Overexpression, purification, biochemical characterization, and molecular modeling of recombinant GDP-mannosyltransferase (GumH) from Xylella fastidiosa

Muniz, J.R.C.; Alves, Cláudia; Pieri, Celina de; Beltramini, Leila Maria; Selistre-de-Araújo, Heloísa Sobreiro; Vettore, André Luiz; Silva, Felipe Rodrigues da; Arruda, Paulo; Garratt, R.C.; Oliva, Glaucius; Souza, Dulce Helena Ferreira de

doi:10.1016/j.bbrc.2004.01.077

Cited by 11 publications

(3 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…An alignment of the amino acid sequence of PelF with glycosyltransferases from the GT-4 family showed the presence of conserved amino acid residues and an EX 7 E motif in the C-terminal domain of the protein. Previous studies proposed that these conserved residues are involved in stabilizing the donor substrate and in glycosidic bond formation as a nucleophile (45)(46)(47)(48)(49)(50). Here, it was shown that replacement of E405 in this motif with alanine caused a significant reduction in PelF activity, whereas mutation of the second glutamic acid, E413, had almost no impact on PelF activity, as shown by pellicle production at the air-liquid interface (Fig.…”

Section: Discussionmentioning

confidence: 55%

Role of PelF in Pel Polysaccharide Biosynthesis in Pseudomonas aeruginosa

Ghafoor

Jordens

Rehm

2013

Appl Environ Microbiol

View full text Add to dashboard Cite

Pseudomonas aeruginosa produces three exopolysaccharides, Psl, Pel, and alginate, that play vital roles in biofilm formation. Pel is a glucose-rich, cellulose-like exopolysaccharide. The essential Pel biosynthesis proteins are encoded by seven genes, pelA to pelG. Bioinformatics analysis suggests that PelF is a cytosolic glycosyltransferase. Here, experimental evidence was provided to support this PelF function. A UDP-glucose dehydrogenase-based assay was developed to quantify UDP-glucose. UDP-glucose was proposed as the substrate for PelF. The isogenic pelF deletion mutant accumulated 1.8 times more UDP-glucose in its cytosol than the wild type. This suggested that PelF, which was found localized in the cystosol, uses UDP-glucose as substrate. Additionally, in vitro experiments confirmed that PelF uses UDP-glucose as substrate. To analyze the functional roles of conserved residues in PelF, site-directed mutagenesis was performed. The presence of the EX 7 E motif is characteristic for various glycosyltransferase families, and in PelF, E405/E413 are the conserved residues in this motif. Replacement of E405 with A resulted in a reduction of PelF activity to 30.35% ؎ 3.15% (mean ؎ standard deviation) of the wild-type level, whereas replacement of the second E, E413, with A did not produce a significant change in the activity of PelF. Moreover, replacement of both E residues did not result in a loss of PelF function, but replacement of the conserved R325 or K330 with A resulted in a complete loss of PelF activity. Overall, our data show that PelF is a soluble glycosyltransferase that uses UDP-glucose as the substrate for Pel synthesis and that conserved residues R325 and K330 are important for the activity of PelF.

show abstract

Section: Discussionmentioning

confidence: 55%

Role of PelF in Pel Polysaccharide Biosynthesis in Pseudomonas aeruginosa

Ghafoor

Jordens

Rehm

2013

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…Together, there is a wealth of new knowledge of X. fastidiosa and its interactions with plants and insect vectors. The availability of genomic information on X. fastidiosa has led to numerous reports of proteins and other biochemical processes in this species (30,35,36,45,70,84,89,103,120), but these studies are not emphasized in this review. We also do not address the many studies of detection methods and strain variation (26,39,56,58,81).…”

Section: Introductionmentioning

confidence: 99%

Living in two Worlds: The Plant and Insect Lifestyles ofXylella fastidiosa

Chatterjee

Almeida

Lindow

2008

Annu. Rev. Phytopathol.

361

391

View full text Add to dashboard Cite

Diseases caused by Xylella fastidiosa have attained great importance worldwide as the pathogen and its insect vectors have been disseminated. Since this is the first plant pathogenic bacterium for which a complete genome sequence was determined, much progress has been made in understanding the process by which it spreads within the xylem vessels of susceptible plants as well as the traits that contribute to its acquisition and transmission by sharpshooter vectors. Although this pathogen shares many similarities with Xanthomonas species, such as its use of a small fatty acid signal molecule to coordinate virulence gene expression, the traits that it utilizes to cause disease and the manner in which they are regulated differ substantially from those of related plant pathogens. Its complex lifestyle as both a plant and insect colonist involves traits that are in conflict with these stages, thus apparently necessitating the use of a gene regulatory scheme that allows cells expressing different traits to co-occur in the plant.

show abstract

“…Glycosyltransferases are carbohydrate acting enzymes that transfer glycosyl residues from activated donor molecules to acceptor, which is essential for exopolysaccharide biosynthesis and thus plays an important role in cell surface properties and cellecell interactions of bacteria [11,12].…”

Section: Introductionmentioning

confidence: 99%

Biochemical characterization of a novel bifunctional glycosyl-1-phosphate transferase involved in the exopolysaccharide biosynthesis

Wang

et al. 2015

Biochemical and Biophysical Research Communications

View full text Add to dashboard Cite

Overexpression, purification, biochemical characterization, and molecular modeling of recombinant GDP-mannosyltransferase (GumH) from Xylella fastidiosa

Cited by 11 publications

References 45 publications

Role of PelF in Pel Polysaccharide Biosynthesis in Pseudomonas aeruginosa

Role of PelF in Pel Polysaccharide Biosynthesis in Pseudomonas aeruginosa

Living in two Worlds: The Plant and Insect Lifestyles ofXylella fastidiosa

Biochemical characterization of a novel bifunctional glycosyl-1-phosphate transferase involved in the exopolysaccharide biosynthesis

Contact Info

Product

Resources

About