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4-O-β-D-Mannosyl-D-glucose phosphorylase (MGP), found in anaerobes, converts 4-O-β-D-mannosyl-D-glucose (Man-Glc) to α-D-mannosyl phosphate and D-glucose. It participates in mannan metabolism with cellobiose 2-epimerase (CE), which converts β-1,4-mannobiose to Man-Glc. A putative MGP gene is present in the genome of the thermophilic aerobe Rhodothermus marinus (Rm) upstream of the gene encoding CE. Konjac glucomannan enhanced production by R. marinus of MGP, CE, and extracellular mannan endo-1,4-β-mannosidase. Recombinant RmMGP catalyzed the phosphorolysis of Man-Glc through a sequential bi-bi mechanism involving ternary complex formation. Its molecular masses were 45 and 222 kDa under denaturing and nondenaturing conditions, respectively. Its pH and temperature optima were 6.5 and 75 °C, and it was stable between pH 5.5-8.3 and below 80 °C. In the reverse reaction, RmMGP had higher acceptor preferences for 6-deoxy-D-glucose and D-xylose than R. albus NE1 MGP. In contrast to R. albus NE1 MGP, RmMGP utilized methyl β-D-glucoside and 1,5-anhydro-D-glucitol as acceptor substrates.

Edited by Judit Ov adiIn Ruminococcus albus, 4-O-b-D-mannosyl-D-glucose phosphorylase (RaMP1) and b-(1,4)-mannooligosaccharide phosphorylase (RaMP2) belong to two subfamilies of glycoside hydrolase family 130. The two enzymes phosphorolyze b-mannosidic linkages at the nonreducing ends of their substrates, and have substantially diverse substrate specificity. The differences in their mechanism of substrate binding have not yet been fully clarified. In the present study, we report the crystal structures of RaMP1 with/without 4-O-b-D-mannosyl-Dglucose and RaMP2 with/without b-(1?4)-mannobiose. The structures of the two enzymes differ at the +1 subsite of the substrate-binding pocket. Three loops are proposed to determine the different substrate specificities. One of these loops is contributed from the adjacent molecule of the oligomer structure. In RaMP1, His245 of loop 3 forms a hydrogen-bond network with the substrate through a water molecule, and is indispensible for substrate binding.Keywords: 4-O-b-D-mannosyl-D-glucose phosphorylase; glycoside hydrolase family 130; hydrogen bond-network; substrate recognition; X-ray crystallography; b-1,4-mannooligosaccharide phosphorylase Highlights• Reveals the structures of RaMP1 and RaMP2, and their complexes with substrates.• Structural insight into substrate recognition of RaMP1 and RaMP2 is discussed.• RaMP1 is the first GH130 enzyme exhibiting as a homotrimer in its native state.• Three loops are important in substrate recognition at the +1 subsite in RaMP1 and RaMP2.• A hydrogen-bond network in RaMP1 is considered to be important for substrate binding.

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