Article

Molecular and Functional Characterization of a Unique Sucrose Hydrolase from Xanthomonas axonopodis pv. glycines

School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, South Korea.
Journal of Bacteriology (Impact Factor: 2.81). 02/2004; 186(2):411-8. DOI: 10.1128/JB.186.2.411-418.2004
Source: PubMed

ABSTRACT

A novel sucrose hydrolase (SUH) from Xanthomonas axonopodis pv. glycines, a causative agent of bacterial pustule disease on soybeans, was studied at the functional and molecular levels.
SUH was shown to act rather specifically on sucrose (Km = 2.5 mM) but not on sucrose-6-phosphate. Protein analysis of purified SUH revealed that, in this monomeric enzyme with an
estimated molecular mass of 70,223 ± 12 Da, amino acid sequences determined for several segments have corresponding nucleotide
sequences in XAC3490, a protein-coding gene found in the genome of X. axonopodis pv. citri. Based on this information, the SUH gene, consisting of an open reading frame of 1,935 bp, was cloned by screening
a genomic library of X. axonopodis pv. glycines 8ra. Database searches and sequence comparison revealed that SUH has significant homology to some family 13
enzymes, with all of the crucial invariant residues involved in the catalytic mechanism conserved, but it shows no similarity
to known invertases belonging to family 32. suh expression in X. axonopodis pv. glycines requires sucrose induction, and insertional mutagenesis resulted in an absence of sucrose-inducible sucrose
hydrolase activity in crude protein extracts and a sucrose-negative phenotype. Recombinant SUH, overproduced in Escherichia coli and purified, was shown to have the same enzymatic characteristics in terms of kinetic parameters.

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    • "glycines is a Gram-negative, aerobic , motile, and plant pathogenic bacterium causing bacterial pustules (BP) diseases in soybean (Jones and Fett, 1987; Vauterin et al., 1995). The strain 8ra was first isolated from Iowa, USA in 1980 and has been widely used as a model strain for various genetic studies (Groth and Braun, 1989; Han et al., 2007; Kim et al., 2003, 2004; Pham et al., 2004). The transcription activator-like (TAL) effectors of this species are important because they specifically regulate host gene expression (especially, resistance/susceptibility-related genes) and are highly useful in transcription activator-like effector nuclease (TALEN) technology by customizing their amino acid repeat region. "
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    ABSTRACT: Xanthomonas axonopodis pv. glycines 8ra is a causal agent of bacterial pustule disease in soybean. This bacterium possesses transcription activator-like (TAL) effectors which are useful for genetic/protein engineering applications in higher organisms including plants and humans. Here, we report that the draft genome sequence consists of 5,337,885-bp double-stranded DNA encoding 4,674 open reading frames (ORFs) in 13 different contigs. This genome sequence would be useful in applications of TAL effectors in genetic engineering and in elucidating virulence factors against plants.
    Full-text · Article · Jun 2014 · Journal of Biotechnology
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    • "The lack of expression of tvsuC suggests that this putative transporter is not related to Suc uptake in the conditions assayed. Sequence analyses of the T. virens genome revealed no homologous sequences to bacterial-type Suc carriers previously described, such as Suc permease or Suc-specific phosphotransferase transporters (Slee & Tanzer, 1982; Kakinuma & Unemoto, 1985; Gunasekaran et al., 1990; Postma et al., 1993, 1996; Jahreis et al., 2002; Kim et al., 2004). Additionally, no homologous sequences to UmSrt were identified in T. virens genome. "
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    ABSTRACT: • Sucrose exuded by plants into the rhizosphere is a crucial component for the symbiotic association between the beneficial fungus Trichoderma and plant roots. In this article we sought to identify and characterize the molecular basis of sucrose uptake into the fungal cells. • Several bioinformatics tools enabled us to identify a plant-like sucrose transporter in the genome of Trichoderma virens Gv29-8 (TvSut). Gene expression profiles in the fungal cells were analyzed by Northern blotting and quantitative real-time PCR (qRT-PCR). Biochemical and physiological studies were conducted on Gv29-8 and fungal strains impaired in the expression of TvSut. • TvSut exhibits biochemical properties similar to those described for sucrose symporters from plants. The null expression of tvsut caused a detrimental effect on fungal growth when sucrose was the sole source of carbon in the medium, and also affected the expression of genes involved in the symbiotic association. • Similar to plants, T. virens contains a highly specific sucrose/H(+) symporter that is induced in the early stages of root colonization. Our results suggest an active sucrose transference from the plant to the fungal cells during the beneficial associations. In addition, our expression experiments suggest the existence of a sucrose-dependent network in the fungal cells that regulates the symbiotic association.
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    ABSTRACT: La synthèse d’oligosides complexes et glycoconjugués reste difficilement réalisable par voie chimique, freinant ainsi l’exploitation de ces molécules à grande échelle. Le recours aux catalyseurs enzymatiques apparaît comme une alternative prometteuse mais les enzymes naturelles ne présentent pas toujours les propriétés adéquates, en particulier sur le plan de leurs spécificités, pour être intégrées dans des voies de synthèse chimio-enzymatiques. Le défi de cette thèse a consisté à mettre à profit les techniques d’ingénierie enzymatique pour concevoir des glyco-enzymes déployant de nouvelles spécificités, produire à façon des intermédiaires de synthèse chimique et ouvrir la voie à de nouveaux procédés d’obtention d’oligosides d’intérêt thérapeutique. Il s’agissait de développer de nouvelles transglucosidases capables de glucosyler des dérivés de la N-acétyl-D-glucosamine et du L-rhamnose pour produire des disaccharides n’existant pas dans la nature mais pouvant entrer dans une voie de synthèse chimique destinées à synthétiser des haptènes oligosaccharidiques mimant les motifs antigéniques des lipopolysaccharides des sérotypes 1b et 3a de Shigella flexneri. A terme, l’objectif est de développer un vaccin multivalent de troisième génération contre ces pathogènes, responsables du décès de milliers de personnes chaque année dans le monde. Pour relever ce défi, nous avons tout d’abord évalué le potentiel de quatre glucane-saccharases sauvages, spécifiques de la synthèse de liaisons osidiques distinctes, à glucosyler les accepteurs cibles. Les résultats obtenus ont montré qu’aucune des enzymes testées ne catalysait efficacement la réaction attendue. Cette étude préliminaire a cependant permis de sélectionner l’amylosaccharase de Neisseria polysaccharea, comme enzyme candidate au développement de biocatalyseurs à stéréo- et régio-spécificités contrôlées. Le remodelage du catalyseur a été guidé par l’analyse des modèles des complexes enzyme:accepteur. Sept acides aminés situés dans le site de fixation de l’accepteur (sous-site +1) ont été identifiés et individuellement remplacés par les 19 acides aminés possibles par mutagénèse dirigée. Une banque de 133 mutants a été créée qui renfermait 43 mutants d’intérêt pour les réactions de glucosylation considérées. Notamment, le mutant I228Y a révélé une toute nouvelle régio-spécificité vis-à-vis de l’α-methyl L-rhamnopyranose. Il s’agit du premier exemple de création de nouvelle spécificité pour les enzymes de cette famille et plus largement pour des α-transglycosidases. Le mutant F290K, également isolé dans cette première librairie, s’est montré capable de glucosyler l’α-allyl N-acétyl-D-glucoamine avec une efficacité catalytique 130 fois plus élevée que celle observée avec l’enzyme sauvage. Ces deux nouvelles enzymes ont été employées pour la synthèse des disaccharides ciblés et la validation de la voie chimio-enzymatique proposée pour la synthèse des haptènes saccharidiques. Cette stratégie a été poursuivie par la construction de banques de double-mutants permettant de générer une diversité plus importante, focalisée sur ces positions, et rechercher des effets synergiques entre les différentes mutations pour améliorer la réaction de glucosylation des accepteurs cibles. Vingt double-mutants ont pu être ainsi identifiés.------------------------------------------------------------------------------------------------------- The difficult access to complex carbohydrates and glycoconjugates by chemical synthesis impairs their development on a large scale. Therefore, the use of biocatalysts appears as an appealing alternative, yet poorly exploited in spite of the fast-growing development of engineering technologies allowing the search and the construction of better performing enzymes, displaying novel specificities. The objective of this thesis aimed to apply enzyme engineering techniques to conceive novel glyco-enzymes well-adapted for the glucosylation of naturally non- or poorly recognized molecules (N-acetyl-D-glucosamine and L-rhamnose derivatives fulfilling the subsequent chemical step requisites). The glucosylated motives obtained via the enzymatic route, and corresponding to the serotype-specific branched α-D-glucopyranosyl linkages of Shigella flexneri 2a, 1b and 3a, are subsequently elongated through a chemical synthetic pathway to synthesize the antigenic oligosaccharides. This chemo-enzymatic road could open the way to oligosaccharide haptens at low cost, that could in the future be used in the development of a third-generation vaccine against S. flexneri 1b and 3a. In a first study, we have evaluated the potential of four wild-type glucansucrases, specific for the synthesis of distinct osidic linkages, to glucosylate both types of targeted acceptors. Our preliminary results demonstrated that none of the tested glucansucrases allowed an efficient glucosylation of the target acceptors with the desired linkage specificity. On the basis of these results, amylosucrase from Neisseria polysaccharea was chosen as a candidate enzyme for the development of biocatalysts with controlled stereo- and regio-specificities that could enable the glucosylation of both types of acceptors of interest. A semi-rational approach based on the engineering of the acceptor binding site (subsite +1) was undertaken to evolve this enzyme. By individually exploring 7 positions of the active site using site-directed mutagenesis, the strategy led to the isolation of 47 mutants of interest for the considered glucosylation reactions (over the 133 generated mono-mutants) and to the identification of 2 key positions (228 and 290). Noteworthy, I228Y mutant displayed an entirely novel regio-specificity towards the α-methyl L-rhamnopyranose and in the presence of allyl N-acetyl-D-glucosamine, F290K mutant permitted a 130 fold enhancement of catalityc efficiency compared to wild-type amylosucrase. This strategy was further pursued with the construction of double-mutant libraries enabling the generation of an enlarged diversity, focused on the identified positions, to investigate the synergistic effects between the different mutations in order to improve the glucosylation reactions of the target acceptors. Twenty double-mutants were thus identified
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