Structural and mutational analysis of substrate recognition in kojibiose phosphorylase

Abstract

Glycoside hydrolase (GH) family 65 contains phosphorylases acting on maltose (Glc-a1,4-Glc), kojibiose (Glc-a1,2-Glc), trehalose (Glc-a1,a1,-Glc), and nigerose (Glc-a1,3-Glc). These phosphorylases can efficiently catalyze the reverse reactions with high specificities, and thus can be applied to the practical synthesis of a-glucosyl oligosaccharides. Here, we determined the crystal structures of kojibiose phosphorylase from Caldicellulosiruptor saccharolyticus in complex with glucose and phosphate and in complex with kojibiose and sulfate, providing the first structural insights into the substrate recognition of a glycoside hydrolase family 65 enzyme. The loop 3 region comprising the active site of kojibiose phosphorylase is significantly longer than the active sites of other enzymes, and three residues around this loop, Trp391, Glu392, and Thr417, recognize kojibiose. Various mutants mimicking the residue conservation patterns of other phosphorylases were constructed by mutation at these three residues. Activity measurements of the mutants against four substrates indicated that Trp391 and Glu392, especially the latter, are required for the kojibiose activity.

Impact of antibiotic treatment and host innate immune pressure on enterococcal adaptation in the human bloodstream

“…The strongest hit was the structure of the kojibiose phosphorylase from Caldicellulosiruptor saccharolyticus, which has been solved in a complex with glucose or kojibiose (PDB no. 3wir and 3wiq, respectively; 28% sequence identity across the complete protein sequence) (29). These structures also included a phosphate/sulfate ion in the active site, mimicking the substrate-bound complex.…”

Diversity of phosphorylases in glycoside hydrolase families

“…(Jung et al 2 0 1 4 ) . T h e 3 D s t r u c t u r e o f t h e e n z y m e f r o m C. saccharolyticus was solved, which enhanced understanding of substrate recognition in this family (Okada et al 2014).…”

Structural Basis for Reversible Phosphorolysis and Hydrolysis Reactions of 2-O-α-Glucosylglycerol Phosphorylase

“…The crystal packing of GGP lacks the disulfide bond-linked dimer interfaces between the N-domains that were observed in the CsKP crystals (Fig. 1C) (22).…”

“…2A. Loop 1 corresponds to "loop 3" in reports for Lactobacillus acidophilus maltose phosphorylase and CsKP (22,29), and it is a key determinant of the substrate specificity of these enzymes. These loops completely cover the active site of GGP.…”

“…The reaction of GH-type inverting phosphorylases, including GH65, GH94, and GH112, is thought to proceed via a single displacement mechanism with the assistance of a general acid residue (13), which is probably Glu-475 in the case of GGP (11 (20), and GGP. However, the crystal structures of only two enzymes in the GH65 family, Lactobacillus brevis maltose phosphorylase (LbMP) in complex with inorganic phosphate (P i ) (21) and Caldicellulosiruptor saccharolyticus kojibiose phosphorylase (CsKP) in complex with glucose and P i and in complex with kojibiose and sulfate (22), have been reported from a structural point of view.…”