Table 1.
List of known phosphorylases.
Figure 1.
Phylogenetic tree of the GH65 enzymes characterized.
Sequences used are: 1, Bsel_2816 of B. selenitireducens MLS10 (GenBank ID: ADI00307.1); 2, All1058 of Nostoc sp. PCC 7120; 3, All4989 of Nostoc sp. PCC 7120; 4, Csac_0439 of Caldicellulosiruptor saccharolyticus DSM 8903 (ABP66077.1); 5, KojP of Thermoanaerobacter brockii ATCC 35047 (AAE30762.1); 6, Bsel_2056 of B. selenitireducens MLS10 (ADH99560.1); 7, MapA of Paenibacillus sp. SH-55 (BAD97810.1); 8, maltose phosphorylase of Lactobacillus sanfranciscensis DSM 20451 (CAA11905.1); 9, MPase of Bacillus sp. RK-1 (BAC54904.1); 10, EF0957 of Enterococcus faecalis V583 (AAO80764.1); 11, LBA1870 of Lactobacillus acidophilus NCFM (AAV43670.1); 12, maltose phosphorylase from Lactobacillus brevis ATCC 8287 (Q7SIE1); 13, TreP of T. brockii ATCC 35047 (AAE18727.1); 14, Bsel_1207 of B. selenitireducens MLS10 (ADH98720.1); 15, TPase of Geobacillus stearothermophilus SK-1 (BAC20640.1); 16, Cphy_1874 of Clostridium phytofermentans ISDg (ABX42243.1); 17, Cphy_1019 of C. phytofermentans ISDg (ABX41399.1); 18, TrePP of Lactococcus lactis subsp. lactis Il1403 (AAK04526.1); 19, Atm1 of Metarhizium acridum CQMA102 (ABB51158.1); 20, TreA of Aspergillus nidulans FGSCA4 (EAA66407.1); 21, Ath1 of Saccharomyces cerevisiae S288c (CAA58961.1); 22, Atc1 of Candida albicans (AAV05390.1). Proteins from B. selenitireducens MLS10 are underlined.
Figure 2.
Double-reciprocal plot of the Pi-dependent hydrolysis of kojibiose by Bsel_2816 at various concentrations of Pi.
Open circles, 1 mM Pi; closed circles, 2 mM Pi; open squares, 3 mM Pi; closed squares, 5 mM Pi; and open triangles, 10 mM Pi. The kinetic parameters were kcat = 0.43 ± 0.02 (s−1), KmA = 8.9 ± 0.6 (mM), KmB = 0.19 ± 0.12 (mM), and KiA = 41 ± 29 (mM). The values were determined by regressing the data with the following equation using the Grafit Version 7.02: v = kcat[E]0[A][B]/(KiAKmB+KmA[B]+KmB[A]+[A][B]).
Figure 3.
Linkages cleaved in the hydrolysis of βGlc1P.
A: Cleavage positions by the action of phosphatases and glycosidases. B: Results of the enzymatic hydrolysis of βGlc1P by Bsel_2816 in H218O.
Figure 4.
The anomeric specificity during the hydrolysis of βGlc1P.
Figure 5.
Screening for the suitable acceptor within polyols in the reverse phosphorolysis by Bsel_2816.
The acceptors used are: 1, ethylene glycol; 2, glycerol; 3, erythritol; 4, no acceptor. S1 and S2, Glc and as βGlc1P as standards, respectively. The TLC plate was developed twice with acetonitrile-water (9∶1 v/v).
Table 2.
Assignments of 1H and 13C NMR signals of 2-O-α-d-glucosylglycerol.
Figure 6.
The reaction rates of the generations of GG and Glc from 10βGlc1P and various concentrations of glycerol.
Open circles, d[GG]/dt; closed circles, d[Glc]/dt.
Figure 7.
Time course of the increase of βGlc1P and Glc during the phosphorolysis of GG.
Open circles, [βGlc1P]; closed circles, [Glc].
Figure 8.
Double-reciprocal plot of the phosphorolysis of GG by Bsel_2816 at various concentrations of Pi.
Open circles, 0.5; closed circles, 1 mM Pi; open squares, 2 mM Pi; closed squares, 3 mM Pi; open triangles, 5 mM Pi; and closed triangles, 10 mM Pi. The kinetic parameters were kcat = 95±2 (s−1), KmA = 1.1±0.1 (mM), KmB = 0.57±0.07 (mM), and KiA = 18±3 (mM). The values were determined by regressing the data with the following equation using Grafit Version 7.02: v = kcat[E]0[A][B]/(KiAKmB+KmA[B]+KmB[A]+[A][B]).
Figure 9.
The reactions catalyzed by GGP.
A, The reaction mechanism of GGP; B, Detectable reactions catalyzed by GGP.