Crystal structure of maltose phosphorylase from Lactobacillus brevis: Unexpected evolutionary relationship with glucoamylases

Background: Maltose phosphorylase (MP) is a dimeric enzyme that catalyzes t he conversion of maltose and inorganic phosphate into beta -D-glucose-1-pho sphate and glucose without requiring any cofactors, such as pyridoxal phosp hate. The enzyme is part of operons that are involved in maltose/malto-olig osaccharide metabolism. Maltose phosphorylases have been classified in fami ly 65 of the glycoside hydrolases. No structure is available for any member of this family. Results: We report here the 2.15 Angstrom resolution crystal structure of t he MP from Lactobacillus brevis in complex with the cosubstrate phosphate. This represents the first structure of a disaccharide phosphorylase. The st ructure consists of an N-terminal complex beta sandwich domain, a helical l inker, an (alpha/alpha)(6) barrel catalytic domain, and a C-terminal beta s heet domain. The (alpha/alpha)(6) barrel has an unexpected strong structura l and functional analogy with the catalytic domain of glucoamylase from Asp ergillus awamori. The only conserved glutamate of MP (Glu487) superposes on to the catalytic residue Glu179 of glucoamylase and likely represents the g eneral acid catalyst. The phosphate ion is bound in a pocket facing the car boxylate of Glu487 and is ideally positioned for nucleophilic attack of the anomeric carbon atom. This site is occupied by the catalytic base carboxyl ate in glucoamylase. Conclusions: These observations strongly suggest that maltose phosphorylase has evolved from glucoamylase. MP has probably conserved one carboxylate g roup for acid catalysis and has exchanged the catalytic base for a phosphat e binding pocket. The relative positions of the acid catalytic group and th e bound phosphate are compatible with a direct-attack mechanism of a glycos idic bond by phosphate, in accordance with inversion of configuration at th e anomeric carbon as observed for this enzyme.