Enzymatic Characteristics of Cellobiose Phosphorylase fromRuminococcus albusNE1 and Kinetic Mechanism of Unusual Substrate Inhibition in Reverse Phosphorolysis

Abstract

Cellobiose phosphorylase (CBP) catalyzes the reversible phosphorolysis of cellobiose to produceD -glucopyranosyl phosphate (Glc1P) and D-glucose. It is an essential enzyme for the metabolism of cello-oligosaccharides in a ruminal bacterium, Ruminococcus albus. In this study, recombinant R. albus CBP (RaCBP) produced in Escherichia coli was characterized. It showed highest activity at pH 6.2 at 50 C, and was stable in a pH range of 5.5-8.8 and at below 40 C. It phosphorolyzed only cellobiose efficiently, and the reaction proceeded through a random-ordered bi bi mechanism, by which inorganic phosphate and cellobiose bind in random order and D-glucose is released before Glc1P. In the synthetic reaction, RaCBP showed highest activity to D-glucose, followed by 6-deoxy-Dglucose. D-Mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, 1,5-anhydro-D-glucitol, and gentiobiose also served as acceptors, although the activities for them were much lower than for D-glucose. D-Glucose acted as a competitive-uncompetitive inhibitor of the reverse synthetic reaction, which bound not only the Glc1P site (competitive) but also the ternary enzyme-Glc1P-Dglucose complex (uncompetitive).

Metabolic Mechanism of Mannan in a Ruminal Bacterium, Ruminococcus albus, Involving Two Mannoside Phosphorylases and Cellobiose 2-Epimerase

“…Man1P acted as a competitive inhibitor of rRaMP1 and rRaMP2 against both Man-Glc and inorganic phosphate, as observed for several cellobiose phosphorylases (3,29), indicating that these substrates bind to the enzymes in random order, and Man1P can be the second product (the first substrate in the reverse reaction). Therefore, a possible kinetic mechanism for these enzymes is a random order Bi Bi mechanism or a randomordered Bi Bi mechanism (3,29). In the reverse reaction of rRaMP1, Man-Glc served as a competitive inhibitor against both Man1P and D-glucose, indicating that rRaMP1 catalyzes phosphorolysis of Man-Glc through a random order Bi Bi mechanism.…”

“…5). Both rRaMP1 and rRaMP2 catalyzed phosphorolytic and synthetic reactions of Man-Glc through a random Bi Bi mechanism in contrast to other known inverting carbohydrate phosphorylases (3,(32)(33)(34). Both rRaMP1 and rRaMP2 specifically catalyzed the phosphorolysis of a ␤-1,4-mannosidic linkage at the non-reducing end of a substrate, but they had acceptor specificities that were clearly different from each other.…”

Characterization of a thermophilic 4-O-β-d-mannosyl-d-glucose phosphorylase fromRhodothermus marinus

“…These data indicate that RmMGP catalyzed the phosphorolysis of Man-Glc through a sequential bi-bi mechanism involving the formation of a ternary complex, as observed for RaMGP and other inverting carbohydrate phosphorylases. 5,6,[31][32][33][34][35][36][37] The calculated kinetic parameters are as follows: k cat = 20.5 ± 0.1 s −1 , K mA = 0.994 ± 0.051 mM, K mB = 1.07 ± 0.03 mM, and K iA = 5.78 ± 0.43 mM (A, Man-Glc; B, Pi).…”

Overcoming inefficient cellobiose fermentation by cellobiose phosphorylase in the presence of xylose

“…The reported catalytic efficiency (k cat,app /K M,app ) of RaCBP for xylose in the reverse phosphorolysis reaction is only 1% of that for glucose [18]. However, with SdCBP, a substantial amount of GX formation is observed (Figure  1E).…”

“…However, they are inefficient in terms of sugar consumption and ethanol production rates [16], or the systems remain to be fully optimized [17]. CBP from Ruminococcus albus NE1 (RaCBP) uses xylose as a substrate for the reverse of the phosphorolytic reaction [18]. We therefore hypothesized that the inefficiency in cellobiose and xylose co-fermentation previously observed was due to xylose interference with cellobiose consumption via CBP.…”