Pseudomonas syringae DC3000 levaansukraasi Lsc3 katalüütilise ...

Identification of catalytic triad amino acids in Pseudomonas syringae pv. tomato
levansucrase Lsc3 by mutational analysis
Triin Elmi
SUMMARY
Levansucrases are bacterial exoenzymes belonging to glycoside hydrolase family 68. They
catalyse the transfer of fructosyl residue from substrate to a variety of acceptors including
water (sucrose hydrolysis), sucrose (oligofructose synthesis) and oligofructans (polymerase
reaction). It has been shown that fructans, like polysaccharidic levan, are involved in fitness
and survival of bacteria in the environment and in phytopathogenesis, e.g. in development of
the dental plaque and biofilm. Moreover, fructooligosaccharides act as health-promoting
prebiotics in the gut of humans and animals.
Levansucrases and other fructosyl transferases and also invertases have a common five-blade
β-propeller fold containing a catalytic pocket with three conserved acidic amino acids. The
catalytic triad is composed of catalytic nucleophile (Asp), transition state stabilizer (Asp) and
general acid/base catalyst (Glu).
The aim of this study was to identify catalytic triad amino acids in the active centre of P.
syringae pv. tomato DC3000 levansucrase Lsc3. Using sequence alignment of Lsc3 and
levansucrases from other Gram-negative bacteria, Asp62, Asp219 and Glu303 were predicted
to form the catalytic triad. We performed site-directed mutagenesis of the lsc3 gene to
substitute Asp62 and Glu303 residues in Lsc3 protein with alanins and cloned the mutated
lsc3 genes to pURI3 expression vector. Previously constructed mutant lsc3 genes (encoding
substitutions Asp219Ala, Asp219Asn, Asp225Ala, Asp225Asn) were also cloned into pURI3
vector. As a result of this study, several mutant levansucrases (Asp62Ala, Asp219Ala,
Asp219Asn, Pro222Leu, Asp225Ala, Asp225Asn, Glu303Ala) were expressed in Escherichia
coli and their properties were characterized. Mutation of Asp62, Asp219 and Glu303 virtually
abolished catalytic activity of Lsc3. Substitution of Pro222 and Asp225 had no significant
effect on catalytic properties of Lsc3.
We conclude that Asp62, Asp219 and Glu303 are at key position in Lsc3 protein of P.
syringae pv. tomato and most probably comprise catalytic triad of the active site.
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