Acetobacter diazotrophicus, a nitrogen-fixing bacterium associated with sugar cane, secretes a levansucrase (sucrose-2,6-beta-D-fructan 6-beta-D-fructosyltransferase; EC 2.4.1.10). This enzyme is constitutively expressed and represents more than 70% of the total proteins secreted by strain SRT4. The purified protein consists of a single 58 kDa polypeptide with an isoelectric point of 5.5. Its activity is optimal at pH 5.0. It catalyses transfructosylation from sucrose to a variety of acceptors including water (sucrose hydrolysis), glucose (exchange reaction), fructan (polymerase reaction) and sucrose (oligofructoside synthesis). In vivo the polymerase activity leads to synthesis of a high-molecular-mass fructan of the levan type. A. diazotrophicus levansucrase catalyses transfructosylation via a Ping Pong mechanism involving the formation of a transient fructosyl-enzyme intermediate. The catalytic mechanism is very similar to that of Bacillus subtilis levansucrase. The kinetic parameters of the two enzymes are of the same order of magnitude. The main difference between the two enzyme specificities is the high yield of oligofructoside, particularly 1-kestotriose and kestotetraose, accumulated by A. diazotrophicus levansucrase during sucrose transformation. We discuss the hypothesis that these catalytic features may serve the different biological functions of each enzyme.

The levansucrase (sucrose:2,6-beta-D-fructan 6-beta-D-fructosyltransferase, EC 2.4.1.10) structural gene from a Bacillus subtilis mutant strain displaying a low polymerase activity was sequenced. Only one missense mutation changing Arg331 to His was responsible for this modified catalytic property. From this allele we created new mutations by directed mutagenesis, which modified the charge and polarity of site 331. Examination of the kinetics of the purified levansucrase variants revealed that transfructosylation activities are affected differently by the substitution chosen. His331----Arg completely restored the properties of the wild-type enzyme. The most striking feature of the other variants, namely Lys331, Ser331 and Leu331, was that they lost the ability of the wild-type enzyme to synthesize levan from sucrose alone. They were only capable of catalysing the first step of levan chain elongation, which is the formation of the trisaccharide ketose. The variant His331----Lys presented a higher kcat. for sucrose hydrolysis than the wild-type, and only this hydrolase activity was preserved in a solvent/water mixture in which the wild-type acted as a true polymerase. The two other substitutions reduced the efficiency of transfructosylation activities of the enzyme via the decrease of the rate of fructosyl-enzyme intermediate formation. For all variants, the sucrose affinity was slightly affected. This strong modulation of the enzyme specificities from a single amino acid substitution led us to postulate the hypothesis that bacterial levansucrases and plant fructosyltransferases involved in fructan synthesis may possess a common ancestral form.

The mechanism of action of levansucrase of Bacillus subtilis was investigated by initial velocity measurements of reactions catalyzed by this enzyme and initial velocity of isotopic exchange at equilibrium. Comparison of experimental results with theoretical results derived from various postulated mechanisms, supports a "ping-pong" mechanism, involving the intermediate participation of a fructosyl-enzyme. Rate constants for each step of this sequential mechanism were estimated.Levansucrase of Bacillus subtilis catalyses mainly the following reaction:According to the experimental conditions [l], water, alcohols, monosaccharides, sucrose, oligosaccharides and levans may act as fructosyl acceptors. In the presence of sucrose alone the enzymatic activity leads to the formation of free fructose, oligosaccharides and levans.Small levans accelerate the rate of transfructosylation from sucrose and increase the ratio of levans formed to free fructose. In the presence of levans the reversibility of the reaction (1) was demonstrated :Levansucrase has also an hydrolytic activity towards small levans Levan, + H,O -+ Levann-l + Fructose and it catalyzes the following exchange reaction :Preliminary studies [I -41 of these enzymatic activities led the authors to assume the formation of a fructosyl enzyme as an intermediate in the catalytic process.Abbreviations. In kinetic equations and symbols the letters G, F, L, S and E, represent glucose, fructose, levans, sucrose and total enzyme, respectively.Enzyme. Levansucrase or P-2.6-fructan : D-glucose I-fructosyltransferase (EC 2.4.1.10).In the present work the mechanism of action of the levansucrase of B. subtilis was reinvestigated from careful kinetic examination of catalytic activities in steady-state conditions.First of all, owing to the complex activity of levansucrase, we attempted to define conditions in which we can describe with simple rate equations, the catalytic activities of levansucrase. Under such conditions we tried, from analysis of kinetic results, to answer the following questions: Which is the sequence of elementary steps in the catalytic process of levansucrase? Is there a fructosyl-enzyme intermediate in this sequence? What are the magnitudes of the rate constants associated with each step in this sequence Z MATERIALS AND METHODS LevansucraseLevansucrase was prepared from culture supernatant of a constitutive strain of B. subtilis [5] according to the procedure of Dedonder [ 13. SubstratesUniformly labelled [14C]sucrose and [14C]glucose were purchased from C.E.A. (Saclay) and purified by paper chromatography before use. With purified samples no retention of radioactivity was observed at the origin of the paper chromatogram.Levans of low molecular weight, obtained according to Dedonder et al. [6] were used throughout this work. Molecular dispersity was reduced by filtration on a molecular sieve. The molecular weight was determined by chromatography on Biogel P60 Eur. J. Biochem. 41 (1974)