Aromatic interactions at the catalytic subsite of sucrose phosphorylase: Their roles in enzymatic glucosyl transfer probed with Phe → Ala and Phe → Asn mutants

Mutants of Leuconostoc mesenteroides sucrose phosphorylase having active-site Phe⁵² replaced by Ala (F52A) or Asn (F52N) were characterized by free energy profile analysis for catalytic glucosyl transfer from sucrose to phosphate. Despite large destabilization (?3.5 kcal/mol) of the transition states for enzyme glucosylation and deglucosylation in both mutants as compared to wild-type, the relative stability of the glucosyl enzyme intermediate was weakly affected by substitution of Phe⁵². In reverse reaction where fructose becomes glucocylated, “error hydrolysis” was the preponderant path of breakdown of the covalent intermediate of F52A and F52N. It is proposed, therefore, that Phe⁵² facilitates reaction of the phosphorylase through (1) positioning of the transferred glucosyl moiety at the catalytic subsite and (2) strong cation-π stabilization of the oxocarbenium ion-like transition states flanking the covalent enzyme intermediate.     

On the donor substrate dependence of group-transfer reactions by hydrolytic enzymes: Insight from kinetic analysis of sucrose phosphorylase-catalyzed transglycosylation

Chemical group-transfer reactions by hydrolytic enzymes have considerable importance in biocatalytic synthesis and are exploited broadly in commercial-scale chemical production. Mechanistically, these reactions have in common the involvement of a covalent enzyme intermediate which is formed upon enzyme reaction with the donor substrate and is subsequently intercepted by a suitable acceptor. Here, we studied the glycosylation of glycerol from sucrose by sucrose phosphorylase (SucP) to clarify a peculiar, yet generally important characteristic of this reaction: partitioning between glycosylation of glycerol and hydrolysis depends on the type and the concentration of the donor substrate used (here: sucrose, α-d -glucose 1-phosphate (G1P)). We develop a kinetic framework to analyze the effect and provide evidence that, when G1P is used as donor substrate, hydrolysis occurs not only from the β-glucosyl-enzyme intermediate (E-Glc), but additionally from a noncovalent complex of E-Glc and substrate which unlike E-Glc is unreactive to glycerol. Depending on the relative rates of hydrolysis of free and substrate-bound E-Glc, inhibition (Leuconostoc mesenteroides SucP) or apparent activation (Bifidobacterium adolescentis SucP) is observed at high donor substrate concentration. At a G1P concentration that excludes the substrate-bound E-Glc, the transfer/hydrolysis ratio changes to a value consistent with reaction exclusively through E-Glc, independent of the donor substrate used. Collectively, these results give explanation for a kinetic behavior of SucP not previously accounted for, provide essential basis for design and optimization of the synthetic reaction, and establish a theoretical framework for the analysis of kinetically analogous group-transfer reactions by hydrolytic enzymes.     

Cellobiose phosphorylase from Cellulomonas uda: gene cloning and expression in Escherichia coli, and application of the recombinant enzyme in a ‘glycosynthase-type’ reaction

We have cloned and sequenced the gene encoding cellobiose phosphorylase from Cellulomonas uda and report high yield production in Escherichia coli of a functional recombinant enzyme containing an N-terminal metal affinity fusion peptide. Use of heterologous gene expression increases the space-time yield of active phosphorylase by three orders of magnitude, compared to production of the enzyme with the natural organism. The full-length phosphorylase is a 91.3 kDa protein that consists of 821 amino acids and whose primary structure shares significant residue identity with different members of glycosyltransferase family 36. Purified enzyme was obtained in 39% overall yield by using copper-chelate and hydroxyapatite chromatographies. A comparative steady-state kinetic analysis for enzymatic reactions in the directions of phosphorolysis and synthesis of cellobiose at 30 °C and pH 6.6 demonstrates that the catalytic properties of the natural enzyme are retained completely in the recombinant cellobiose phosphorylase. The ability of the phosphorylase to utilize - -glucose 1-fluoride (G1F) as alternate glucosyl donor in place of - -glucose 1-phosphate (G1P) is exploited for the synthesis of β-1,4-glucosides under thermodynamic control in close to 100% yield.     

Catalytic mechanism of C-C hydrolase MhpC from Escherichia coli: kinetic analysis of His263 and Ser110 site-directed mutants

C-C hydrolase MhpC (2-hydroxy-6-keto-nona-1,9-dioic acid 5,6-hydrolase) from Escherichia coli catalyses the hydrolytic C-C cleavage of the meta-ring fission product on the phenylpropionic acid catabolic pathway. The crystal structure of E. coli MhpC has revealed a number of active-site amino acid residues that may participate in catalysis. Site-directed mutants of His263, Ser110, His114, and Ser40 have been analysed using steady-state and stopped-flow kinetics. Mutants H263A, S110A and S110G show 10(4)-fold reduced catalytic efficiency, but still retain catalytic activity for C-C cleavage. Two distinct steps are observed by stopped-flow UV/Vis spectrophotometry, corresponding to ketonisation and C-C cleavage: H263A exhibits very slow ketonisation and C-C cleavage, whereas S110A and S110G exhibit fast ketonisation, an intermediate phase, and slow C-C cleavage. H114A shows only twofold-reduced catalytic efficiency, ruling out a catalytic role, but shows a fivefold-reduced K(M) for the natural substrate, and an ability to process an aryl-containing substrate, implying a role for His114 in positioning of the substrate. S40A shows only twofold-reduced catalytic efficiency, but shows a very fast (500 s(-1)) interconversion of dienol (317 nm) to dienolate (394 nm) forms of the substrate, indicating that the enzyme accepts the dienol form of the substrate. These data imply that His263 is responsible for both ketonisation of the substrate and for deprotonation of water for C-C cleavage, a novel catalytic role in a serine hydrolase. Ser110 has an important but non-essential role in catalysis, which appears not to be to act as a nucleophile. A catalytic mechanism is proposed involving stabilisation of reactive intermediates and activation of a nucleophilic water molecule by Ser110.     

Probing the active site of cellodextrin phosphorylase from Clostridium stercorarium: kinetic characterization, ligand docking, and site-directed mutagenesis

Cellodextrin phosphorylase from Clostridium stercorarium has been recombinantly expressed in Escherichia coli for the first time. Kinetic characterization of the purified enzyme has revealed that aryl and alkyl β-glucosides can be efficiently glycosylated, an activity that has not yet been described for this enzyme class. To obtain a better understanding of the factors that determine the enzyme's specificity, homology modeling and ligand docking were applied. Residue W168 has been found to form a hydrophobic stacking interaction with the substrate in subsite +2, and its importance has been examined by means of site-directed mutagenesis. The mutant W168A retains about half of its catalytic activity, indicating that other residues also contribute to the binding affinity of subsite +2. Finally, residue D474 has been identified as the catalytic acid, interacting with the glycosidic oxygen between subsites -1 and +1. Mutating this residue results in complete loss of activity. These results, for the first time, provide an insight in the enzyme-substrate interactions that determine the activity and specificity of cellodextrin phosphorylases.