Active site studies and mechanism of action of Leuconostoc mesenteroides B-512FM dextransucrase

• Main Author: Fu, Daotian
• Format: Dissertation
• Language: English
• Subjects: chemistry, biochemistry

Leuconostoc mesenteroides NRRL B-512FM dextransucrase was treated extensively with Sigma crude dextranase, followed by column chromatography on Bio-Gel A-5m. The purified dextransucrase has a specific activity of 84 IU/mg, and was shown by SDS-PAGE to have a single protein of 158 kDa with dextransucrase activity. The procedure gave a 100-fold purification with 42% yield, and was used to produce purified enzyme for sequencing.
The active site of dextransucrase was studied by chemical modification with diethyl pyrocarbonate and photo-dye-oxidation. Kinetic analysis of the inactivation data showed that two histidines are essential for the dextransucrase activity. We proposed that the two imidazolium groups of the histidines at the active site donate protons to the leaving fructose molecules during dextran synthesis.
The specificity of the acceptor binding site was studied with $\alpha$-methyl-D-glucopyranoside analogues modified at C-2, C-3, and C-4 positions. It was found that the enzyme transfers D-glucopyranose to the C-6 hydroxyl group of analogues modified at C-2 and C-3, and to the C-4 hydroxyl of 4-inverted, and to the C-3 hydroxyl of 4-deoxy analogues. The hydroxyl at C-2 is not as important as the hydroxyls at C-3 and C-4 for acceptor binding. The hydroxyl at C-4 is particularly important since it determines the binding orientation of the $\alpha$-methyl-D-glucopyranoside ring.
The acceptor product structures of maltodextrins with dextransucrase have been studied by using known specificities of porcine pancreatic $\alpha$-amylase and $\alpha$-glucosidase, and by methylation analysis. It was found that dextransucrase transfer a D-glucosyl group to either the nonreducing end residue or the reducing end residue of maltodextrins. When a D-glucose was transferred to the nonreducing residue, the first product thus formed then serves as an acceptor to give the second product, which serves as an acceptor to give the third product, etc. When a D-glucose was transferred to the reducing residue, however, the first product thus formed will not serve as an acceptor to give further products or serves as a very poor acceptor.