Structure and activity of exo-1,3/1,4-ß-glucanase from marine bacterium Pseudoalteromonas sp. BB1 showing a novel C-terminal domain.

UNLABELLED: Following the discovery of an exo-1,3/1,4-ß-glucanase (glycoside hydrolase family 3) from a seaweed-associated bacterium Pseudoalteromonas sp. BB1, the recombinant three-domain protein (ExoP) was crystallized and its structure solved to 2.3 Å resolution. The first two domains of ExoP, both of which contribute to the architecture of the active site, are similar to those of the two-domain barley homologue ß-d-glucan exohydrolase (ExoI) with a distinctive Trp-Trp clamp at the +1 subsite, although ExoI displays broader specificity towards ß-glycosidic linkages. Notably, excision of the third domain of ExoP results in an inactive enzyme. Domain 3 has a ß-sandwich structure and was shown by CD to be more temperature stable than the native enzyme. It makes relatively few contacts to domain 1 and none at all to domain 2. Two of the domain 3 residues involved at the interface, Q683 (forming one hydrogen bond) and Q676 (forming two hydrogen bonds) were mutated to alanine. Variant Q676A retained about half the activity of native ExoP, but the Q683A variant was severely attenuated. The crystal structure of Q683A-ExoP indicated that domain 3 was highly mobile and that Q683 is critical to the stabilization of ExoP by domain 3. Small-angle X-ray scattering data lent support to this proposal. Domain 3 does not appear to be an obvious carbohydrate-binding domain and is related neither in sequence nor structure to the additional domains characterized in other glycoside hydrolase 3 subgroups. Its major role appears to be for protein stability but it may also help orient substrate. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 3UT0, 3USZ, 3F95 and 3RRX.

Carbohydrate binding sites in Candida albicans exo-ß-1,3-glucanase and the role of the Phe-Phe 'clamp' at the active site entrance.

Candida albicans exo-ß-1,3-glucanase (Exg; EC 3.2.1.58) is implicated in cell wall ß-D-glucan remodelling through its glucosyl hydrolase and/or transglucosylase activities. A pair of antiparallel phenylalanyl residues (F144 and F258) flank the entrance to the active site pocket. Various Exg mutants were studied using steady-state kinetics and crystallography aiming to understand the roles played by these residues in positioning the ß-1,3-D-glucan substrate. Mutations at the Phe-Phe entranceway demonstrated the requirement for double-sided CH/π interactions at the +1 subsite, and the necessity for phenylalanine rather than tyrosine or tryptophan. The Tyr-Tyr double mutations introduced ordered water molecules into the entranceway. A third Phe residue (F229) nearby was evaluated as a possible +2 subsite. The inactive double mutant E292S/F229A complexed with laminaritriose has provided the first picture of substrate binding to Exg and demonstrated how the Phe-Phe arrangement acts as a clamp at the +1 subsite. The terminal sugar at the -1 site showed displacement from the position of a monosaccharide analogue with interchange of water molecules and sugar hydroxyls. An unexpected additional glucose binding site, well removed from the active site, was revealed. This site may enable Exg to associate with the branched glucan structure of the C. albicans cell wall.