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Starch-branching enzyme catalyzes the cleavage of α-1, 4-linkages and the subsequent transfer of α-1,4 glucan to form an α-1,6 branch point in amylopectin. Sequence analysis of the rice-branching enzyme I (BEI) indicated a modular structure in which the central α-amylase domain is flanked on each side by the N-terminal carbohydrate-binding module 48 and the α-amylase C-domain. We determined the crystal structure of BEI at a resolution of 1.9 Å by molecular replacement using the Escherichia coli glycogen BE as a search model. Despite three modular structures, BEI is roughly ellipsoidal in shape with two globular domains that form a prominent groove which is proposed to serve as the α-polyglucan-binding site. Amino acid residues Asp344 and Glu399, which are postulated to play an essential role in catalysis as a nucleophile and a general acid/base, respectively, are located at a central cleft in the groove. Moreover, structural comparison revealed that in BEI, extended loop structures cause a narrowing of the substrate-binding site, whereas shortened loop structures make a larger space at the corresponding subsite in the Klebsiella pneumoniae pullulanase. This structural difference might be attributed to distinct catalytic reactions, transglycosylation and hydrolysis, respectively, by BEI and pullulanase.

The rice branching enzyme I (BEI) overproduced in Escherichia coli cells was investigated with respect to action on starches. BEI treatment decreased the turbidity of starch suspensions with distinct pasting behaviors from a native starch. This result suggests the great potential of BEI as a molecular tool for the production of a novel glucan polymer.

Human Sulfotranferase 1A1 (hSULT1A1) catalyzes the transfer of a sulforyl group from a 3'-phosphoadenosine 5'-phosphosulfate (PAPS) donor to a variety of substrates (acceptors) containing either an amine or a hydroxyl group, leading to modification of the acceptor's biological activity. In order to gain insight into the molecular mechanism underlying the broad specificity of hSULT1A1, we have crystallized the enzyme and determined its structure in the presence of 3' phosphoadenosine 5'-phosphate (PAP) alone, PAP and 3-Cyano-7-Coumarin (3CyC), and PAP and 2-Napthol (2NAP). These structures demonstrated high plasticity of the acceptor binding site which was mainly attributed to substantial movements of the gating loop (residues 86-90) that enabled the binding of large and elongated phenol substrates. We were also interested in increasing the SULT1A1 specificity to para-nitrophenol (pNP) on the account of the other substrates through the use of directed evolution methodology. This method allowed the generation of a hSULT1A1 variant mutated at position D249G which exhibits a marked increase in activity toward pNP while decreasing its activity toward 3CyC and 2NAP. The determined crystal structure of D249G revealed the effect of the mutation on surface electrostatic potential and loop stability in the proximity of the active site pocket.

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