Substitution of Asp-309 by Asn in the Arg-Asp-Pro (Rdp) Motif of Acetobacter Diazotrophicus Levansucrase Affects Sucrose Hydrolysis, but Not Enzyme Specificity

Biochem. J. (1999) 337, 503–506 (Printed in Great Britain)

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Substitution of Asp-309 by Asn in the Arg-Asp-Pro (RDP) motif of Acetobacter diazotrophicus levansucrase affects sucrose hydrolysis, but not enzyme specificity
; ; ; ; Frank R. BATISTA, La! zaro HERNANDEZ1, Julio R. FERNANDEZ, Juan ARRIETA, Carmen MENENDEZ, Rau! l GOMEZ, ; Yanet TAMBARA and Tirso PONS
Centre for Genetic Engineering and Biotechnology (CIGB), P.O. Box 6162, Havana 10600, Cuba

β-Fructofuranosidases share a conserved aspartic acid-containing motif (Arg-Asp-Pro ; RDP) which is absent from α-glucopyranosidases. The role of Asp-309 located in the RDP motif of levansucrase (EC 2.4.1.10) from Acetobacter diazotrophicus SRT4 was studied by site-directed mutagenesis. Substitution of Asp309 by Asn did not affect enzyme secretion. The kcat of the mutant levansucrase was reduced 75-fold, but its Km was similar to that of the wild-type enzyme, indicating that Asp-309 plays a major role in catalysis. The two levansucrases showed optimal

activity at pH 5.0 and yielded similar product profiles. Thus the mutation D309N affected the efficiency of sucrose hydrolysis, but not the enzyme specificity. Since the RDP motif is present in a conserved position in fructosyltransferases, invertases, levanases, inulinases and sucrose-6-phosphate hydrolases, it is likely to have a common functional role in β-fructofuranosidases. Key words : enzyme activity, β-fructofuranosidase, fructosyltransferase, LsdA, site-directed mutagenesis.

INTRODUCTION
The endophytic sugar-cane bacterium Acetobacter diazotrophicus SRT4 produces a constitutive levansucrase (LsdA ; EC 2.4.1.10), which is secreted into the medium [1]. The enzyme catalyses transfructosylation from sucrose to a variety of acceptors, including water (sucrose hydrolysis), glucose (sucrose exchange), sucrose (1-kestose synthesis) and levan (polymerization). The reaction occurs via a Ping-Pong mechanism involving the formation of a transient...