Probing enzyme–substrate interactions at the catalytic subsite ofLeuconostoc mesenteroidessucrose phosphorylase with site-directed mutagenesis: the roles of Asp⁴⁹and Arg³⁹⁵

Abstract

Sucrose phosphorylase is a bacterial a -transglucosidase that catalyses glucosyl transfer from sucrose to phosphate, releasing D-fructose and a -D-glucose 1-phosphate as the product of the fi rst ( enzyme glucosylation ) and second ( enzyme deglucosylation ) step of the enzymatic reaction, respectively. The transferred glucosyl moiety of sucrose is accommodated at the catalytic subsite of the phosphorylase through a network of charged hydrogen bonds whereby a highly conserved residue pair of Asp and Arg points towards the equatorial hydroxyl at C 4 . To examine the role of this ' hyperpolar ' binding site for the substrate 4-OH, we have mutated Asp 49 and Arg 395 of Leuconostoc mesenteroides sucrose phosphorylase individually to Ala (D49A) and Leu (R395L), respectively, and also prepared an ' uncharged ' double mutant harbouring both site-directed substitutions. The effi ciency for enzyme glucosylation from sucrose was massively decreased in purifi ed preparations of D49A (10 7 -fold) and R395L (10 5 -fold) as compared to wild-type enzyme. The double mutant was not active above the detection limit. Enzyme deglucosylation to phosphate proceeded relatively effi cient in D49A as well as R395L, about 500fold less than in the wild-type phosphorylase. Substrate inhibition by phosphate and a loss in selectivity for reaction with phosphate as compared to water were new features in the two mutants. Asp 49 and Arg 395 are both essential in the catalytic reaction of L. mesenteroides sucrose phosphorylase.

Analysis of the diversity of the glycoside hydrolase family 130 in mammal gut microbiomes reveals a novel mannoside-phosphorylase function

“…H is replaced by tyrosine (Y). This variation in phosphatebinding residues has already been described in other GP-containing families, such as members of the family GH13_18 [46]. In the sequences belonging to the metanode UC4, the catalytic and phosphate-binding residues are not conserved, but the presence of a signal peptide in some of these sequences suggests that they could be hydrolases.…”

Phosphoryl Transfer from α-d-Glucose 1-Phosphate Catalyzed by Escherichia coli Sugar-Phosphate Phosphatases of Two Protein Superfamily Types

“…Agp was isolated from the crude extract with a Strep-Tactin sepharose column (IBA, Göttingen, Germany), using a general protocol described previously (51). Had13 was isolated using a Cu 2ϩ -loaded IMAC Sepharose High Performance column (GE Healthcare, Little Chalfont, United Kingdom) operated according to standard procedures.…”

Interplay of catalytic subsite residues in the positioning of α-d-glucose 1-phosphate in sucrose phosphorylase

“…A two-step purification procedure was used. Chromatography on Strep -Tactin Sepharose column was as described previously [22] . Pooled protein fractions were concentrated, loaded onto Fractogel EMD-DEAE and purified according to reported procedures [8] .…”

“…The following compounds were analyzed using protocols described previously [11] , [22] . Inorganic phosphate was measured colorimetrically at 850 nm (detection limit: 2.5 µM).…”

“…αGlc 1- P and Glc 6- P were assayed enzymatically using PGM and G6PDH (detection limit: 50 µM). Sucrose synthesized in enzymatic reactions was measured using high performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD; detection limit of sucrose: 5 µM) [22] . Protein concentration was measured with the BioRad dye-binding method referenced against BSA.…”