ABSTRACT
Xylella fastidiosa is a gram-negative bacterium that causes serious diseases in economically important crops, including grapevine, coffee, and citrus fruits. X. fastidiosa colonizes the xylem vessels of the infected plants, thereby blocking water and nutrient transport. The genome sequence of X. fastidiosa has revealed an operon containing nine genes possibly involved in the synthesis of an exopolisaccharide (EPS) named fastidian gum that can be related with the pathogenicity of this bacterium. The α-1,3-mannosyltransferase (GumH) enzyme from X. fastidiosa is involved in fastidian gum production. GumH is responsible for the transfer of mannose from guanosine diphosphate mannose (GDP-man) to the cellobiose-pyrophosphate-polyprenol carrier lipid (CPP-Lip) during the assembly and biosynthesis of EPS. In this work, a method for real-time detection of recombinant GumH enzymatic activity was successfully developed using a Quartz Crystal Microbalance with dissipation monitoring (QCM-D). The QCM-D transducer was strategically modified with CPP-Lip by using a solid-supported lipid bilayer that makes use of a self-assembled monolayer of 1-undecanethiol. Monitoring the real-time CPP-Lip QCM-D transducer in the presence of GDP-man and GumH enzyme shows a mass increase, indicating the transfer of mannose. The real-time QCM-D determination of mannosyltransferase function was validated by a High Performance Liquid Chromatography (LC) method developed for determination of GDP produced by enzymatic reaction. LC results confirmed the activity of recombinant GumH protein, which is the first enzyme involved in the biosynthesis of the EPS from X. fastidiosa enzymatically characterized.
Subject(s)
Bacterial Proteins/chemistry , Mannosyltransferases/chemistry , Quartz Crystal Microbalance Techniques/methods , Xylella/enzymology , Bacterial Proteins/genetics , Enzymes, Immobilized/chemistry , Mannosyltransferases/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Xylella/geneticsABSTRACT
The GumH enzyme from Xylella fastidiosa catalyzes the transfer reaction of a mannose from GDP-mannose to the carrier lipid cellobiose-pyrophosphate-polyprenol (Glc(2)-PP-Lip), an intermediary in the reaction for the synthesis of the exopolysaccharide (EPS) fastidian gum. The gumH gene was subcloned in the pMal-c2x vector, allowing the expression of the GumH-MBP fusion protein. Various attempts were made to obtain protein with the necessary degree of purity for crystallographic studies but the yield was very low. The gumH gene was then subcloned in the pET28a vector allowing the expression of the GumH enzyme in fusion with a histidine-rich peptide. The protein was purified and characterized. The three-dimensional structure of the X. fastidiosa GumH enzyme was modeled by threading studies. The model consists of N- and C-terminal domains similar in size and topology and separated by a deep cleft, which includes the EX(7)E motif that can be involved in the catalysis of GumH.
Subject(s)
Bacterial Proteins/chemistry , Mannosyltransferases/chemistry , Recombinant Proteins/chemistry , Xylella/enzymology , Amino Acid Motifs , Amino Acid Sequence , Catalysis , Circular Dichroism , Cloning, Molecular , Crystallography, X-Ray , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Genetic Vectors , Histidine/chemistry , Lipid Metabolism , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Peptides/chemistry , Protein Conformation , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Sequence Homology, Amino AcidABSTRACT
Este trabalho estudou o comportamento de soluções de glutaraldeído utilizadas na fixação de biopróteses, por meio das relações 280/235 nm. Os resultados mostraram que este índice de controle analítico não é adequado, porque grandes variações nesta relação são devidas aos elevados valores de coeficientes de extinção molares das formas poliméricas. Foram detectadas também variações significativas nas absorbâncias em 280nm, sugerindo a ocorrência de alterações químicas do glutaraldeído em função do tempo (< 7 dias). Estes resultados mostram que estas soluções de glutaraldeído podem estar longe do ideal a obtenção de biopróteses homogêneas.
Abstract - This work studied the behavior of aqueous or buffered glutaraldehyde solutions used for the fixation of cardiac bioprostheses by means of the 280/235 nm ratios. The results showed that this analytical index may not be adequate since great variations are mainly due to the high extinction coefficients for the polymeric forms. Besides, variations were also observed in the 280 nm absorbancies, suggesting that significative changes may also occur in glutaraldehyde effective concentration (7 days). These results suggests that this type of glutaraldehyde solutions may be far from ideal for the manufacture of homogeneou
