Shigeyuki Kawai

Molecular Identification of Oligoalginate Lyase of Sphingomonas sp. Strain A1 as One of the Enzymes Required for Complete Depolymerization of Alginate

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 degrees 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.

Transformation of Saccharomyces cerevisiae and other fungi: Methods and possible underlying mechanism

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.

Bioethanol production from marine biomass alginate by metabolically engineered bacteria

Bioethanol production from algae is a promising approach that resolves problems associated with biofuel production from land biomass, such as bioethanol–food conflicts and the indirect land use change. However, it presents several technical difficulties because existing ethanologenic microbes can neither degrade alginate, a major component of brown algae, nor assimilate alginate degradation products. We developed an integrated bacterial system for converting alginate to ethanol using a metabolically modified, alginate-assimilating, pit-forming bacterium, Sphingomonas sp. A1 (strain A1). Overexpression of Zymomonas mobilis pdc and adhB was achieved using a strong constitutive expression promoter newly identified in strain A1 and by inserting multiple gene copies using the methylation sensitivity of XbaI. Metabolome analysis revealed by-product accumulation, and its synthesis pathway was blocked by gene disruption. The ethanologenic recombinant strain A1 accumulated 13.0 g L−1ethanol in 3 d using alginate as the sole carbon source.

Identification of ATP-NADH kinase isozymes and their contribution to supply of NADP(H) in Saccharomyces cerevisiae

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 °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 β‐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.

Strategies for the production of high concentrations of bioethanol from seaweeds: production of high concentrations of bioethanol from seaweeds.

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.

Structure and Function of NAD Kinase and NADP Phosphatase: Key Enzymes That Regulate the Intracellular Balance of NAD(H) and NADP(H)

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.

Identification and characterization of a human mitochondrial NAD kinase

NAD kinase is the sole NADP+ biosynthetic enzyme. Despite the great significance of NADP+, to date no mitochondrial NAD kinase has been identified in human, and the source of human mitochondrial NADP+ remains elusive. Here we present evidence demonstrating that a human protein of unknown function, C5orf33, is a human mitochondrial NAD kinase; this protein likely represents the missing source of human mitochondrial NADP+. The C5orf33 protein exhibits NAD kinase activity, utilizing ATP or inorganic polyphosphate, and is localized in the mitochondria of human HEK293A cells. C5orf33 mRNA is more abundant than human cytosolic NAD kinase mRNA in almost all tissues examined. We further show by database searches that some animals and protists carry C5orf33 homologues as their sole NADP+ biosynthetic enzyme, whereas plants and fungi possess no C5orf33 homologue. These observations provide insights into eukaryotic NADP+ biosynthesis, which has pivotal roles in cells and organelles.

Antifungal activity of plant extracts against Arthrinium sacchari and Chaetomium funicola.

Various plant extracts were examined for antifungal activity with the objective of improving the commercial sterility of aseptically filled tea beverage products in PET bottles. When the hot water extract and the methanol extract of 29 samples were measured for their antifungal activity against Arthrinium sacchari M001 and Chaetomium funicola M002 strains, five samples, Acer nikoense, Glycyrrhiza glabra, Lagerstroemia speciosa, Psidium guajava and Thea sinensis, showed high activity. Of these, the extracts from A. nikoense, G. glabra and T. sinensis were fractionated by extraction with CHCl3, and the CHCl3-soluble fractions from G. glabra showed antifungal activity with minimum inhibitory concentrations (MICs) between 62.5 and 125 microg/ml against the above-mentioned two fungi. When the EtOAc-soluble fraction of A. nikoense was used, the MIC against A. sacchari M001 was 62.5 microg/ml. However, none of the fractions from A. nikoense or T. sinensis showed high activity against C. funicola M002 and their MICs were greater than 500 microg/ml. A licorice preparation made from the commercially available oil-based extract of G. glabra showed a low MIC of 25 microg/ml against five tested strains of filamentous fungi, but not against Aspergillus fumigatus M008, in a blended tea. Consequently, the possibility of adding a licorice preparation made from the oil-based extract of G. glabra to tea beverages (aseptically filled into PET bottles) was suggested.

Crystal Structure of Bacterial Inorganic Polyphosphate/ATP-glucomannokinase: INSIGHTS INTO KINASE EVOLUTION

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 Å 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.

Characterization and molecular cloning of a novel enzyme, inorganic polyphosphate/ATP-glucomannokinase, of Arthrobacter sp. strain KM.

ABSTRACT 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 Km 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.