Structural features of a bacterial cyclic α-maltosyl-(1→6)-maltose (CMM) hydrolase critical for CMM recognition and hydrolysis

Abstract

Cyclic α-maltosyl-(1→6)-maltose (CMM, -{→6)-α-d-Glc-(1→4)-α-d-Glc-(1→6)-α-d-Glc-(1→4)-α-d-Glc-(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 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.

Molecular analysis of cyclic α-maltosyl-(1→6)-maltose binding protein in the bacterial metabolic pathway

“…In the case of maltose transport system of E. coli, the substrate specificity is conveyed by the interface between maltose-binding protein in a closed conformation and the membrane transporter (MalFGK 2 ) in a pre-translocation state [32], and the structure of maltose-binding protein complexed with a non-physiological ligand (β-cyclodextrin) was in a fully open conformation [33]. Therefore, the present study suggested that CMMBP plays a key role in the "three-stage" metabolic pathway of A. globiformis [14] because this protein is tuned for specific binding and intake of the cyclic compound, CMM. While CNN is formed from partial hydrolysate of starch (maltodextrin) by the actions of two enzymes of L. monocytogenes (α-6-glucosyltransferase and 3-α-isomaltosyltransferase) [25], the metabolic system of A. globiformis forms CMM directly from starch by the two-step actions of a single extracellular enzyme, 6-α-maltosyltransferase (CmmA) [10].…”

“…Therefore, the present study suggested that CMMBP plays a key role in the “three-stage” metabolic pathway of A . globiformis [ 14 ] because this protein is tuned for specific binding and intake of the cyclic compound, CMM. While CNN is formed from partial hydrolysate of starch (maltodextrin) by the actions of two enzymes of L .…”

“…An extracellular enzyme, 6-α-maltosyltransferase (CmmA), produces CMM by inter- and intramolecular α-1,6-transglucosylation [ 10 ], and two intracellular enzymes, CMM hydrolase (CmmF) [ 12 ] and α-glucosidase (CmmB), synergistically degrade CMM to glucose [ 13 ]. We previously reported the mechanistic details of the hydrolysis of CMM by CMM hydrolase with its crystal structures [ 14 ]. Gene products encoded by cmmC , cmmD , and cmmE were suggested to form an ABC transporter system of an SBP (CmmC) and TMDs (CmmD and CmmE) [ 11 ].…”

A Novel Subfamily GH13_46 of the α-Amylase Family GH13 Represented by the Cyclomaltodextrinase from Flavobacterium sp. No. 92

“…Despite the separated position of the entire cluster of the novel subfamily in the evolutionary tree (Figure 4), from the structural point of view, it is possible to point out that the members of the so-called neopullulanase subfamily [22,30] could be considered the most closely related structural homologs of the new subfamily. They are represented here by various enzyme specificities from CAZy subfamilies GH13_20 and GH13_21 [31][32][33][34][72][73][74][75][76][77][78], and eventually GH13_39 (Table S3). However, since the Flavobacterium sp.…”