The genes encoding the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum VPI1718 were characterized from a molecular and a biochemical point of view. This operon is composed of three genes, dhaB1, dhaB2, and dhaT. When grown in a vitamin B12-free mineral medium with glycerol as carbon source, Escherichia coli expressing dhaB1, dhaB2, and dhaT produces 1,3-PD and high glycerol dehydratase and 1,3-PD dehydrogenase activities. dhaB1 and dhaB2 encode, respectively, a new type of glycerol dehydratase and its activator protein. The deduced proteins DhaB1 and DhaB2, with calculated molecular masses of 88,074 and 34,149 Da, respectively, showed no homology with the known glycerol dehydratases that are all B12 dependent but significant similarity with the pyruvate formate lyases and pyruvate formate lyases activating enzymes and their homologues. The 1,158-bp dhaT gene codes for a 1,3-PD dehydrogenase with a calculated molecular mass of 41,558 Da, revealing a high level of identity with other DhaT proteins from natural 1,3-PD producers. The expression of the 1,3-PD operon in C. butyricum is regulated at the transcriptional level, and this regulation seems to involve a two-component signal transduction system DhaAS͞DhaA, which may have a similar function to DhaR, a transcriptional regulator found in other natural 1,3-PD producers. The discovery of a glycerol dehydratase, coenzyme B12 independent, should significantly influence the development of an economical vitamin B12-free biological process for the production of 1,3-PD from renewable resources.

Amylosucrase (E.C. 2.4.1.4) is a member of Family 13 of the glycoside hydrolases (the ␣-amylases), although its biological function is the synthesis of amylose-like polymers from sucrose. The structure of amylosucrase from Neisseria polysaccharea is divided into five domains: an all helical N-terminal domain that is not similar to any known fold, a (␤/␣) 8 -barrel A-domain, B-and B-domains displaying ␣/␤-structure, and a C-terminal eightstranded ␤-sheet domain. In contrast to other Family 13 hydrolases that have the active site in the bottom of a large cleft, the active site of amylosucrase is at the bottom of a pocket at the molecular surface. A substrate binding site resembling the amylase 2 subsite is not found in amylosucrase. The site is blocked by a salt bridge between residues in the second and eight loops of the (␤/␣) 8 -barrel. The result is an exo-acting enzyme. Loop 7 in the amylosucrase barrel is prolonged compared with the loop structure found in other hydrolases, and this insertion (forming domain B) is suggested to be important for the polymer synthase activity of the enzyme. The topology of the B-domain creates an active site entrance with several ravines in the molecular surface that could be used specifically by the substrates/ products (sucrose, glucan polymer, and fructose) that have to get in and out of the active site pocket.

Amylosucrase is a glucosyltransferase that synthesises an insoluble alpha-glucan from sucrose. The catalytic properties of the highly purified amylosucrase from Neisseria polysaccharea were characterised. Contrary to previously published results, it was demonstrated that in the presence of sucrose alone, several reactions are catalysed, in addition to polymer synthesis: sucrose hydrolysis, maltose and maltotriose synthesis by successive transfers of the glucosyl moiety of sucrose onto the released glucose, and finally turanose and trehalulose synthesis - these two sucrose isomers being obtained by glucosyl transfer onto fructose. The effect of initial sucrose concentration on initial activity demonstrated a non-Michaelian profile never previously described.