Nuno Empadinhas

Structure of mycobacterial maltokinase, the missing link in the essential GlgE-pathway

A novel four-step pathway identified recently in mycobacteria channels trehalose to glycogen synthesis and is also likely involved in the biosynthesis of two other crucial polymers: intracellular methylglucose lipopolysaccharides and exposed capsular glucan. The structures of three of the intervening enzymes - GlgB, GlgE, and TreS - were recently reported, providing the first templates for rational drug design. Here we describe the structural characterization of the fourth enzyme of the pathway, mycobacterial maltokinase (Mak), uncovering a eukaryotic-like kinase (ELK) fold, similar to methylthioribose kinases and aminoglycoside phosphotransferases. The 1.15 Å structure of Mak in complex with a non-hydrolysable ATP analog reveals subtle structural rearrangements upon nucleotide binding in the cleft between the N- and the C-terminal lobes. Remarkably, this new family of ELKs has a novel N-terminal domain topologically resembling the cystatin family of protease inhibitors. By interfacing with and restraining the mobility of the phosphate-binding region of the N-terminal lobe, Maks unusual N-terminal domain might regulate its phosphotransfer activity and represents the most likely anchoring point for TreS, the upstream enzyme in the pathway. By completing the gallery of atomic-detail models of an essential pathway, this structure opens new avenues for the rational design of alternative anti-tubercular compounds.

Biosynthesis of Mannosylglycerate in the Thermophilic Bacterium Rhodothermus marinus BIOCHEMICAL AND GENETIC CHARACTERIZATION OF A MANNOSYLGLYCERATE SYNTHASE

The biosynthetic reaction scheme for the compatible solute mannosylglycerate in Rhodothermus marinusis proposed based on measurements of the relevant enzymatic activities in cell-free extracts and in vivo 13C labeling experiments. The synthesis of mannosylglycerate proceeded via two alternative pathways; in one of them, GDP mannose was condensed withd-glycerate to produce mannosylglycerate in a single reaction catalyzed by mannosylglycerate synthase, in the other pathway, a mannosyl-3-phosphoglycerate synthase catalyzed the conversion of GDP mannose and d-3-phosphoglycerate into a phosphorylated intermediate, which was subsequently converted to mannosylglycerate by the action of a phosphatase. The enzyme activities committed to the synthesis of mannosylglycerate were not influenced by the NaCl concentration in the growth medium. However, the combined mannosyl-3-phosphoglycerate synthase/phosphatase system required the addition of NaCl or KCl to the assay mixture for optimal activity. The mannosylglycerate synthase enzyme was purified and characterized. Based on partial sequence information, the corresponding mgs gene was identified from a genomic library of R. marinus. In addition, the mgs gene was overexpressed inEscherichia coli with a high yield. The enzyme had a molecular mass of 46,125 Da, and was specific for GDP mannose andd-glycerate. This is the first report of the characterization of a mannosylglycerate synthase.

The Bacterium Thermus thermophilus, Like Hyperthermophilic Archaea, Uses a Two-Step Pathway for the Synthesis of Mannosylglycerate

ABSTRACT The biosynthetic pathway for the synthesis of the compatible solute α-mannosylglycerate (MG) in the thermophilic bacterium Thermus thermophilus HB27 was identified based on the activities of recombinant mannosyl-3-phosphoglycerate synthase (MPGS) (EC 2.4.1.217) and mannosyl-3-phosphoglycerate phosphatase (MPGP) (EC 3.1.3.70). The sequences of homologous genes from the archaeon Pyrococcus horikoshii were used to identify MPGS and MPGP genes in T. thermophilus HB27 genome. Both genes were separately cloned and overexpressed in Escherichia coli, yielding 3 to 4 mg of pure recombinant protein per liter of culture. The molecular masses were 43.6 and 28.1 kDa for MPGS and MPGP, respectively. The recombinant MPGS catalyzed the synthesis of α-mannosyl-3-phosphoglycerate (MPG) from GDP-mannose and d-3-phosphoglycerate, while the recombinant MPGP catalyzed the dephosphorylation of MPG to MG. The recombinant MPGS had optimal activity at 80 to 90°C and a pH optimum near 7.0; MPGP had maximal activity between 90 and 95°C and at pH 6.0. The activities of both enzymes were strictly dependent on divalent cations; Mn2+ was most effective for MPGS, while Mn2+, Co2+, Mg2+, and to a lesser extent Ni2+ activated MPGP. The organization of MG biosynthetic genes in T. thermophilus HB27 is different from the P. horikoshii operon-like structure, since the genes involved in the conversion of fructose-6-phosphate to GDP-mannose are not found immediately downstream of the contiguous MPGS and MPGP genes. The biosynthesis of MG in the thermophilic bacterium T. thermophilus HB27, proceeding through a phosphorylated intermediate, is similar to the system found in hyperthermophilic archaea.

Albidovulum inexpectatum gen. nov., sp. nov., a Nonphotosynthetic and Slightly Thermophilic Bacterium from a Marine Hot Spring That Is Very Closely Related to Members of the Photosynthetic Genus Rhodovulum

ABSTRACT Several bacterial isolates, with an optimum growth temperature of about 50°C, were recovered from the marine hot spring at Ferraria on the island of São Miguel in the Azores. The geothermal water emerged from a porous lava flow and rapidly cooled in contact with seawater except at low tide. The bacterial species represented by strains FRR-10T and FRR-11 was nonpigmented, strictly aerobic, and organotrophic. Several genes, bchZ, pufB, pufA, pufL, or pufM, encoding the photosynthetic reaction center proteins and the core light-harvesting complexes were not detected in these strains. The organism oxidized thiosulfate to sulfate with enhancement of growth. The organism did not require additional NaCl in the culture medium for growth, but NaCl at 1.0% enhanced growth. Phylogenetic analyses using the 16S rRNA gene sequence of strain FRR-10T indicated that the new organism represented a new species of the α-3 subclass of the Proteobacteria and that it branches within the species of the genus Rhodovulum. The contradiction of classifying an organism which branches within the radiation of the genus Rhodovulum but does not possess the hallmark characteristics of this genus is discussed. However, the absence of several of these characteristics, namely, the lack of photosynthesis and pigmentation, which could be related to colonization of dark environments, and growth at high temperatures, leads to our proposal that strains FRR-10T and FRR-11 should be classified as a new species of a novel genus, Albidovulum inexpectatum, representing, at present, the most thermophilic organism within the α-3 subclass of the Proteobacteria.

Organic solutes in Rubrobacter xylanophilus: the first example of di-myo-inositol-phosphate in a thermophile.

The thermophilic and halotolerant nature of Rubrobacter xylanophilus led us to investigate the accumulation of compatible solutes in this member of the deepest lineage of the Phylum Actinobacteria. Trehalose and mannosylglycerate (MG) were the major compounds accumulated under all conditions examined, including those for optimal growth. The addition of NaCl to a complex medium and a defined medium had a slight or negligible effect on the accumulation of these compatible solutes. Glycine betaine, di-myo-inositol-phosphate (DIP), a new phosphodiester compound, identified as di-N-acetyl-glucosamine phosphate and glutamate were also detected but in low or trace levels. DIP was always present, except at the highest salinity examined (5% NaCl) and at the lowest temperature tested (43°C). Nevertheless, the levels of DIP increased with the growth temperature. This is the first report of MG and DIP in an actinobacterium and includes the identification of the new solute di-N-acetyl-glucosamine phosphate.

A Gene from the Mesophilic Bacterium Dehalococcoides ethenogenes Encodes a Novel Mannosylglycerate Synthase

Mannosylglycerate (MG) is a common compatible solute found in thermophilic and hyperthermophilic prokaryotes. In this study we characterized a mesophilic and bifunctional mannosylglycerate synthase (MGSD) encoded in the genome of the bacterium Dehalococcoides ethenogenes. mgsD encodes two domains with extensive homology to mannosyl-3-phosphoglycerate synthase (MPGS, EC 2.4.1.217) and to mannosyl-3-phosphoglycerate phosphatase (MPGP, EC 3.1.3.70), which catalyze the consecutive synthesis and dephosphorylation of mannosyl-3-phosphoglycerate to yield MG in Pyrococcus horikoshii, Thermus thermophilus, and Rhodothermus marinus. The bifunctional MGSD was overproduced in Escherichia coli, and we confirmed the combined MPGS and MPGP activities of the recombinant enzyme. The optimum activity of the enzyme was at 50 degrees C. To examine the properties of each catalytic domain of MGSD, we expressed them separately in E. coli. The monofunctional MPGS was unstable, while the MPGP was stable and was characterized. Dehalococcoides ethenogenes cannot be grown sufficiently to identify intracellular compatible solutes, and E. coli harboring MGSD did not accumulate MG. However, Saccharomyces cerevisiae expressing mgsD accumulated MG, confirming that this gene product can synthesize this compatible solute and arguing for a role in osmotic adjustment in the natural host. We did not detect MGSD activity in cell extracts of S. cerevisiae. Here we describe the first gene and enzyme for the synthesis of MG from a mesophilic microorganism and discuss the possible evolution of this bifunctional MGSD by lateral gene transfer from thermophilic and hyperthermophilic organisms.

Rubritepida flocculans gen. nov., sp. nov., a New Slightly Thermophilic Member of the α-1 Subclass of the Proteobacteria

A bacterial isolate, with an optimum growth temperature of about 50 degrees C, was recovered from the hot spring at Egerszalók in Hungary. Phylogenetic analyses using the 16S rRNA gene sequence of strain H-8T indicated that the new organism represented a new genus and species of alpha-1 subclass of the Proteobacteria. The major fatty acids of strain H-8T are 16:0, 18:1 omega7c; the rare fatty acid 19:0 20H cyclo 11,12 is also present. Ubiquinone 9 is the major respiratory quinone, the polar lipids are phosphatidylcholine, phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol in addition to two unidentified aminolipids. The new isolate forms red-colored colonies, flocculates in liquid media, is heterotrophic and strictly aerobic. Thiosulfate is oxidized to sulfate, but an increase in biomass could not be measured because of the flocculating behavior. Bacteriochloropyll a was detected by direct spectrophotometric analysis when the organism was grown at 30 degrees C, but could not be detected after growth at 50 degrees C. pufL and pufM genes were present. Heterotrophic growth of strain H-8T occurs on a few carbohydrates, amino acids and organic acids. On the basis of the phylogenetic analyses, physiological and biochemical characteristics, we propose that strain H-8T represents a new genus and a new species most closely related to Roseococcus thiosulfatophilus for which we propose the name Rubritepida flocculans.

Glucosylglycerate Biosynthesis in the Deepest Lineage of the Bacteria: Characterization of the Thermophilic Proteins GpgS and GpgP from Persephonella marina

The pathway for the synthesis of glucosylglycerate (GG) in the thermophilic bacterium Persephonella marina is proposed based on the activities of recombinant glucosyl-3-phosphoglycerate (GPG) synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP). The sequences of gpgS and gpgP from the cold-adapted bacterium Methanococcoides burtonii were used to identify the homologues in the genome of P. marina, which were separately cloned and overexpressed as His-tagged proteins in Escherichia coli. The recombinant GpgS protein of P. marina, unlike the homologue from M. burtonii, which was specific for GDP-glucose, catalyzed the synthesis of GPG from UDP-glucose, GDP-glucose, ADP-glucose, and TDP-glucose (in order of decreasing efficiency) and from d-3-phosphoglycerate, with maximal activity at 90 degrees C. The recombinant GpgP protein, like the M. burtonii homologue, dephosphorylated GPG and mannosyl-3-phosphoglycerate (MPG) to GG and mannosylglycerate, respectively, yet at high temperatures the hydrolysis of GPG was more efficient than that of MPG. Gel filtration indicates that GpgS is a dimeric protein, while GpgP is monomeric. This is the first characterization of genes and enzymes for the synthesis of GG in a thermophile.

Identification of the mycobacterial glucosyl-3-phosphoglycerate synthase

Mycobacteria synthesize unique polysaccharides that regulate fatty acid synthesis, namely the methylglucose lipopolysaccharide (MGLP) and the methylmannose polysaccharide. Glucosyl-(1-->2)-glycerate is found at the reducing end of MGLP. The mycobacterial gene encoding a glucosyl-3-phosphoglycerate synthase (GpgS), primarily found in actinobacteria and sharing very low amino acid identity with known homo-functional GpgSs, has been identified. This gene has been annotated as an inverting family 2 glycosyltransferase of unknown function. The gpgS genes from the fast-growing Mycobacterium smegmatis strain 1102 and from the slow-growing Mycobacterium bovis BCG in Escherichia coli were expressed, and the recombinant enzymes were purified and characterized. The substrates for optimal activity were UDP-glucose and d-3-phosphoglycerate but ADP-glucose was also an efficient donor. The enzymes had maximal activity around 45 degrees C, pH 8.0, and were strictly dependent on Mg(2+). In Mycobacterium tuberculosis H37Rv, the gene encoding GpgS (Rv1208) is identical to the homologue in Mycobacterium bovis BCG and was considered to be essential for growth. It is shown that these genes encode retaining family 81 glycosyltransferases regardless of the low amino acid identity with other known enzymes of this family.

A unique combination of genetic systems for the synthesis of trehalose in Rubrobacter xylanophilus: properties of a rare actinobacterial TreT.

Trehalose is the primary organic solute in Rubrobacter xylanophilus under all conditions tested, including those for optimal growth. We detected genes of four different pathways for trehalose synthesis in the genome of this organism, namely, the trehalose-6-phosphate synthase (Tps)/trehalose-6-phosphate phosphatase (Tpp), TreS, TreY/TreZ, and TreT pathways. Moreover, R. xylanophilus is the only known member of the phylum Actinobacteria to harbor TreT. The Tps sequence is typically bacterial, but the Tpp sequence is closely related to eukaryotic counterparts. Both the Tps/Tpp and the TreT pathways were active in vivo, while the TreS and the TreY/TreZ pathways were not active under the growth conditions tested and appear not to contribute to the levels of trehalose observed. The genes from the active pathways were functionally expressed in Escherichia coli, and Tps was found to be highly specific for GDP-glucose, a rare feature among these enzymes. The trehalose-6-phosphate formed was specifically dephosphorylated to trehalose by Tpp. The recombinant TreT synthesized trehalose from different nucleoside diphosphate-glucose donors and glucose, but the activity in R. xylanophilus cell extracts was specific for ADP-glucose. The TreT could also catalyze trehalose hydrolysis in the presence of ADP, but with a very high K(m). Here, we functionally characterize two systems for the synthesis of trehalose in R. xylanophilus, a representative of an ancient lineage of the actinobacteria, and discuss a possible scenario for the exceptional occurrence of treT in this extremophilic bacterium.