P. B. Høj

Identification and characterization of a fruit-specific, thaumatin-like protein that accumulates at very high levels in conjunction with the onset of sugar accumulation and berry softening in grapes.

The protein composition of the grape (Vitis vinifera cv Muscat of Alexandria) berry was examined from flowering to ripeness by gel electrophoresis. A protein with an apparent molecular mass of 24 kD, which was one of the most abundant proteins in extracts of mature berries, was purified and identified by amino acid sequence to be a thaumatin-like protein. Combined cDNA sequence analysis and electrospray mass spectrometry revealed that this protein, VVTL1 (for V. vinifera thaumatin-like protein 1), is synthesized with a transient signal peptide as seen for apoplastic preproteins. Apart from the removal of the targeting signal and the formation of eight disulfide bonds, VVTL1 undergoes no other posttranslational modification. Southern, northern, and western analyses revealed that VVTL1 is found in the berry only and is encoded by a single gene that is expressed in conjunction with the onset of sugar accumulation and softening. The exact role of VVTL1 is unknown, but the timing of its accumulation correlates with the inability of the fungal pathogen powdery mildew (Uncinula necator) to initiate new infections of the berry. Western analysis revealed that the presence of thaumatin-like proteins in ripening fruit might be a widespread phenomenon.

Cloning and characterization of Vitis vinifera UDP-glucose:flavonoid 3-O-glucosyltransferase, a homologue of the enzyme encoded by the maize Bronze-1 locus that may primarily serve to glucosylate anthocyanidins in vivo.

We report here the cloning and optimized expression at 16 °C and the characterization of a Vitis vinifera UDP-glucose:flavonoid 3-O-glucosyltransferase, an enzyme responsible for a late step in grapevine anthocyanin biosynthesis. The properties of this and other UDP-glucose:flavonoid 3-O-glucosyltransferases, homologues of the product encoded by the maize Bronze-1locus, are a matter of conjecture. The availability of a purified recombinant enzyme allowed for the unambiguous determination of the characteristics of a flavonoid 3-O-glucosyltransferase. Kinetic analyses showed that k cat for glucosylation of cyanidin, an anthocyanidin substrate, is 48 times higher than for glucosylation of the flavonol quercetin, whereasK m values are similar for both substrates. Activity toward other classes of substrates is absent. Cu2+ ions strongly inhibit the action of this and other glucosyltransferases; however, we suggest that this phenomenon in large part is due to Cu2+-mediated substrate degradation rather than inhibition of the enzyme. Additional lines of complementary biochemical data also indicated that in the case of V. vinifera, the principal, if not only, role of UDP-glucose:flavonoid 3-O-glucosyltransferases is to glucosylate anthocyanidins in red fruit during ripening. Other glucosyltransferases with a much higher relative activity toward quercetin are suggested to glucosylate flavonols in a distinct spatial and temporal pattern. It should be considered whether gene products homologous to Bronze-1 in some cases more accurately should be referred to as UDP-glucose:anthocyanidin 3-O-glucosyltransferases.

The UDP-glucose:p-Hydroxymandelonitrile-O-Glucosyltransferase That Catalyzes the Last Step in Synthesis of the Cyanogenic Glucoside Dhurrin in Sorghum bicolor ISOLATION, CLONING, HETEROLOGOUS EXPRESSION, AND SUBSTRATE SPECIFICITY

The final step in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor is the transformation of the labile cyanohydrin into a stable storage form byO-glucosylation of (S)-p-hydroxymandelonitrile at the cyanohydrin function. The UDP-glucose:p-hydroxymandelonitrile-O-glucosyltransferase was isolated from etiolated seedlings of S. bicoloremploying Reactive Yellow 3 chromatography with UDP-glucose elution as the critical step. Amino acid sequencing allowed the cloning of a full-length cDNA encoding the glucosyltransferase. Among the few characterized glucosyltransferases, the deduced translation product showed highest overall identity to Zea maysflavonoid-glucosyltransferase (Bz-Mc-2 allele). The substrate specificity of the enzyme was established using isolated recombinant protein. Compared with endogenousp-hydroxymandelonitrile, mandelonitrile, benzyl alcohol, and benzoic acid were utilized at maximum rates of 78, 13, and 4%, respectively. Surprisingly, the monoterpenoid geraniol was glucosylated at a maximum rate of 11% compared withp-hydroxymandelonitrile. The picture that is emerging regarding plant glucosyltransferase substrate specificity is one of limited but extended plasticity toward metabolites of related structure. This in turn ensures that a relatively high, but finite, number of glucosyltransferases can give rise to the large number of glucosides found in plants.

Genetic Determinants of Volatile-Thiol Release by Saccharomyces cerevisiae during Wine Fermentation

ABSTRACT Volatile thiols, particularly 4-mercapto-4-methylpentan-2-one (4MMP), make an important contribution to the aroma of wine. During wine fermentation, Saccharomyces cerevisiae mediates the cleavage of a nonvolatile cysteinylated precursor in grape juice (Cys-4MMP) to release the volatile thiol 4MMP. Carbon-sulfur lyases are anticipated to be involved in this reaction. To establish the mechanism of 4MMP release and to develop strains that modulate its release, the effect of deleting genes encoding putative yeast carbon-sulfur lyases on the cleavage of Cys-4MMP was tested. The results led to the identification of four genes that influence the release of the volatile thiol 4MMP in a laboratory strain, indicating that the mechanism of release involves multiple genes. Deletion of the same genes from a homozygous derivative of the commercial wine yeast VL3 confirmed the importance of these genes in affecting 4MMP release. A strain deleted in a putative carbon-sulfur lyase gene, YAL012W, produced a second sulfur compound at significantly higher concentrations than those produced by the wild-type strain. Using mass spectrometry, this compound was identified as 2-methyltetrathiophen-3-one (MTHT), which was previously shown to contribute to wine aroma but was of unknown biosynthetic origin. The formation of MTHT in YAL012W deletion strains indicates a yeast biosynthetic origin of MTHT. The results demonstrate that the mechanism of synthesis of yeast-derived wine aroma components, even those present in small concentrations, can be investigated using genetic screens.

Purification of (1→3)-β-glucan endohydrolase isoenzyme II from germinated barley and determination of its primary structure from a cDNA clone

A (1→3)-β-D-glucan 3-glucanonydrolase (EC 3.2.1.39) of apparent Mr 32 000, designated GII, has been purified from germinated barley grain and characterized. The isoenzyme is resolved from a previously purified isoenzyme (GI) on the basis of differences in their isoelectric points; (1→3)-β-glucanases GI and GII have pI values of 8.6 and ≥ 10.0, respectively. Comparison of the sequences of their 40 NH2-terminal amino acids reveals 68% positional identity. A 1265 nucleotide pair cDNA encoding (1→3)-β-glucanase isoenzyme GII has been isolated from a library prepared with mRNA of 2-day germinated barley scutella. Nucleotide sequence analysis of the cDNA has enabled the complete primary structure of the 306 amino acid (1→3)-β-glucanase to be deduced, together with that of a putative NH2-terminal signal peptide of 28 amino acid residues. The (1→3)-β-glucanase cDNA is characterized by a high (G+C) content, which reflects a strong bias for the use of G or C in the wobble base position of codons. The amino acid sequence of the (1→3)-β-glucanase shows highly conserved internal domains and 52% overall positional identity with barley (1→3, 1→4)-β-glucanase isoenzyme EII, an enzyme of related but quite distinct substrate specificity. Thus, the (1→3)-β-glucanases, which may provide a degree of protection against microbial invasion of germinated barley grain through their ability to degrade fungal cell wall polysaccharides, appear to share a common evolutionary origin with the (1→3, 1→4)-β-glucanases, which function to depolymerize endosperm cell walls in the germinated grain.

Generation of a stable folding intermediate which can be rescued by the chaperonins GroEL and GroES

Pig heart mitochondrial malate dehydrogenase was chemically denatured in guanidine HCl. Upon 50‐fold dilution of the denaturant spontaneous refolding could be observed in the temperature range 12–32°C. At 36°C spontaneous refolding was not observed but a stable folding intermediate that is fairly resistant to aggregation was formed. This intermediate is readily refolded by the chaperonins GroEL and GroES and may prove useful in future attempts to describe several aspects of chaperonin action at physiological temperatures.

Cellobiohydrolase I from Trichoderma reesei: Identification of an active-site nucleophile and additional information on sequence including the glycosylation pattern of the core protein.

(R,S)-3,4-Epoxybutyl beta-cellobioside, but not the corresponding propyl and pentyl derivatives, inactivates specifically and irreversibly cellobiohydrolase I from Trichoderma reesei by covalent modification of Glu212, the putative active-site nucleophile. The position and identity of the modified amino acid residue were determined using a combination of comparative liquid chromatography coupled on-line to electrospray ionization mass spectrometry, tandem mass spectrometry and microsequencing. It was found that the core protein corresponds to the N-terminal sequence pyrGlu1-Gly434 (Gly435) of intact cellobiohydrolase I. In the particular enzyme samples investigated, the asparagine residues in positions 45, 270 and 384 are each linked to a single 2-acetamido-2-deoxy-D-glucopyranose residue.

The Role of Molecular Chaperones in Mitochondrial Protein Import and Folding

Molecular chaperones play a critical role in many cellular processes. This review concentrates on their role in targeting of proteins to the mitochondria and the subsequent folding of the imported protein. It also reviews the role of molecular chaperons in protein degradation, a process that not only regulates the turnover of proteins but also eliminates proteins that have folded incorrectly or have aggregated as a result of cell stress. Finally, the role of molecular chaperones, in particular to mitochondrial chaperonins, in disease is reviewed. In support of the endosymbiont theory on the origin of mitochondria, the chaperones of the mitochondrial compartment show a high degree of similarity to bacterial molecular chaperones. Thus, studies of protein folding in bacteria such as Escherichia coli have proved to be instructive in understanding the process in the eukaryotic cell. As in bacteria, the molecular chaperone genes of eukaryotes are activated by a variety of stresses. The regulation of stress genes involved in mitochondrial chaperone function is reviewed and major unsolved questions regarding the regulation, function, and involvement in disease of the molecular chaperones are identified.

THE GENES ENCODING MAMMALIAN CHAPERONIN 60 AND CHAPERONIN 10 ARE LINKED HEAD-TO-HEAD AND SHARE A BIDIRECTIONAL PROMOTER

Chaperonins are a class of stress-inducible molecular chaperones involved in protein folding. We report the cloning, sequencing and characterisation of the rat mitochondrial chaperonin 60 and chaperonin 10 genes. The two genes are arranged in a head-to-head configuration and together comprise 14 kb and contain 14 introns. The genes are linked together by a region of approximately 280 bp, which constitutes a bidirectional promoter and includes a common heat-shock element. Insertion of the shared promoter region between two reporter genes is sufficient to drive their expression under both constitutive and heat-shock conditions. The arrangement of the mammalian chaperonin genes suggests the potential to provide the coordinated regulation of their products in a manner that is mechanistically distinct from, yet conceptually similar to, that employed by the bacterial chaperonin (groE) operon.

Reducing haziness in white wine by overexpression of Saccharomyces cerevisiae genes YOL155c and YDR055w

Grape proteins aggregate in white wine to form haze. A novel method to prevent haze in wine is the use of haze protective factors (Hpfs), specific mannoproteins from Saccharomyces cerevisiae, which reduce the particle size of the aggregated proteins. Hpf1p was isolated from white wine and Hpf2p from a synthetic grape juice fermentation. Putative structural genes, YOL155c and YDR055w, for these proteins were identified from partial amino acid sequences of Hpf1p and Hpf2p, respectively. YOL155c also has a homologue, YIL169c, in S. cerevisiae. Comparison of the partial amino acid sequence of deglycosylated-Hpf2p with the deduced protein sequence of YDR055w, confirmed five of the 15 potential N-linked glycosylation sites in this sequence were occupied. Methylation analysis of the carbohydrate moieties of Hpf2p indicated that this protein contained both N- and O-linked mannose chains. Material from fermentation supernatant of deletion strains had significantly less activity than the wild type. Moreover, YOL155c and YIL169c overexpressing strains and a strain overexpressing 6xHis-tagged Hpf2p produced greater haze protective activity than the wild type strains. A storage trial demonstrated the short to midterm stability of 6xHis-tagged Hpf2p in wine.