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A bacterium with a "mouth"-like pit structure isolated for the first time in the history of microbiology was a Gram-negative rod, containing glycosphingolipids in the cell envelope, and named Sphingomonas sp. strain A1. The pit was dynamic, with repetitive opening and closing during growth on alginate, and directly included alginate concentrated around the pit, particularly by flagellins, an alginate-binding protein localized on the cell surface. Alginate incorporated into the periplasm was subsequently transferred to the cytoplasm by cooperative interactions of periplasmic solute-binding proteins and an ATP-binding cassette transporter in the cytoplasmic membrane. The mechanisms of assembly, functions, and interactions between the above-mentioned molecules were clarified using structural biology. The pit was transplanted into other strains of sphingomonads, and the pitted recombinant cells were effectively applied to the production of bioethanol, bioremediation for dioxin removal, and other tasks. Studies of the function of the pit shed light on the biological significance of cell surface structures and macromolecule transport in bacteria.

4-Deoxy-l-erythro-5-hexoseulose uronate (DEH), DEH reductase, and alginate lyase have key roles in the metabolism of alginate, a promising carbon source in brown macroalgae for biorefinery. In contrast to the widely reviewed alginate lyase, DEH and DEH reductase have not been previously reviewed. Here, we summarize the current understanding of DEH and DEH reductase, with emphasis on (i) the non-enzymatic and enzymatic formation and structure of DEH and its reactivity to specific amino groups, (ii) the molecular identification, classification, function, and structure, as well as the structural determinants for coenzyme specificity of DEH reductase, and (iii) the significance of DEH for biorefinery. Improved understanding of this and related fields should lead to the practical utilization of alginate for biorefinery.

Alginate is a linear polyuronate in brown macroalgae. It is also a promising marine biopolymer that can be degraded by exo-type alginate lyase into an unsaturated uronate that is non-enzymatically or enzymatically converted to 4-deoxy-l-erythro-5-hexoseulose uronate (DEH). In a bioengineered yeast Saccharomyces cerevisiae (DEH++) strain that utilizes DEH, DEH is not only an important physiological metabolite but also a promising carbon source for biorefinery systems. In this study, we uncovered the essential chemical nature of DEH. In particular, we showed that DEH non-enzymatically reacts with specific amino groups in Tris, ammonium salts [(NH4)2SO4 and NH4Cl], and certain amino acids (e.g., Gly, Ser, Gln, Thr, and Lys) at 30 °C and forms other compounds, one of which we tentatively named DEH-related product-1 (DRP-1). In contrast, Asn, Met, Glu, and Arg were almost inert and Ala, Pro, Leu, Ile, Phe, Val, and Asp, as well as sodium nitrate (NaNO3), were inert in the presence of DEH. Some of the above amino acids (Asn, Glu, Ala, Pro, Phe, and Asp) were suitable nitrogen sources for the DEH++ yeast strain, whereas ammonium salts and Ser, Gln, and Thr were poor nitrogen sources owing to their high reactivity to DEH. Nutrient-rich YP medium with 1% (w/v) Yeast extract and 2% (w/v) Tryptone, as well as 10-fold diluted YP medium, could also be effectively used as nitrogen sources. Finally, we identified DRP-1 as a 2-furancarboxylic acid and showed that it has a growth-inhibitory effect on the DEH++ yeast strain. These results show the reactive nature of DEH and suggest a basis for selecting nitrogen sources for use with DEH and alginate in biorefineries. Our results also provide insight into the physiological utilization of DEH. The environmental source of 2-furancarboxylic acid is also discussed.

Tup1-Cyc8 (also known as Tup1-Ssn6) is a general transcriptional corepressor. D-Mannitol (mannitol) and D-sorbitol (sorbitol) are the major polyols in nature. Budding yeastSaccharomyces cerevisiaeis unable to assimilate mannitol or sorbitol, but acquires the ability to assimilate mannitol due to a spontaneous mutation inTUP1orCYC8. In this study, we found that spontaneous mutation ofTUP1orCYC8also permitted assimilation of sorbitol. Some spontaneous nonsense mutations ofCYC8produced a truncated Cyc8 with a C-terminal polyglutamine. The effects were guanidine hydrochloride-sensitive and were dependent on Hsp104, but were complemented by introduction ofCYC8, ruling out involvement of a prion. Assimilation of mannitol and sorbitol conferred by other mutations ofTUP1orCYC8was guanidine hydrochloride-tolerant. It is physiologically reasonable thatS.cerevisiaecarries this mechanism to acquire the ability to assimilate major polyols in nature.

Alginate, a major acidic polysaccharide in brown macroalgae, has attracted attention as a carbon source for production of ethanol and other chemical compounds. Alginate is monomerized by exo-type alginate lyase into an unsaturated uronate; thus, this enzyme is critical for the saccharification and utilization of alginate. Although several exo-type alginate lyases have been characterized independently, their activities were not assayed under the same conditions or using the same unit definition, making it difficult to compare enzymatic properties or to select the most suitable enzyme for saccharification of alginate. In this study, we characterized the three bacterial exo-type alginate lyases under the same conditions: A1-IV of Sphingomonas sp. strain A1, Atu3025 of Agrobacterium tumefaciens, and Alg17c of Saccharophagus degradans. A1-IV had the highest specific activity as well as the highest productivity of uronate, whereas Alg17c had the lowest activity and productivity. Only dialyzed Atu3025 and Alg17c were tolerant to freezing. Alg17c exhibited a remarkable halotolerance, which may be advantageous for monomerization of alginate from marine brown algae. Thus, each enzyme exhibited particular desirable and undesirable properties. Our results should facilitate further utilization of the promising polysaccharide alginate.

Mannitol is contained in brown macroalgae up to 33% (w/w, dry weight), and thus is a promising carbon source for white biotechnology. However, Saccharomyces cerevisiae, a key cell factory, is generally regarded to be unable to assimilate mannitol for growth. We have recently succeeded in producing S. cerevisiae that can assimilate mannitol through spontaneous mutations of Tup1-Cyc8, each of which constitutes a general corepressor complex. In this study, we demonstrate production of pyruvate from mannitol using this mannitol-assimilating S. cerevisiae through deletions of all 3 pyruvate decarboxylase genes. The resultant mannitol-assimilating pyruvate decarboxylase-negative strain produced 0.86 g/L pyruvate without use of acetate after cultivation for 4 days, with an overall yield of 0.77 g of pyruvate per g of mannitol (the theoretical yield was 79%). Although acetate was not needed for growth of this strain in mannitol-containing medium, addition of acetate had a significant beneficial effect on production of pyruvate. This is the first report of production of a valuable compound (other than ethanol) from mannitol using S. cerevisiae, and is an initial platform from which the productivity of pyruvate from mannitol can be improved.

In brown macroalgae, alginate and D-mannitol are promising carbohydrates for biorefinery. Saccharomyces cerevisiae is widely used as a microbial cell factory, but this budding yeast is unable to utilize either alginate or D-mannitol. Alginate can be depolymerized by both endo-type and exo-type alginate lyases, yielding a monouronate, 4-deoxy-L-erythro-5-hexoseulose uronate (DEH), a key intermediate in the metabolism of alginate. Here, we constructed engineered two S. cerevisiae strains that are able to utilize both DEH and D-mannitol on two different strain backgrounds, and we also improved their aerobic growth in a DEH liquid medium through adaptive evolution. In both evolved strains, one of the causal mutations was surprisingly identical, a c.50A > G mutation in the codon-optimized NAD(P)H-dependent DEH reductase gene, one of the 4 genes introduced to confer the capacity to utilize DEH. This mutation resulted in an E17G substitution at a loop structure near the coenzyme-binding site of this reductase, and enhanced the reductase activity and aerobic growth in both evolved strains. Thus, the crucial role for this reductase reaction in the metabolism of DEH in the engineered S. cerevisiae is demonstrated, and this finding provides significant information for synthetic construction of a S. cerevisiae strain as a platform for alginate utilization., 代謝改変酵母のアルギン酸モノマー代謝能向上メカニズムの解明--国産海洋バイオマス資源から代替ガソリンや合成ゴム原料を生産するための重要な第一歩--. 京都大学プレスリリース. 2017-06-26.

Acetate kinase (AK) generally utilizes ATP as a phosphoryl donor, but AK from Entamoeba histolytica (PPi-ehiAK) uses pyrophosphate (PPi), not ATP, and is PPi-specific. The determinants of the phosphoryl donor specificity are unknown. Here, we inferred 5 candidate amino acid residues associated with this specificity, based on structural information. Each candidate residue in Escherichia coli ATP-specific AK (ATP-ecoAK), which is unable to use PPi, was substituted with the respective PPi-ehiAK amino acid residue. Each variant ATP-ecoAK had an increased Km for ATP, indicating that the 5 residues are the determinants for the specificity to ATP in ATP-ecoAK. Moreover, Asn-337 of ATP-ecoAK was shown to be particularly significant for the specificity to ATP. The 5 residues are highly conserved in 2625 PPi-ehiAK homologs, implying that almost all organisms have ATP-dependent, rather than PPi-dependent, AK.

Marine brown macroalgae is a promising source of material for biorefining, and alginate is one of the major components of brown algae. Despite the huge potential availability of alginate, no system has been reported for the production of valuable compounds other than ethanol from alginate, hindering its further utilization. Here we report that a bacterium, Sphingomonas sp. strain A1, produces pyruvate from alginate and secretes it into the medium. High aeration and deletion of the gene for d-lactate dehydrogenase are critical for the production of high concentrations of pyruvate. Pyruvate concentration and productivity were at their maxima (4.56 g/l and 95.0 mg/l/h, respectively) in the presence of 5% (w/v) initial alginate, whereas pyruvate produced per alginate consumed and % of theoretical yield (0.19 g/g and 18.6%, respectively) were at their maxima at 4% (w/v) initial alginate. Concentration of pyruvate decreased after it reached its maximum after cultivations for 2 or 3 days at 145 strokes per minute. Our study is the first report to demonstrate the production of other valuable compounds than ethanol from alginate, a promising marine macroalgae carbon source.

Marine macroalgae is a promising carbon source that contains alginate and mannitol as major carbohydrates. A bioengineered ethanologenic strain of the bacterium Sphingomonas sp. A1 can produce ethanol from alginate, but not mannitol, whereas the yeast Saccharomyces paradoxus NBRC 0259-3 can produce ethanol from mannitol, but not alginate. Thus, one practical approach for converting both alginate and mannitol into ethanol would involve two-step fermentation, in which the ethanologenic bacterium initially converts alginate into ethanol, and then the yeast produces ethanol from mannitol. In this study, we found that, during fermentation from alginate, the ethanologenic bacterium lost viability and secreted toxic byproducts into the medium. These toxic byproducts inhibited bacterial growth and killed bacterial cells and also inhibited growth of S. paradoxus NBRC 0259-3. We discovered that adjusting the pH of the culture supernatant or the culture medium containing the toxic byproducts to 6.0 attenuated the toxicity toward both bacteria and yeast, and also extended the period of viability of the bacterium. Although continuous adjustment of pH to 6.0 failed to improve the ethanol productivity of this ethanologenic bacterium, this pH adjustment worked very well in the two-step fermentation due to the attenuation of toxicity toward S. paradoxus NBRC 0259-3. These findings provide information critical for establishment of a practical system for ethanol production from brown macroalgae.

Mannitol is a promising marine macroalgal carbon source. However, organisms that produce ethanol from mannitol are limited; to date, only the yeast Pichia angophorae and the bacterium Escherichia coli KO11 have been reported to possess this capacity. In this study, we searched a yeast strain with a high capacity to produce ethanol from mannitol and selected Saccharomyces paradoxus NBRC 0259 for its ability to produce ethanol from mannitol. This ability was enhanced after a 3-day cultivation of this strain in medium containing mannitol; the enhanced strain was renamed S. paradoxus NBRC 0259-3. We compared the ability of strain NBRC 0259-3 to produce ethanol from mannitol and glucose, under several conditions, with those of P. angophorae and E. coli KO11. As a result, we concluded that S. paradoxus NBRC 0259-3 strain is the most suitable yeast strain for the production of ethanol from mannitol.

Bioethanol has attracted attention as an alternative to petroleum-derived fuel. Seaweeds have been proposed as some of the most promising raw materials for bioethanol production because they have several advantages over lignocellulosic biomass. However, because seaweeds contain low contents of glucans, i.e., polysaccharides composed of glucose, the conversion of only the glucans from seaweed is not sufficient to produce high concentrations of ethanol. Therefore, it is also necessary to produce ethanol from other specific carbohydrate components of seaweeds, including sulfated polysaccharides, mannitol, alginate, agar and carrageenan. This review summarizes the current state of research on the production of ethanol from seaweed carbohydrates for which the conversion of carbohydrates to sugars is a key step and makes comparisons with the production of ethanol from lignocellulosic biomass. This review provides valuable information necessary for the production of high concentrations of ethanol from seaweeds.

NAD kinase catalyzes the phosphorylation of NAD(+) to synthesize NADP(+), whereas NADH kinase catalyzes conversion of NADH to NADPH. The mitochondrial protein Pos5 of Saccharomyces cerevisiae shows much higher NADH kinase than NAD kinase activity and is therefore referred to as NADH kinase. To clarify the structural determinant underlying the high NADH kinase activity of Pos5 and its selectivity for NADH over NAD(+), we determined the tertiary structure of Pos5 complexed with NADH at a resolution of 2.0 Å. Detailed analysis, including a comparison of the tertiary structure of Pos5 with the structures of human and bacterial NAD kinases, revealed that Arg-293 of Pos5, corresponding to His-351 of human NAD kinase, confers a positive charge on the surface of NADH-binding site, whereas the corresponding His residue does not. Accordingly, conversion of the Arg-293 into a His residue reduced the ratio of NADH kinase activity to NAD kinase activity from 8.6 to 2.1. Conversely, simultaneous changes of Ala-330 and His-351 of human NAD kinase into Ser and Arg residues significantly increased the ratio of NADH kinase activity to NAD kinase activity from 0.043 to 1.39; human Ala-330 corresponds to Pos5 Ser-272, which interacts with the side chain of Arg-293. Arg-293 and Ser-272 were highly conserved in Pos5 homologs (putative NADH kinases), but not in putative NAD kinases. Thus, Arg-293 of Pos5 is a major determinant of NADH selectivity. Moreover, Ser-272 appears to assist Arg-293 in achieving the appropriate conformation.

Transformation is an indispensable method for the genetic manipulation of cells. Saccharomyces cerevisiae can be transformed by incubating intact cells and plasmid DNA in the presence of polyethylene glycol alone. Lithium acetate (LiAc) and single-stranded carrier DNA (ssDNA) enhance the transformation efficiency, but the mechanism underlying this enhancement has remained elusive. In this study, we first confirmed that LiAc and ssDNA synergistically improve the transformation efficiency of S. cerevisiae intact cells. We then used transmission electron microscopy to observe the cell walls of yeast incubated with both LiAc and ssDNA in the presence of negatively charged Nanogold (in this context, a mimic of DNA). Under these conditions, the cell walls exhibited protruded, loose, and porous structures. The Nanogold was observed within the cell wall, rather than on the surface. We also made observations using YOYO-1, a fluorescent DNA probe. Based on the transmission electron microscopy and fluorescence data, we speculated that ssDNA covers the whole cell and enters, at least partially, into the cell wall structure, causing the cell wall to become protruded, loose, and porous; meanwhile, LiAc produces effect on the cell wall. Together, the two compounds synergistically enhance the transformation efficiency and frequency.

NAD kinase (NADK, EC 2.7.1.23) is the sole NADP(+)-biosynthetic enzyme that catalyzes phosphorylation of NAD(+) to yield NADP(+) using ATP as a phosphoryl donor, and thus, plays a vital role in the cell and represents a potentially powerful antimicrobial drug target. Although methods for expression and purification of human NADK have been previously established (Lerner et al. Biochem Biophys Res Commun 288:69-74, 2001), the purification procedure could be significantly improved. In this study, we improved the method for expression and purification of human NADK in Escherichia coli and obtained a purified homogeneous enzyme only through heat treatment and single column chromatography. Using the purified human NADK, we revealed a sigmoidal kinetic behavior toward ATP and the inhibitory effects of NADPH and NADH, but not of NADP(+), on the catalytic activity of the enzyme. These inhibitory effects provide insight into the regulation of intracellular NADPH synthesis. Furthermore, these attributes may provide a clue to design a novel drug against Mycobacterium tuberculosis in which this bacterial NADK is potently inhibited by NADP(+).

Transformation (i.e., genetic modification of a cell by the incorporation of exogenous DNA) is indispensable for manipulating fungi. Here, we review the transformation methods for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris and Aspergillus species and discuss some common modifications to improve transformation efficiency. We also present a model of the mechanism underlying S. cerevisiae transformation, based on recent reports and the mechanism of transfection in mammalian systems. This model predicts that DNA attaches to the cell wall and enters the cell via endocytotic membrane invagination, although how DNA reaches the nucleus is unknown. Polyethylene glycol is indispensable for successful transformation of intact cells and the attachment of DNA and also possibly acts on the membrane to increase the transformation efficiency. Both lithium acetate and heat shock, which enhance the transformation efficiency of intact cells but not that of spheroplasts, probably help DNA to pass through the cell wall.

Intact cells of the yeast Saccharomyces cerevisiae were transformed with exogenous DNA by incubating the cells with plasmid in the presence of polyethylene glycol (PEG), which has been shown to be required, although the underlying has not been elucidated. In this study, we found that incubation of the S. cerevisiae cells with PEG was not only required for the PEG-dependent transformation but also enhanced transformation, suggesting that PEG might cause an intracellular response. To understand the response, microarray and metabolome analyses were conducted. We found that incubation of the cells without PEG caused up-regulation of several genes, including those which are involved in carbon source metabolisms, e.g. fatty acid metabolism, yielding acetyl-CoA and those involved in stress-response. Contrary to this, incubation of the cells with PEG gave no transcriptional change. These microarray data were supported by the results of metabolome analysis for anionic metabolites, implying that the physical effect of PEG on cell membrane, rather than the effect of PEG itself on the intracellular response, could cause high transformation in the PEG-dependent transformation. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

The functions of NAD(H) (NAD(+) and NADH) and NADP(H) (NADP(+) and NADPH) are undoubtedly significant and distinct. Hence, regulation of the intracellular balance of NAD(H) and NADP(H) is important. The key enzymes involved in the regulation are NAD kinase and NADP phosphatase. In 2000, we first succeeded in identifying the gene for NAD kinase, thereby facilitating worldwide studies of this enzyme from various organisms, including eubacteria, archaea, yeast, plants, and humans. Molecular biological study has revealed the physiological function of this enzyme, that is to say, the significance of NADP(H), in some model organisms. Structural research has elucidated the tertiary structure of the enzyme, the details of substrate-binding sites, and the catalytic mechanism. Research on NAD kinase also led to the discovery of archaeal NADP phosphatase. In this review, we summarize the physiological functions, applications, and structure of NAD kinase, and the way we discovered archaeal NADP phosphatase.

Sphingomonas species A1 is a newly identified pit-forming bacterium that directly incorporates a macromolecule (alginate) into its cytoplasm through a pit-dependent transport system, which we termed a superchannel. A pit is a novel, high-dimensional organ acquired through the fluidity and reconstitution of cell surface molecules, including flagellin, and through cooperation with the transport machinery in the cells, which confers upon bacterial cells a more efficient way to secure and assimilate macromolecules. The analysis of the superchannel changes general ideas regarding the fluidity and function of the cell surface, evolution and origin of cell-surface organs, including flagella, transport, and assimilation systems of macromolecules, and the divergence and energetics of metabolism.

Saccharomyces cerevisiae normally cannot assimilate mannitol, a promising brown macroalgal carbon source for bioethanol production. The molecular basis of this inability remains unknown. We found that cells capable of assimilating mannitol arose spontaneously from wild-type S. cerevisiae during prolonged culture in mannitol-containing medium. Based on microarray data, complementation analysis, and cell growth data, we demonstrated that acquisition of mannitol-assimilating ability was due to spontaneous mutations in the genes encoding Tup1 or Cyc8, which constitute a general corepressor complex that regulates many kinds of genes. We also showed that an S. cerevisiae strain carrying a mutant allele of CYC8 exhibited superior salt tolerance relative to other ethanologenic microorganisms; this characteristic would be highly beneficial for the production of bioethanol from marine biomass. Thus, we succeeded in conferring the ability to assimilate mannitol on S. cerevisiae through dysfunction of Tup1-Cyc8, facilitating production of ethanol from mannitol.

During the latter half of the 1970s, first spheroplasts transformation method was established in the yeast Saccharomyces cerevisiae. With the advent of synthetic biology and genome technologies, which require handling of very large sized DNA molecules, the spheroplast method has once again become important. We present here the spheroplast method that achieved in the highest transformation efficiency of S. cerevisiae.

Since it was initially established, the lithium method for transforming Saccharomyces cerevisiae intact cells has been modified, and a possible underlying mechanism has been elucidated. This method requires polyethylene glycol (PEG) and transformation efficiency is enhanced by lithium and single-stranded carrier DNA (ssDNA). Here, we describe the original lithium method, the modified method, and the possible underlying mechanism, in which plasmid DNA that was absorbed onto the cell wall enters into the cells via endocytotic membrane invagination and lithium and ssDNA synergistically alter cell wall structure and enhance transformation efficiency.

The alginate-assimilating bacterium, Sphingomonas sp. strain A1, degrades the polysaccharides to monosaccharides through four alginate lyase reactions. The resultant monosaccharide, which is nonenzymatically converted to 4-deoxy-L-erythro-5-hexoseulose uronate (DEH), is further metabolized to 2-keto-3-deoxy-D-gluconate by NADPH-dependent reductase A1-R in the short-chain dehydrogenase/reductase (SDR) family. A1-R-deficient cells produced another DEH reductase, designated A1-R', with a preference for NADH. Here, we show the identification of a novel NADH-dependent DEH reductase A1-R' in strain A1, structural determination of A1-R' by x-ray crystallography, and structure-based conversion of a coenzyme requirement in SDR enzymes, A1-R and A1-R'. A1-R' was purified from strain A1 cells and enzymatically characterized. Except for the coenzyme requirement, there was no significant difference in enzyme characteristics between A1-R and A1-R'. Crystal structures of A1-R' and A1-R'·NAD(+) complex were determined at 1.8 and 2.7 Å resolutions, respectively. Because of a 64% sequence identity, overall structures of A1-R' and A1-R were similar, although a difference in the coenzyme-binding site (particularly the nucleoside ribose 2' region) was observed. Distinct from A1-R, A1-R' included a negatively charged, shallower binding site. These differences were caused by amino acid residues on the two loops around the site. The A1-R' mutant with the two A1-R-typed loops maintained potent enzyme activity with specificity for NADPH rather than NADH, demonstrating that the two loops determine the coenzyme requirement, and loop exchange is a promising method for conversion of coenzyme requirement in the SDR family.

NADP(H) phosphatase has not been identified in eubacteria and eukaryotes. In archaea, MJ0917 of hyperthermophilic Methanococcus jannaschii is a fusion protein comprising NAD kinase and an inositol monophosphatase homologue that exhibits high NADP(H) phosphatase activity (S. Kawai, C. Fukuda, T. Mukai, and K. Murata, J. Biol. Chem. 280:39200-39207, 2005). In this study, we showed that the other archaeal inositol monophosphatases, MJ0109 of M. jannaschii and AF2372 of hyperthermophilic Archaeoglobus fulgidus , exhibit NADP(H) phosphatase activity in addition to the already-known inositol monophosphatase and fructose-1,6-bisphosphatase activities. Kinetic values for NADP + and NADPH of MJ0109 and AF2372 were comparable to those for inositol monophosphate and fructose-1,6-bisphosphate. This implies that the physiological role of the two enzymes is that of an NADP(H) phosphatase. Further, the two enzymes showed inositol polyphosphate 1-phosphatase activity but not 3′-phosphoadenosine 5′-phosphate phosphatase activity. The inositol polyphosphate 1-phosphatase activity of archaeal inositol monophosphatase was considered to be compatible with the similar tertiary structures of inositol monophosphatase, fructose-1,6-bisphosphatase, inositol polyphosphate 1-phosphatase, and 3′-phosphoadenosine 5′-phosphate phosphatase. Based on this fact, we found that 3′-phosphoadenosine 5′-phosphate phosphatase (CysQ) of Escherichia coli exhibited NADP(H) phosphatase and fructose-1,6-bisphosphatase activities, although inositol monophosphatase (SuhB) and fructose-1,6-bisphosphatase (Fbp) of E. coli did not exhibit any NADP(H) phosphatase activity. However, the kinetic values of CysQ and the known phenotype of the cysQ mutant indicated that CysQ functions physiologically as 3′-phosphoadenosine 5′-phosphate phosphatase rather than as NADP(H) phosphatase.

ATP-NAD kinase phosphorylates NAD to produce NADP by using ATP, whereas ATP-NADH kinase phosphorylates both NAD and NADH. Three NAD kinase homologues, namely, ATP-NAD kinase (Utr1p), ATP-NADH kinase (Pos5p) and function-unknown Yel041wp (Yef1p), are found in the yeast Saccharomyces cerevisiae. In this study, Yef1p was identified as an ATP-NADH kinase. The ATP-NADH kinase activity of Utr1p was also confirmed. Thus, the three NAD kinase homologues were biochemically identified as ATP-NADH kinases. The phenotypic analysis of the single, double and triple mutants, which was unexpectedly found to be viable, for UTR1, YEF1 and POS5 demonstrated the critical contribution of Pos5p to mitochondrial function and survival at 37 degrees C and the critical contribution of Utr1p to growth in low iron medium. The contributions of the other two enzymes were also demonstrated; however, these were observed only in the absence of the critical contributor, which was supported by complementation for some pos5 phenotypes by the overexpression of UTR1 and YEF1. The viability of the triple mutant suggested that a 'novel' enzyme, whose primary structure is different from those of all known NAD and NADH kinases, probably catalyses the formation of cytosolic NADP in S. cerevisiae. Finally, we found that LEU2 of Candida glabrata, encoding beta-isopropylmalate dehydrogenase and being used to construct the triple mutant, complemented some pos5 phenotypes; however, overexpression of LEU2 of S. cerevisiae did not. The complementation was putatively attributed to an ability of Leu2p of C. glabrata to use NADP as a coenzyme and to supply NADPH.

Glucose kinase, which we tentatively use in this review, represents the enzymes catalyzing the phosphorylation of glucose and other hexoses by means of phosphoryl donors (ATP, ADP, and inorganic polyphosphate [poly(P)]). Except for glucose kinases utilizing ADP, all other glucose kinases belong to the hexokinase (HK) family and are classified into three groups based on primary structural information, i.e., groups HK, A, and B. The structural and evolutionary relationships of glucose kinases belonging to the above three groups have been controversial due to the lack of tertiary structural information on those in groups A and B. However, recent studies on the tertiary structures of poly(P)/ATP-glucomannokinase (GMK: a glucose kinase in group B) from Arthrobacter sp. strain KM and glucokinase (GK) (ecoGK: a glucose kinase in group A) from Escherichia coli have shed light on this problem. A comparison of the tertiary structures of GMK and ecoGK with those of glucose kinases in group HK demonstrated that both GMK and ecoGK are structurally homologous with glucose kinases in group HK, and that glucose kinases belonging to groups HK, A, and B in the HK family evolved divergently from a common ancestor. Based on the simple structure of GMK compared to those of ecoGK and glucose kinases in group HK, and the putative poly(P)-binding site in GMK, we propose that the ancestor of glucose kinases in the HK family was similar to GMK and used poly(P). We also discuss the ancestor and evolutionary process of ROK proteins, whose primary structures are homologous with those of glucose kinases in group B, in connection with the ancestor and evolutionary process of glucose kinases in the HK family.

NAD kinase is a key enzyme in NADP biosynthesis. We solved the crystal structure of polyphosphate/ATP-NAD kinase from Mycobacterium tuberculosis (Ppnk) complexed with NAD (Ppnk-NAD) at 2.6A resolution using apo-Ppnk structure solved in this work, and revealed the details of the structure and NAD-binding site. Superimposition of tertiary structures of apo-Ppnk and Ppnk-NAD demonstrated a substantial conformational difference in a loop (Ppnk-flexible loop). As a quaternary structure, these Ppnk structures exhibited tetramer as in solution condition. Notably, the Ppnk-flexible loop was involved in the intersubunit contact and probably related to the NAD-binding of the other subunit. Furthermore, the two residues (Asp189, His226) substantially contributed to creating NAD-binding site on the other subunit. The two residues and the residues involved in NAD-binding were conserved. However, residues corresponding to the Ppnk-flexible loop were not conserved, making us to speculate that the Ppnk-flexible loop may be Ppnk-specific.

Inorganic polyphosphate (poly(P)) is a biological high energy compound presumed to be an ancient energy carrier preceding ATP. Several poly(P)-dependent kinases that use poly(P) as a phosphoryl donor are known to function in bacteria, but crystal structures of these kinases have not been solved. Here we present the crystal structure of bacterial poly(P)/ATP-glucomannokinase, belonging to Gram-positive bacterial glucokinase, complexed with 1 glucose molecule and 2 phosphate molecules at 1.8 A resolution, being the first among poly(P)-dependent kinases and bacterial glucokinases. The poly(P)/ATP-glucomannokinase structure enabled us to understand the structural relationship of bacterial glucokinase to eucaryotic hexokinase and ADP-glucokinase, which has remained a matter of debate. These comparisons also enabled us to propose putative binding sites for phosphoryl groups for ATP and especially for poly(P) and to obtain insights into the evolution of kinase, particularly from primordial poly(P)-specific to ubiquitous ATP-specific proteins.

Gluconacetobacter diazotrophicus is a gram-negative and endophytic nitrogen-fixing bacterium that has several beneficial effects in host plants; thus, utilization of this bacterium as a biofertilizer in agriculture may be possible. G. diazotrophicus synthesizes levan, a D-fructofuranosyl polymer with β-(2→6) linkages, as an exopolysaccharide and the synthesized levan improves the stress tolerance of the bacterium. In this study, we found that phosphate enhances levan production by G. diazotrophicus Pal5, a wild type strain that showed a stronger mucous phenotype on solid medium containing 28 mM phosphate than on solid medium containing 7 mM phosphate. A G. diazotrophicus Pal5 levansucrase disruptant showed only a weak mucous phenotype regardless of the phosphate concentration, indicating that the mucous phenotype observed on 28 mM phosphate medium was caused by levan. To our knowledge, this is the first report of the effect of a high concentration of phosphate on exopolysaccharide production.

Understanding of the molecular system for DNA delivery into eucaryotic cells, a key to human DNA therapy, remains obscure. To understand this system, we undertook a study using the Saccharomyces cerevisiae model into which DNA delivery is easily assessed through competence (transformability) and for which all nonessential gene mutants (about 5000 strains) are available. We analyzed the competence of each of these mutants and identified three low-competence mutants, i.e., sin3Delta, she4Delta, and arc18Delta, and three high-competence mutants, i.e., pde2Delta, spf1Delta, and pmr1Delta. Through further studies using the six mutants, we concluded that the Arp2/3 activation machinery involving the Myo3/5p, Vrp1p, Las17p, Pan1p, and Arp2/3 complex is crucial to delivery (competence), and that high cAMP enhances competence via protein kinase A installing Tpk3p. We also propose that DNA is taken up via an endocytosis-like event, being at least partially different from well-known endocytosis.

TORC1-dependent phosphorylation of Saccharomyces cerevisiae Sch9 was dramatically reduced upon exposure to a protonophore or in respiration-incompetent ρ 0 cells but not in respiration-incompetent pet mutants, providing important insight into the molecular mechanisms governing interorganellar signaling in general and retrograde signaling in particular.

NAD kinase (NADK) is a crucial enzyme for production of NADP(+). ATP-specific NADK prefers ATP to inorganic polyphosphate [poly(P)] as a phosphoryl donor, whereas poly(P)/ATP-NADK utilizes both ATP and poly(P), and is employed in industrial mass production of NADP(+). Poly(P)/ATP-NADKs are distributed throughout Gram-positive bacteria and Archaea, whereas ATP-specific NADKs are found in Gram-negative α- and γ-proteobacteria and eukaryotes. In this study, we succeeded in conferring the ability to utilize poly(P) on γ-proteobacterial ATP-specific NADKs through a single amino-acid substitution; the substituted amino-acid residue is therefore important in determining the phosphoryl-donor specificity of γ-proteobacterial NADKs. We also demonstrate that a poly(P)/ATP-NADK created through this method is suitable for the poly(P)-dependent mass production of NADP(+). Moreover, based on our results, we provide insight into the evolution of bacterial NADKs, in particular, how NADKs evolved from poly(P)/ATP-NADKs into ATP-specific NADKs., ATP特異性の獲得メカニズムの解明 : 新薬と新しい物質生産系の開発に期待. 京都大学プレスリリース. 2013-09-11.

A bacterium exhibiting activities of several inorganic polyphosphate [poly(P)]- and ATP-dependent kinases, including glucokinase, NAD kinase, mannokinase, and fructokinase, was isolated, determined to belong to the genus Arthrobacter , and designated Arthrobacter sp. strain KM. Among the kinases, a novel enzyme responsible for the poly(P)- and ATP-dependent mannokinase activities was purified 2,200-fold to homogeneity from a cell extract of the bacterium. The purified enzyme was a monomer with a molecular mass of 30 kDa. This enzyme phosphorylated glucose and mannose with a high affinity for glucose, utilizing poly(P) as well as ATP, and was designated poly(P)/ATP-glucomannokinase. The K m values of the enzyme for glucose, mannose, ATP, and hexametaphosphate were determined to be 0.50, 15, 0.20, and 0.02 mM, respectively. The catalytic sites for poly(P)-dependent phosphorylation and ATP-dependent phosphorylation of the enzyme were found to be shared, and the poly(P)-utilizing mechanism of the enzyme was shown to be nonprocessive. The gene encoding the poly(P)/ATP-glucomannokinase was cloned from Arthrobacter sp. strain KM, and its nucleotide sequence was determined. This gene contained an open reading frame consisting of 804 bp coding for a putative polypeptide with a calculated molecular mass of 29,480 Da. The deduced amino acid sequence of the polypeptide exhibited homology to the amino acid sequences of the poly(P)/ATP-glucokinase of Mycobacterium tuberculosis H37Rv (level of homology, 45%), ATP-dependent glucokinases of Corynebacterium glutamicum (45%), Renibacterium salmoninarum (45%), and Bacillus subtilis (35%), and proteins of bacteria belonging to the order Actinomyces whose functions are not known. Alignment of these homologous proteins revealed seven conserved regions. The mannose and poly(P) binding sites of poly(P)/ATP-glucomannokinase are discussed.

The gene encoding an inorganic polyphosphate/ATP-NAD kinase was cloned from Micrococcus flavus, and its primary structure was analyzed. Alignment of the primary structure with those of other characterized NAD kinases revealed candidate amino acid residues, mainly charged ones, that would be related to inorganic polyphosphate use. The alignment also showed that the primary structure found carried a protruding C-terminal polypeptide. Although the C-terminal polypeptide was demonstrated to be dispensable for the kinase activities, and was proposed to be removed in M. flavus, the entire primary structure including the C-terminal polypeptide was homologous with that of the ATP synthase beta chain. The inorganic polyphosphate used by the inorganic polyphosphate/ATP-NAD kinase as a phosphoryl donor was isolated from cells of M. flavus, suggesting that the ability of the enzyme to use inorganic polyphosphate is of physiological significance and is not an evolutionary trait alone.

In the presence of polyethylene glycol (PEG), budding cells of Saccharomyces cerevisiae in the early log phase were transformed by exogenous plasmid DNA without additional specific chemical or physical treatments. This capacity of the yeast cells to become competent was strictly dependent on the growth phase, being induced in the early log phase, becoming maximum between the early and mid log phases and then disappearing rapidly in the mid log phase. The transformation was most efficient at pH 6 and the frequency increased with increasing DNA and cell concentrations. PEGs with average molecular sizes between 1000 and 3500 showed almost the same effects and were used most efficiently at 35%. The transformation frequency of S. cerevisiae was markedly enhanced when the oxidized form of glutathione (GSSG), but not the reduced form, was included in the mixture comprising early log phase cells, plasmid DNA, and PEG, and the transformation system with GSSG could be used as a convenient transformation method for the yeast S. cerevisiae.

NAD kinase was purified to homogeneity from Escherichia coli MG1655. The enzyme was a hexamer consisting of 30 kDa subunits and utilized ATP or other nucleoside triphosphates as phosphoryl donors for the phosphorylation of NAD, most efficiently at pH 7.5 and 60 degrees C. The enzyme could not use inorganic polyphosphates as phosphoryl donors and was designated as ATP-NAD kinase. The N-terminal amino-acid sequence of the purified enzyme was encoded by yfjB, which had been deposited as a gene of unknown function in the E. coli whole genomic DNA sequence database. yfjB was cloned and expressed in E. coli BL21(DE3)pLysS. The purified product (YfjB) showed NAD kinase activity, and was identical to ATP-NAD kinase purified from E. coli MG1655 in molecular structure and other enzymatic properties. The deduced amino-acid sequence of YfjB exhibited homology with that of Mycobacterium tuberculosis inorganic polyphosphate/ATP-NAD kinase [Kawai, S., Mori, S., Mukai, T., Suzuki, S., Hashimoto, W., Takeshi, Y. & Murata, K. (2000) Biochem. Biophys. Res. Commun. 276, 57-63], and those of many hypothetical proteins for which functions have not yet been revealed. The YfjB homologues were considered to be NAD kinases and alignment of their sequences revealed highly conserved regions, XXX-XGGDG-XL and DGXXX-TPTGSTAY, where X represents a hydrophobic amino-acid residue.

UTR1 of the yeast Saccharomyces cerevisiae was cloned from the genomic DNA by polymerase chain reaction and expressed in Escherichia coli. Characterization of the purified UTR1p revealed that UTR1p is a NAD kinase consisting of six identical subunits with a molecular mass of 60 kDa. UTR1p specifically phosphorylated NAD in the presence of ATP, dATP, or CTP as phosphoryl donors, and was most active at pH 8.0, 30 degrees C. Km values of UTR1p for NAD and ATP were determined to be 0.50 mM and 0.60 mM, respectively.

Since the oil-based extract of licorice (Glycyrrhiza glabra L.) (OEL) has been found to have high fungicidal effect against Arthrinium sacchari M001 and Chaetomium funicola M002, we studied the structure of an antifungal substance derived from OEL which showed effectiveness against the above two filamentous fungi. Consequently, glabridin (glabridin, 3-(2', 4'-dihydroxyphenyl)-8-dimethylpyrano [8, 7-e] chroman) was found to be an antifungal component in the OEL and its fungicidal effect was confirmed. In order to prevent fungicaused spoilage, the possibility of adding glabridin or a licorice preparation made from OEL to tea beverages was suggested.

The inorganic polyphosphate/ATP-dependent NAD kinase (Ppnk) of Mycobacterium tuberculosis was applied to the mass-production of NADP from NAD and inorganic polyphosphate (metaphosphate). When Ppnk purified from recombinant Escherichia coli cells overexpressing the M. tuberculosis Ppnk was used, 30 mM (27 g/l) NADP was produced from 50 mM NAD and 100 mg/ml metaphosphate at 37 degrees C and pH 7.0. The recombinant E. coli cells were immobilized in polyacrylamide gel, and treated with acetone to render the cells permeable to substrates and products. When acetone-treated immobilized cells were used, 16 mM (14 g/l) NADP was produced from 50 mM NAD and 100 mg/ml metaphosphate at 37 degrees C and pH 7.0. The isolation of NADP formed in the reaction mixture was easy because of the absence of by-products (ATP degradation compounds), and this NADP production system using purified Ppnk or immobilized recombinant E. coli cells expressing Ppnk is thought to be feasible in the production of NADP on an industrial scale.

A bacterium, Sphingomonas sp. strain A1, can incorporate alginate into cells through a novel ABC (ATP-binding cassette) transporter system specific to the macromolecule. The transported alginate is depolymerized to di- and trisaccharides by three kinds of cytoplasmic alginate lyases (A1-I [66 kDa], A1-II [25 kDa], and A1-III [40 kDa]) generated from a single precursor through posttranslational autoprocessing. The resultant alginate oligosaccharides were degraded to monosaccharides by cytoplasmic oligoalginate lyase. The enzyme and its gene were isolated from the bacterial cells grown in the presence of alginate. The purified enzyme was a monomer with a molecular mass of 85 kDa and cleaved glycosidic bonds not only in oligosaccharides produced from alginate by alginate lyases but also in polysaccharides (alginate, polymannuronate, and polyguluronate) most efficiently at pH 8.0 and 37°C. The reaction catalyzed by the oligoalginate lyase was exolytic and thought to play an important role in the complete depolymerization of alginate in Sphingomonas sp. strain A1. The gene for this novel enzyme consisted of an open reading frame of 2,286 bp encoding a polypeptide with a molecular weight of 86,543 and was located downstream of the genes coding for the precursor of alginate lyases ( aly ) and the ABC transporter ( algS , algM1 , and algM2 ). This result indicates that the genes for proteins required for the transport and complete depolymerization of alginate are assembled to form a cluster.

Feeding studies on rice genetically modified with soybean glycinin were performed on rats for four weeks. The rats were divided into three groups, each being fed on (I) only a commercial diet, (II) this diet plus control rice and (III) this diet plus rice genetically modified with glycinin. The rats were fed with 10 g/kg-weight of rice every day by oral administration. During the test period, the rats in every group grew well without marked differences in appearance, food intake, body weight, or cumulative body weight gain. There were also no significant differences in the blood count, blood composition or internal organ weights among the rats. Necropsy at the end of the experiment indicated neither pathological symptoms nor histopathological abnormalities in the liver and kidney. Judging from these results, the rice genetically modified with glycinin is considered to have been essentially the same in nutritional and biochemical characteristics as the control rice.