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.     

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.     

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.     

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.     

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.     

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.     

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。     

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.     

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.     

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.     

Galactosylononitol and Stachyose Synthesis in Seeds of Adzuki Bean : Purification and Characterization of Stachyose Synthase

Stachyose synthase (STS) (EC 2.4.1.67) was purified to homogeneity from mature seeds of adzuki bean (Vigna angularis). Electrophoresis under denaturing conditions revealed a single polypeptide of 90 kD. Size-exclusion chromatography of the purified enzyme yielded two activity peaks with apparent molecular masses of 110 and 283 kD. By isoelectric focusing and chromatofocusing the protein was separated into several active forms with isoelectric point values between pH 4.7 and 5.0. Purified STS catalyzed the transfer of the galactosyl group from galactinol to raffinose and myo-inositol. Additionally, the enzyme catalyzed the galactinol-dependent synthesis of galactosylononitol from d-ononitol. The synthesis of a galactosylcyclitol by STS is a new oberservation. Mutual competitive inhibition was observed when the enzyme was incubated with both substrates (raffinose and ononitol) simultaneously. Galactosylononitol could also substitute for galactinol in the synthesis of stachyose from raffinose. Although galactosylononitol was the less-efficient donor, the Michaelis constant value for raffinose was lower in the presence of galactosylononitol (13.2 mm) compared with that obtained in the presence of galactinol (38.6 mm). Our results indicate that STS catalyzes the biosynthesis of galactosylononitol, but may also mediate a redistribution of galactosyl residues from galactosylononitol to stachyose.     

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.     

MKAN27435 Is Required for the Biosynthesis of Higher Subclasses of Lipooligosaccharides in Mycobacterium kansasii

Lipooligosaccharides are glycolipids found in the cell wall of many mycobacterial species including the opportunistic pathogen Mycobacterium kansasii. The genome of M. kansasii ATCC12478 contains a cluster with genes orthologous to Mycobacterium marinum LOS biosynthesis genes. To initiate a genetic dissection of this cluster and demonstrate its role in LOS biosynthesis in M. kansasii, we chose MKAN27435, a gene encoding a putative glycosyltransferase. Using Specialized Transduction, a phage-based gene knockout tool previously used to generate null mutants in other mycobacteria, we generated a MKAN27435 null mutant. The mutant strain was found to be defective in the biosynthesis of higher LOS subspecies, viz LOS-IV, LOS-V, LOS-VI and LOS-VII. Additionally, a range of low abundance species were detected in the mutant strain and mass spectroscopic analysis indicated that these were shunt products generated from LOS-III by the addition of up to six molecules of a pentose.     

Lipoic acid metabolism in Arabidopsis thaliana: cloning and characterization of a cDNA encoding lipoyltransferase.

Lipoic acid is an essential coenzyme required for activity of several key enzyme complexes, such as the pyruvate dehydrogenase complex, in the central metabolism. In these complexes, lipoic acid must be covalently attached to one of the component proteins for it to have biological activity. We report the cloning and characterization of Arabidopsis thaliana LIP2 cDNA for lipoyltransferase that catalyzes the transfer of the lipoyl group from lipoyl-acyl carrier protein to lipoate-dependent enzymes. This cDNA was shown to code for lipoyltransferase by its ability to complement an Escherichia coli lipB null mutant lacking lipoyltransferase activity. The expressed enzyme in the E. coli mutant efficiently complemented the activity of pyruvate dehydrogenase complex, but less efficiently than that of 2-oxoglutarate dehydrogenase complex. Comparison of the deduced amino acid sequence of LIP2 with those of E. coli and yeast lipoyltransferases showed a marked sequence similarity and the presence of a leader sequence presumably required for import into mitochondria. Southern and northern hybridization analyses suggest that LIP2 is a single-copy gene and is expressed as an mRNA of 860 nt in leaves. Western blot analysis with an antibody against lipoyltransferase demonstrated that a 29 kDa form of lipoyltransferase is located in the mitochondrial compartment of A. thaliana.     

First Discovery of Two Polyketide Synthase Genes for Mitorubrinic Acid and Mitorubrinol Yellow Pigment Biosynthesis and Implications in Virulence of Penicillium marneffei

Background

The genome of P. marneffei, the most important thermal dimorphic fungus causing respiratory, skin and systemic mycosis in China and Southeast Asia, possesses 23 polyketide synthase (PKS) genes and 2 polyketide synthase nonribosomal peptide synthase hybrid (PKS-NRPS) genes, which is of high diversity compared to other thermal dimorphic pathogenic fungi. We hypothesized that the yellow pigment in the mold form of P. marneffei could also be synthesized by one or more PKS genes.

Methodology/Principal Findings

All 23 PKS and 2 PKS-NRPS genes of P. marneffei were systematically knocked down. A loss of the yellow pigment was observed in the mold form of the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants. Sequence analysis showed that PKS11 and PKS12 are fungal non-reducing PKSs. Ultra high performance liquid chromatography-photodiode array detector/electrospray ionization-quadruple time of flight-mass spectrometry (MS) and MS/MS analysis of the culture filtrates of wild type P. marneffei and the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants showed that the yellow pigment is composed of mitorubrinic acid and mitorubrinol. The survival of mice challenged with the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants was significantly better than those challenged with wild type P. marneffei (P<0.05). There was also statistically significant decrease in survival of pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants compared to wild type P. marneffei in both J774 and THP1 macrophages (P<0.05).

Conclusions/Significance

The yellow pigment of the mold form of P. marneffei is composed of mitorubrinol and mitorubrinic acid. This represents the first discovery of PKS genes responsible for mitorubrinol and mitorubrinic acid biosynthesis. pks12 and pks11 are probably responsible for sequential use in the biosynthesis of mitorubrinol and mitorubrinic acid. Mitorubrinol and mitorubrinic acid are virulence factors of P. marneffei by improving its intracellular survival in macrophages.