The sugar donor specificity of plant family 1 glycosyltransferases.

Plant family 1 glycosyltransferases (UGTs) represent a formidable tool to produce valuable natural and novel glycosides. Their regio- and stereo-specific one-step glycosylation mechanism along with their inherent wide acceptor scope are desirable traits in biotechnology. However, their donor scope and specificity are not well understood. Since different sugars have different properties in vivo and in vitro, the ability to easily glycodiversify target acceptors is desired, and this depends on our improved understanding of the donor binding site. In the aim to unlock the full potential of UGTs, studies have attempted to elucidate the structure-function relationship governing their donor specificity. These efforts have revealed a complex phenomenon, and general principles valid for multiple enzymes are elusive. Here, we review the studies of UGT donor specificity, and attempt to group the information into key concepts which can help shape future research. We zoom in on the family-defining PSPG motif, on two loop residues reported to interact with the C6 position of the sugar, and on the role of active site arginines in donor specificity. We continue to discuss attempts to alter and expand the donor specificity by enzyme engineering, and finally discuss future research directions.

A facile and efficient synthesis approach of salidroside esters by whole-cell biocatalysts in organic solvents.

Salidroside, the main bioactive compound isolated from the plant source of Rhodiola rosea L, possesses broad-spectrum pharmacological activities, but suffers from the low cell membranes permeability and alimentary absorption due to its high polarity. Therefore, a whole-cell catalytic strategy for the synthesis of salidroside esters was explored to improve its lipophilicity. The results showed that Aspergillus oryzae demonstrated the highest biocatalytic activity among the microbial strains tested. For the synthesis of salidroside caprylate, the optimum conditions of reaction medium, Aspergillus oryzae amount, molar ratio of vinyl caprylate to salidroside and reaction temperature were acetone, 30 mg/ml, 10°C and 40°C, respectively. Under these conditions, the initial reaction rate was 15.36 mM/h, and substrate conversion and regioselectivity all reached 99%. Moreover, the results indicated that although various 6'-monoesters derivatives of salidroside were exclusively obtained with excellent conversions (96%-99%), the reaction rate varied greatly with different chain-length acyl donors. This study details an efficient and cost-effective biocatalytic approach for the synthesis of salidroside esters by using Aspergillus oryzae as a catalyst for the first time. Considering the whole cell catalytic efficiency and operational stability, this strategy may provide a new opportunity to develop green industrial processes production for ester derivatives of salidroside and its analogues.

Structures of Fission Yeast Inositol Pyrophosphate Kinase Asp1 in Ligand-Free, Substrate-Bound, and Product-Bound States.

Expression of the fission yeast Schizosaccharomyces pombe phosphate regulon is sensitive to the intracellular level of the inositol pyrophosphate signaling molecule 1,5-IP8. IP8 dynamics are determined by Asp1, a bifunctional enzyme consisting of an N-terminal kinase domain and a C-terminal pyrophosphatase domain that catalyze IP8 synthesis and catabolism, respectively. Here, we report structures of the Asp1 kinase domain, crystallized with two protomers in the asymmetric unit, one of which was complexed with ligands (ADPNP, ADP, or ATP; Mg2+ or Mn2+; IP6, 5-IP7, or 1,5-IP8) and the other which was ligand-free. The ligand-free enzyme adopts an "open" conformation that allows ingress of substrates and egress of products. ADPNP, ADP, and ATP and associated metal ions occupy a deep phospho-donor pocket in the active site. IP6 or 5-IP7 engagement above the nucleotide favors adoption of a "closed" conformation, in which surface protein segments undergo movement and a disordered-to-ordered transition to form an inositol polyphosphate-binding site. In a structure mimetic of the kinase Michaelis complex, the anionic 5-IP7 phosphates are encaged by an ensemble of nine cationic amino acids: Lys43, Arg223, Lys224, Lys260, Arg274, Arg285, Lys290, Arg293, and Lys341. Alanine mutagenesis of amino acids that contact the adenosine nucleoside of the ATP donor underscored the contributions of Asp258 interaction with the ribose 3'-OH and of Glu248 with adenine-N6. Changing Glu248 to Gln elicited a gain of function whereby the kinase became adept at using GTP as phosphate donor. Wild-type Asp1 kinase can utilize N6-benzyl-ATP as phosphate donor. IMPORTANCE The inositol pyrophosphate signaling molecule 1,5-IP8 modulates fission yeast phosphate homeostasis via its action as an agonist of RNA 3'-processing and transcription termination. Cellular IP8 levels are determined by Asp1, a bifunctional enzyme composed of an N-terminal kinase and a C-terminal pyrophosphatase domain. Here, we present a series of crystal structures of the Asp1 kinase domain, in a ligand-free state and in complexes with nucleotides ADPNP, ADP, and ATP, divalent cations magnesium and manganese, and inositol polyphosphates IP6, 5-IP7, and 1,5-IP8. Substrate binding elicits a switch from open to closed conformations, entailing a disordered-to-ordered transition and a rearrangement or movement of two peptide segments that form a binding site for the phospho-acceptor. Our structures, along with structure-guided mutagenesis, fortify understanding of the mechanism and substrate specificity of Asp1 kinase, and they extend and complement structural and functional studies of the orthologous human kinase PPIP5K2.

Novel UDP-GalNAc Derivative Structures Provide Insight into the Donor Specificity of Human Blood Group Glycosyltransferase.

Two closely related glycosyltransferases are responsible for the final step of the biosynthesis of ABO(H) human blood group A and B antigens. The two enzymes differ by only four amino acid residues, which determine whether the enzymes transfer GalNAc from UDP-GalNAc or Gal from UDP-Gal to the H-antigen acceptor. The enzymes belong to the class of GT-A folded enzymes, grouped as GT6 in the CAZy database, and are characterized by a single domain with a metal dependent retaining reaction mechanism. However, the exact role of the four amino acid residues in the specificity of the enzymes is still unresolved. In this study, we report the first structural information of a dual specificity cis-AB blood group glycosyltransferase in complex with a synthetic UDP-GalNAc derivative. Interestingly, the GalNAc moiety adopts an unusual yet catalytically productive conformation in the binding pocket, which is different from the "tucked under" conformation previously observed for the UDP-Gal donor. In addition, we show that this UDP-GalNAc derivative in complex with the H-antigen acceptor provokes the same unusual binding pocket closure as seen for the corresponding UDP-Gal derivative. Despite this, the two derivatives show vastly different kinetic properties. Our results provide a important structural insight into the donor substrate specificity and utilization in blood group biosynthesis, which can very likely be exploited for the development of new glycosyltransferase inhibitors and probes.

Donor specificity and regioselectivity in Lipolase mediated acylations of methyl α-D-glucopyranoside by vinyl esters of phenolic acids and their analogues.

Methyl α-D-glucopyranoside as a model acceptor was acylated by several phenolic and non-phenolic vinyl esters using immobilised Lipolase. Donor specificity and regioselectivity of reaction were investigated. Conversion and rate of acylation by structurally varied donors indicates that the synthetic reactivity of Lipolase corresponds to the hydrolytic activity of feruloyl esterase type A. Lipolase exhibited remarkable regioselectivity for primary position of methyl α-D-glucopyranoside. The acylation occurred exclusively at 6-O primary position when vinyl esters of phenolic acids (hydroxybenzoates, hydroxyphenylalkanoates and hydroxycinnamates) served as acyl donors (5-77%). In addition to the major 6-O-acyl products (52-79%), 2,6-di-O-acylated derivatives were isolated from reaction mixtures (2-13%) when non-phenolic donors were used (vinyl esters of fully methoxylated derivatives of phenolic acids, along with vinyl benzoates, cinnamates or some heterocyclic analogues).