Abstract
Cyclic α-maltosyl-(1→6)-maltose (CMM, cyclo-{→6)-α-d-Glcp-(1→4)-α-d-Glcp-(1→6)-α-d-Glcp-(1→4)-α-d-Glcp-(1→})is a cyclic glucotetrasaccharide with alternating α-1,4 and α-1,6 linkages. CMM is composed of two maltose units and is one of the smallest cyclic glucooligosaccharides. Although CMM is resistant to usual amylases, it is efficiently hydrolyzed by CMM hydrolase (CMMase), belonging to subfamily 20 of glycoside hydrolase family 13 (GH13_20). Here, we determined the ligand-free crystal structure of CMMase from the soil-associated bacterium Arthrobacter globiformis and its structures in complex with maltose, panose, and CMM to elucidate the structural basis of substrate recognition by CMMase. The structures disclosed that although the monomer structure consists of three domains commonly adopted by GH13 and other α-amylase-related enzymes, CMMase forms a unique wing-like dimer structure. The complex structure with CMM revealed four specific subsites, namely -3', -2, -1, and +1'. We also observed that the bound CMM molecule adopts a low-energy conformer compared with the X-ray structure of a single CMM crystal, also determined here. Comparison of the CMMase active site with those in other enzymes of the GH13_20 family revealed that three regions forming the wall of the cleft, denoted PYF (Pro-203/Tyr-204/Phe-205), CS (Cys-163/Ser-164), and Y (Tyr-168), are present only in CMMase and are involved in CMM recognition. Combinations of multiple substitutions in these regions markedly decreased the activity toward CMM, indicating that the specificity for this cyclic tetrasaccharide is supported by the entire shape of the pocket. In summary, our work uncovers the mechanistic basis for the highly specific interactions of CMMase with its substrate CMM.
Highlights
Cyclic ␣-maltosyl-(136)-maltose (CMM, cyclo-{36)-␣-DGlcp-(134)-␣-D-Glcp-(136)-␣-D-Glcp-(134)-␣-D-Glcp-(13}) is a cyclic glucotetrasaccharide with alternating ␣-1,4 and ␣-1,6 linkages
The crystal structure of CMM hydrolase (CMMase) was solved by molecular replacement using the Geobacillus stearothermophilus neopullulanase (GsNPL) structure (33.8% sequence identity by EMBOSS Needle Pairwise Alignment, PDB code 1J0H) as a search model
The three-domain architecture is generally present in GH13 and ␣-amylase–related clan glycoside hydrolase (GH)-H enzymes (e.g. GH70 and GH77) [36]
Summary
We have previously identified a novel enzymatically produced cyclic tetrasaccharide, cyclic ␣-maltosyl-(136)-maltose (CMM, cyclo-{36)-␣-D-Glcp-(134)-␣-D-Glcp-(136)-␣-DGlcp-(134)-␣-D-Glcp-(13}) [29]. Further studies showed that the action of a single enzyme contained in the culture supernatant, 6-␣-maltosyltransferase (6MT, EC 3.2.1.-), was responsible for the synthesis of CMM [30]. A further study revealed that an intracellular enzyme, CMM hydrolase (CMMase, EC 3.2.1.-), plays a key role in the CMM catabolism [31,32,33]. CMMase degrades CMM into two maltose molecules by a two-stage hydrolysis of the ␣-1,6 linkages via maltosyl-maltose (MM, Fig. 1). We determined the crystal structures of CMMase in a ligand-free form and in complex forms with three glucooligosaccharide ligands, including CMM. The X-ray structure of a single CMM crystal was determined, and its conformation was compared with the CMMase-bound CMM molecule. A site-directed mutational analysis of the key residues in the substrate-binding site confirmed their importance in the recognition of CMM
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