Dirk Penninga

The raw starch binding domain of cyclodextrin glycosyltransferase from Bacillus circulans strain 251

The E-domain of cyclodextrin glycosyltransferase (CGTase) (EC 2.4.1.19) from Bacillus circulans strain 251 is a putative raw starch binding domain. Analysis of the maltose-dependent CGTase crystal structure revealed that each enzyme molecule contained three maltose molecules, situated at contact points between protein molecules. Two of these maltoses were bound to specific sites in the E-domain, the third maltose was bound at the C-domain. To delineate the roles in raw starch binding and cyclization reaction kinetics of the two maltose binding sites in the E-domain, we replaced Trp-616 and Trp-662 of maltose binding site 1 and Tyr-633 of maltose binding site 2 by alanines using site-directed mutagenesis. Purified mutant CGTases were characterized with respect to raw starch binding and cyclization reaction kinetics on both soluble and raw starch. The results show that maltose binding site 1 is most important for raw starch binding, whereas maltose binding site 2 is involved in guiding linear starch chains into the active site. β-Cyclodextrin causes product inhibition by interfering with catalysis in the active site and the function of maltose binding site 2 in the E-domain. CGTase mutants in the E-domain maltose binding site 1 could no longer be crystallized as maltose-dependent monomers. Instead, the W616A mutant CGTase protein was successfully crystallized as a carbohydrate-independent dimer; its structure has been refined to 2.2 Å resolution. The three-dimensional structure shows that, within the error limits, neither the absence of carbohydrates nor the W616A mutation caused significant further conformational changes. The modified starch binding and cyclization kinetic properties observed with the mutant CGTase proteins thus can be directly related to the amino acid replacements.

Enzymic synthesis of cyclothiomaltins

The effective conversion of 4-thio-α-maltosyl fluoride 1 into cyclothiomaltins 2, 3 and 4, using cyclodextrin glycosyltransferase enzymes from Bacillus circulans, is described.

63 PRENATAL DIAGNOSIS OF CYSTIC FIBEOSIS BY LINKED DNAPROBES

Several groups recently reported DNA markers on chromosome 7 in linkage with the locus of the cystic fibrosis (CF) mutation. From the linkage data collected to date probes pJ3.11 and met appear to be sufficiently close to the CF locus (each with about 1% of recombination in approx. 400 meioses studied) to be used in diagnosis. They can be applied in prenatal diagnosis and carrier detection in informative families with an affected child. As an illustration we present the first diagnosis on CF by DNA markers carried out prenatally in the Netherlands. In the family involved one parent appeared to be heterozygous for met D and the other for pJ3.11, both on TaqI-restricted DNA. Their affected child appeared to be homozygous for both probes, whereas DNA isolated from a chorionic villus biopsy at 11 weeks pregnancy showed heterozygosity for both pJ3.11 and met D. Our risk calculations assuming a 3% recombination frequency between 3.11 and the CF locus, and a 4% between met and CF, resulted in a greater than 99.5% probability for the foetus to be non-affected.