Primary structure of the <i>Saccharomyces cerevisiae GAL7</i> gene

Tajima, M; Nogi, Yasuhisa; Fukasawa, Toshio

doi:10.1002/yea.320010108

Cited by 148 publications

(77 citation statements)

References 43 publications

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“…cerevisiae (88) and E. coli (89) GalT sequences were similar enough to guide cloning of human GalT (9). GalT sequences co-existed with Hint sequences (6,90) in the databases for several years without notice.…”

Section: Galt-related Enzymes Are Specific Nucleoside Monophosphate Tmentioning

confidence: 99%

Hint, Fhit, and GalT: Function, Structure, Evolution, and Mechanism of Three Branches of the Histidine Triad Superfamily of Nucleotide Hydrolases and Transferases

2002

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HIT (histidine triad)1 proteins, named for a motif related to the sequence HφHφHφφ, (φ a hydrophobic amino acid) are a superfamily of nucleotide hydrolases and transferases, which act on the α-phosphate of ribonucleotides, and contain a ∼30 kDa domain that is typically either a homodimer of ∼15 kDa polypeptides with two active-sites or an internally, imperfectly repeated polypeptide that retains a single HIT active site. On the basis of sequence, substrate specificity, structure, evolution and mechanism, HIT proteins can be classified into the Hint branch, which consists of adenosine 5′-monophosphoramide hydrolases, the Fhit branch, which consists of diadenosine polyphosphate hydrolases, and the GalT branch, which consists of specific nucleoside monophosphate transferases including galactose-1-phosphate uridylyltransferase, diadenosine tetraphosphate phosphorylase, and adenylylsulfate:phosphate adenylytransferase. At least one human representative of each branch is lost in human diseases. Aprataxin, a Hint branch hydrolase, is mutated in ataxia-oculomotor apraxia syndrome. Fhit is lost early in development of many epithelially derived tumors. GalT is deficient in galactosemia. Additionally, ASW is an avian Hint family member that has evolved to have unusual gene expression properties and the complete loss of its nucleotide binding-site. The potential roles of ASW and Hint in avian sexual development are discussed in an accompanying manuscript. Here we review what is known about biological activities of HIT proteins, the structural and biochemical bases for their functions, and propose a new enzyme mechanism for Hint and Fhit that may account for the differences between HIT hydrolases and transferases. Mammalian Hint and GalT Structurally Define the HIT SuperfamilyGalactose-1-phosphate uridylyltransferase (GalT), the second enzyme in the Leloir pathway of galactose utilization (1), was crystallized and its structure determined to understand the mechanistic basis for transferring UMP between glucose-1-phosphate and galactose-1-phosphate (2-4). Histidine triad nucleotide-binding protein (Hint), purified as a purine nucleoside/nucleotide-binding protein from rabbit heart cytosol (5) and shown to have a widely conserved sequence (6), was crystallized and its structure determined to establish whether the conserved sequence formed the basis for a new mode of nucleotide-binding (7). GalT (2) and Hint are both dimers (8) but the GalT monomer is more than twice the size of the Hint dimer. Though GalT (9) and Hint (6) homologs could be cloned by similarity and the crystal structures (2,8) co-existed in protein databases, mutual similarity was not noted until inspection of the rabbit Hint crystal structure (7) and computational analysis (10) indicated that a GalT monomer and a Hint dimer can be superimposed and that their nucleotide-binding sites retain some of the same residues for binding a nucleoside monophosphate (7) (Figure 1). The level of sequence similarity of Hint and GalT is difficult to distinguish from n...

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Section: Galt-related Enzymes Are Specific Nucleoside Monophosphate Tmentioning

confidence: 99%

Hint, Fhit, and GalT: Function, Structure, Evolution, and Mechanism of Three Branches of the Histidine Triad Superfamily of Nucleotide Hydrolases and Transferases

2002

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“…S. cerevisiae strain MT8-1 (MATa ade2 his3 leu2 trp1 ura3) (22) was used as the host for protein expression. E. coli was grown in LB medium (10 g/liter tryptone, 5 g/liter yeast extract, 5 g/liter sodium chloride) containing 50 g/ml ampicillin.…”

Section: Strains and Media Escherichia Colimentioning

confidence: 99%

Enhanced Reactivity of Rhizopus oryzae Lipase Displayed on Yeast Cell Surfaces in Organic Solvents: Potential as a Whole-Cell Biocatalyst in Organic Solvents

Shiraga

Kawakami

Ishiguro

et al. 2005

Appl Environ Microbiol

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Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with ␣-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 ؋ 10 4 -fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 ؋ 10 4 -fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H 2 O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three-to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.Lipases (EC 3.1.1.3) catalyze the hydrolysis of acylglycerides and other fatty acid esters under aqueous conditions and the synthesis of esters in organic solvents (5, 25). Lipase-catalyzed reactions have high substrate specificity or regioselectivity, so lipase-catalyzed ester synthesis in organic solvents is potentially an industrially feasible alternative to conventional chemical methods (1, 4). However, organic solvents may alter the structure and activity of enzymes that usually function in an aqueous environment.One alternative is to immobilize the enzymes on solid supports in order to increase their function and stability in response to organic solvents or increased temperatures. Enzymes may be stabilized by chemical and physical processes (6,15,20,24). With chemical methods enzymes are immobilized by strong covalent bonding, but changes in protein structure often result. In physical stabilization processes, the interactions between enzymes and solids usually are weaker, resulting in fewer changes in the enzyme's structure.Yeast cell surface engineering (12, 13) is an alternative approach that immobilizes an enzyme on the yeast cell surface. Proteins are immobilized by using an outer shell cell wall protein, the C-terminal half of ␣-agglutinin (10, 26). The gene encoding Rhizopus oryzae lipase (ROL), which has been used to produce diesel fuels (8), was fused to a DNA sequence encoding the C-terminal half of yeast ␣-agglutinin and expressed in Saccharomyces cerevisiae. The transformants have hydrolytic...

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“…The well known GAL7 gene encodes UDP-galactose pyrophosphorylase (37), and more than a dozen of proteins in eukaryotes are homologous to the Gal7 protein in the data base. UGP1 encodes UDP-glucose pyrophosphorylase (38), and there are also more than a dozen of its homologs in the data base.…”

Section: P]gdp-man From [␣-mentioning

confidence: 99%

Saccharomyces cerevisiae VIG9 Encodes GDP-mannose Pyrophosphorylase, Which Is Essential for Protein Glycosylation

Hashimoto

Sakakibara

Yamasaki

et al. 1997

Journal of Biological Chemistry

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A genomic DNA fragment that complements a newly identified protein glycosylation-defective mutation, vig9, of Saccharomyces cerevisiae was cloned. Chromosomal integration of this fragment by homologous recombination indicated that it contains the wild type VIG9 gene. The nucleotide sequence was determined. A predicted gene product showed significant amino acid sequence homology with several bacterial enzymes that catalyze the synthesis of (deoxy)ribonucleotide diphosphate sugars from sugar phosphates and (deoxy)ribonucleotide triphosphate. We examined the enzyme activity to synthesize GDP-mannose in the cell extracts of the wild type, vig9 -1 mutant, and VIG9 transformant yeasts. Reduction of the activity in the mutant cell and its restoration by VIG9 suggested that the VIG9 gene is the structural gene for GDP-mannose pyrophosphorylase of S. cerevisiae which catalyzes the production of GDPmannose. We demonstrated the enzyme activity of Vig9 protein using a recombinant fusion protein produced in Escherichia coli.Most secretory and membrane proteins of eukaryotic cells are modified by glycosylation. Glycosylation endows protein with a wide variety of characteristics that are important in their cellular functions. Glycosylation occurs in the secretory pathway, and three types of modification are known: (i) attachment of N-linked saccharides to asparagine residues; (ii) attachment of O-linked saccharides to serine or threonine residues; and (iii) attachment of glycosylphosphatidylinositol anchors at the COOH termini. This is also the case with the yeast Saccharomyces cerevisiae, although mannose is the main component of N-and O-linked saccharides, and varieties of sugars are found in those of higher eukaryotes. In yeasts, the fourth type of protein-saccharide linkage was suggested for the covalent linkage between the cell wall proteins and glucans, although details are yet to be studied (1). Several excellent reviews on glycosylation in yeasts have been published (2)(3)(4)(5).N-Linked saccharides are first transferred to proteins as a Glc 3 Man 9 GlcNAc 2 unit from the Dol 1 -PP-intermediate when proteins are translocated into the endoplasmic reticulum. They are then trimmed to form the core N-linked saccharide, Man 8 GlcNAc 2 , which is common in eukaryotes. After proteins are transported from the endoplasmic reticulum to the Golgi apparatus in S. cerevisiae, core N-linked saccharides receive various quantities of mannose, depending on the protein species, one by one from GDP-Man. O-Linked saccharides that are composed exclusively of mannose in S. cerevisiae are also transferred to proteins in the Golgi apparatus sequentially from GDP-Man except for the first mannose, which is transferred from Man-P-Dol in the endoplasmic reticulum. As mannose is the major component of both N-and O-linked saccharides in S. cerevisiae, formation of GDP-Man, the activated form of mannose, should be vitally important.In S. cerevisiae, genetic approaches have been utilized in many scientific studies. To study the mechanism and function...

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Primary structure of the Saccharomyces cerevisiae GAL7 gene

Cited by 148 publications

References 43 publications

Hint, Fhit, and GalT: Function, Structure, Evolution, and Mechanism of Three Branches of the Histidine Triad Superfamily of Nucleotide Hydrolases and Transferases

Hint, Fhit, and GalT: Function, Structure, Evolution, and Mechanism of Three Branches of the Histidine Triad Superfamily of Nucleotide Hydrolases and Transferases

Enhanced Reactivity of Rhizopus oryzae Lipase Displayed on Yeast Cell Surfaces in Organic Solvents: Potential as a Whole-Cell Biocatalyst in Organic Solvents

Saccharomyces cerevisiae VIG9 Encodes GDP-mannose Pyrophosphorylase, Which Is Essential for Protein Glycosylation

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