucleotides to deoxyribonucleotides. The accompanying paper (Makhlynets, O., Boal, A. K., Rhodes, D. V., Kitten, T., Rosenzweig, A. C., and Stubbe, J. (2014) J. Biol. Chem. 289, 6259–6272) indicates that in the presence of O₂, the S. sanguinis class Ib RNR self-assembles an essential diferric-tyrosyl radical (Fe^III₂-Y^•) in vitro, whereas assembly of a dimanganese-tyrosyl radical (Mn^III₂-Y^•) cofactor requires NrdI, and Mn^III₂-Y^• is more active than Fe^III₂-Y^• with the endogenous reducing system of NrdH and thioredoxin reductase (TrxR1). In this study, we have shown that deletion of either nrdHEKF or nrdI completely abolishes virulence in an animal model of endocarditis, whereas nrdD mutation has no effect. The nrdHEKF, nrdI, and trxR1 mutants fail to grow aerobically, whereas anaerobic growth requires nrdD. The nrdJ gene encoding an O₂-independent adenosylcobalamin-cofactored RNR was introduced into the nrdHEKF, nrdI, and trxR1 mutants. Growth of the nrdHEKF and nrdI mutants in the presence of O₂ was partially restored. The combined results suggest that Mn^III₂-Y^•-cofactored NrdF is required for growth under aerobic conditions and in animals. This could explain in part why manganese is necessary for virulence and O₂ tolerance in many bacterial pathogens possessing a class Ib RNR and suggests NrdF and NrdI may serve as promising new antimicrobial targets.     

浙贝母角鲨烯合酶全长cDNA的克隆及功能分析

鲨烯是甾醇和其他三萜类化合物的关键代谢中间体,其生物合成由鲨烯合酶（squalene synthase,SQS）催化,该酶将2分子法呢基焦磷酸转化为鲨烯。浙贝母异甾体生物碱的生物合成途径与三萜类化合物类似。在本研究中,基于cDNA末端的快速扩增（RACE）技术克隆了浙贝母鲨烯合酶 (FtSQS）基因的全长cDNA,GenBank登录号为KF551097.2。通过生物信息学方法对FtSQS进行详细表征,包括保守区检测、序列同源分析、二级和三级结构预测及系统发育树分析。结果表明,其开放阅读框（ORF）为1 230 bp,编码409个氨基酸,FtSQS氨基酸序列与印度甘松、截形苜蓿、紫衫、马铃薯、柴胡、金铁锁和拟南芥的SQS氨基酸同源性分别达到73.84%、73.23%、72.24%、70.66%、70.66%、69.44%和68.14%。启动子分析表明,FtSQS的5′上游区域具有与生理和环境因素相关的各种潜在因素。为了获得可溶性FtSQS表达,从羧基末端截断24个疏水氨基酸,构建了原核表达载体pGEX-2T-FtSQSΔTM,并在大肠杆菌BL21(DE3)中表达。SDS-PAGE检测到约66 kD的重组FtSQSΔTM蛋白。体外酶促反应证明,FtSQS可以催化FPP转化成鲨烯。qRT-PCR分析FtSQS mRNA在叶中的表达量最高,茎、根次之,而在鳞茎中表达水平最低。这提示,叶子是浙贝母碱生物合成的主要活性器官。FtSQS的鉴定及功能研究为浙贝母次生代谢产物的研究提供了重要依据。     

Haloferax volcanii N-Glycosylation: Delineating the Pathway of dTDP-rhamnose Biosynthesis

In the halophilic archaea Haloferax volcanii, the surface (S)-layer glycoprotein can be modified by two distinct N-linked glycans. The tetrasaccharide attached to S-layer glycoprotein Asn-498 comprises a sulfated hexose, two hexoses and a rhamnose. While Agl11-14 have been implicated in the appearance of the terminal rhamnose subunit, the precise roles of these proteins have yet to be defined. Accordingly, a series of in vitro assays conducted with purified Agl11-Agl14 showed these proteins to catalyze the stepwise conversion of glucose-1-phosphate to dTDP-rhamnose, the final sugar of the tetrasaccharide glycan. Specifically, Agl11 is a glucose-1-phosphate thymidylyltransferase, Agl12 is a dTDP-glucose-4,6-dehydratase and Agl13 is a dTDP-4-dehydro-6-deoxy-glucose-3,5-epimerase, while Agl14 is a dTDP-4-dehydrorhamnose reductase. Archaea thus synthesize nucleotide-activated rhamnose by a pathway similar to that employed by Bacteria and distinct from that used by Eukarya and viruses. Moreover, a bioinformatics screen identified homologues of agl11-14 clustered in other archaeal genomes, often as part of an extended gene cluster also containing aglB, encoding the archaeal oligosaccharyltransferase. This points to rhamnose as being a component of N-linked glycans in Archaea other than Hfx. volcanii.     

Ixodes ricinus Tick Lipocalins: Identification,Cloning, Phylogenetic Analysis and Biochemical Characterization

Background

During their blood meal, ticks secrete a wide variety of proteins that interfere with their host''s defense mechanisms. Among these proteins, lipocalins play a major role in the modulation of the inflammatory response.

Methodology/Principal Findings

Screening a cDNA library in association with RT-PCR and RACE methodologies allowed us to identify 14 new lipocalin genes in the salivary glands of the Ixodes ricinus hard tick. A computational in-depth structural analysis confirmed that LIRs belong to the lipocalin family. These proteins were called LIR for “Lipocalin from I. ricinus” and numbered from 1 to 14 (LIR1 to LIR14). According to their percentage identity/similarity, LIR proteins may be assigned to 6 distinct phylogenetic groups. The mature proteins have calculated pM and pI varying from 21.8 kDa to 37.2 kDa and from 4.45 to 9.57 respectively. In a western blot analysis, all recombinant LIRs appeared as a series of thin bands at 50–70 kDa, suggesting extensive glycosylation, which was experimentally confirmed by treatment with N-glycosidase F. In addition, the in vivo expression analysis of LIRs in I. ricinus, examined by RT-PCR, showed homogeneous expression profiles for certain phylogenetic groups and relatively heterogeneous profiles for other groups. Finally, we demonstrated that LIR6 codes for a protein that specifically binds leukotriene B4.

Conclusions/Significance

This work confirms that, regarding their biochemical properties, expression profile, and sequence signature, lipocalins in Ixodes hard tick genus, and more specifically in the Ixodes ricinus species, are segregated into distinct phylogenetic groups suggesting potential distinct function. This was particularly demonstrated by the ability of LIR6 to scavenge leukotriene B4. The other LIRs did not bind any of the ligands tested, such as 5-hydroxytryptamine, ADP, norepinephrine, platelet activating factor, prostaglandins D2 and E2, and finally leukotrienes B4 and C4.     

A lipA (yutB) Mutant,Encoding Lipoic Acid Synthase,Provides Insight into the Interplay between Branched-Chain and Unsaturated Fatty Acid Biosynthesis in Bacillus subtilis

Lipoic acid is an essential cofactor required for the function of key metabolic pathways in most organisms. We report the characterization of a Bacillus subtilis mutant obtained by disruption of the lipA (yutB) gene, which encodes lipoyl synthase (LipA), the enzyme that catalyzes the final step in the de novo biosynthesis of this cofactor. The function of lipA was inferred from the results of genetic and physiological experiments, and this study investigated its role in B. subtilis fatty acid metabolism. Interrupting lipoate-dependent reactions strongly inhibits growth in minimal medium, impairing the generation of branched-chain fatty acids and leading to accumulation of copious amounts of straight-chain saturated fatty acids in B. subtilis membranes. Although depletion of LipA induces the expression of the Δ5 desaturase, controlled by a two-component system that senses changes in membrane properties, the synthesis of unsaturated fatty acids is insufficient to support growth in the absence of precursors for branched-chain fatty acids. However, unsaturated fatty acids generated by deregulated overexpression of the Δ5 desaturase functionally replaces lipoic acid-dependent synthesis of branched-chain fatty acids. Furthermore, we show that the cold-sensitive phenotype of a B. subtilis strain deficient in Δ5 desaturase is suppressed by isoleucine only if LipA is present.Lipoic acid (LA; 6,8-thioctic acid or 1,2-dithiolane-3-pentanoic acid) is a sulfur-containing cofactor required for the function of several key enzymes involved in oxidative and single-carbon metabolism, including pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, branched-chain 2-oxoacid dehydrogenase (BCKADH), acetoin dehydrogenase, and the glycine cleavage system (10). Lipoate-requiring complexes typically contain three protein subunits, E1, E2, and E3. LA is linked through an amide bond to lysine residues in the E2 subunits (42) and acts as a swinging arm, transferring covalently attached reaction intermediates among the active sites of the enzyme complexes (40).Although the general role of LA as a bound cofactor has been known for decades, the mechanisms by which LA is synthesized and becomes linked to its cognate proteins in different organisms continue to be elucidated. The reactions whereby LA-modified proteins are produced are best understood in Escherichia coli. In this organism, lipoylation is mediated by two separate enzymes, lipoyl protein ligase A (LplA) and octanoyl-acyl carrier protein-protein transferase (LipB) (30, 31). While LplA uses exogenous LA, LipB transfers endogenous octanoic acid to the target proteins (19). These octanoylated domains are then converted into lipoylated derivatives by the S-adenosyl-l-methionine-dependent enzyme lipoyl synthase (LipA), which catalyzes the insertion of sulfur atoms into the carbon-6 and -8 positions of the corresponding fatty acids (29). This process bypasses the requirement for an exogenous supply of LA.In contrast to the wealth of knowledge available on LA synthesis and utilization in E. coli, the existing information about these pathways in gram-positive bacteria is scarce. It has been found that Listeria monocytogenes mutants defective in proteins homologous to the E. coli LplA enzymes are unable to scavenge exogenous LA for modification of lipoyl domains (22, 23, 38). However, L. monocytogenes is a natural lipoate auxotroph since it does not encode the enzymes necessary for lipoate biosynthesis (15, 55). Bacillus subtilis synthesizes LA, but the biosynthesis, attachment, and function of this essential nutrient in this model gram-positive organism have not yet been studied in detail (50). Analysis of the genome sequence of B. subtilis (25) revealed that it contains an open reading frame, yutB, encoding a protein with a high degree of homology to E. coli LipA and two open reading frames encoding proteins slightly similar to LplA, while no LipB homolog was detected.LA is a critical cofactor of BCKADH, the enzyme involved in the formation of the primer carbons for the initiation of branched-chain fatty acid (BCFA) synthesis (21). Early work indicated that a bfmB mutant of B. subtilis, defective in both BCKADH and pyruvate dehydrogenase, requires short-branched-chain carboxilic acids for growth (56). However, in our hands, this mutant presented a high percentage of reversion, precluding its use in the study of lipid metabolism. Since BCFAs are the dominant acyl chains found in membrane phospholipids of B. subtilis, the goal of this study was to employ a genetic approach to investigate the role of yutB in the physiology of this organism, in particular in fatty acid metabolism. In addition, we provide compelling evidence showing that Δ5 unsaturated fatty acids (UFA), the products of the B. subtilis desaturase, can fully replace the function of BCFAs. Furthermore, we demonstrate that UFA are essential to provide cryoprotective properties in strains depleted of LipA. This work reports the first characterization of a gram-positive mutant deficient in LA synthesis and its use to study the interplay between BCFAs and UFA metabolism.     

Biosynthesis of 4-Thiouridine in tRNA in the Methanogenic Archaeon Methanococcus maripaludis

4-Thiouridine (s⁴U) is a conserved modified nucleotide at position 8 of bacterial and archaeal tRNAs and plays a role in protecting cells from near-UV killing. Escherichia coli employs the following two enzymes for its synthesis: the cysteine desulfurase IscS, which forms a Cys persulfide enzyme adduct from free Cys; and ThiI, which adenylates U8 and transfers sulfur from IscS to form s⁴U. The C-terminal rhodanese-like domain (RLD) of ThiI is responsible for the sulfurtransferase activity. The mechanism of s⁴U biosynthesis in archaea is not known as many archaea lack cysteine desulfurase and an RLD of the putative ThiI. Using the methanogenic archaeon Methanococcus maripaludis, we show that deletion of ThiI (MMP1354) abolished the biosynthesis of s⁴U but not of thiamine. MMP1354 complements an Escherichia coli ΔthiI mutant for s⁴U formation, indicating that MMP1354 is sufficient for sulfur incorporation into s⁴U. In the absence of an RLD, MMP1354 uses Cys²⁶⁵ and Cys²⁶⁸ located in the PP-loop pyrophosphatase domain to generate persulfide and disulfide intermediates for sulfur transfer. In vitro assays suggest that S²⁻ is a physiologically relevant sulfur donor for s⁴U formation catalyzed by MMP1354 (K_m for Na₂S is ∼1 mm). Thus, methanogenic archaea developed a strategy for sulfur incorporation into s⁴U that differs from bacteria; this may be an adaptation to life in sulfide-rich environments.     

Cloning, Expression in Escherichia coli, and Characterization of Arabidopsis thaliana UMP/CMP Kinase

A cDNA encoding the Arabidopsis thaliana uridine 5′-monophosphate (UMP)/cytidine 5′-monophosphate (CMP) kinase was isolated by complementation of a Saccharomyces cerevisiae ura6 mutant. The deduced amino acid sequence of the plant UMP/CMP kinase has 50% identity with other eukaryotic UMP/CMP kinase proteins. The cDNA was subcloned into pGEX-4T-3 and expressed as a glutathione S-transferase fusion protein in Escherichia coli. Following proteolytic digestion, the plant UMP/CMP kinase was purified and analyzed for its structural and kinetic properties. The mass, N-terminal sequence, and total amino acid composition agreed with the sequence and composition predicted from the cDNA sequence. Kinetic analysis revealed that the UMP/CMP kinase preferentially uses ATP (Michaelis constant [K_m] = 29 μm when UMP is the other substrate and K_m = 292 μm when CMP is the other substrate) as a phosphate donor. However, both UMP (K_m = 153 μm) and CMP (K_m = 266 μm) were equally acceptable as the phosphate acceptor. The optimal pH for the enzyme is 6.5. P¹, P⁵-di(adenosine-5′) pentaphosphate was found to be a competitive inhibitor of both ATP and UMP.