Donor Specificity Research Articles

An association between ABO group and HLA antibody detection.

US blood centers can screen female plateletpheresis donors with a history of one or more pregnancies for both Class I and Class II anti-HLA antibodies using one of two platforms. One is a flow-based assay that yields a quantitative result and the other an enzyme-linked immunosorbent assay (ELISA) that yields either a positive or a negative result (above or below cutoff). The results of HLA antibody screening tests were analyzed by donor ABO group. Results from large and small American blood collection centers using both platforms were analyzed. Positivity rates were compared by chi-square test and the results stratified by parity using the Mann-Whitney test. No differences in parity were noted among donors of different ABO groups, but a significantly higher rate of HLA antibody positivity was observed among group O donors for the ELISA (31% of group O donors vs. 21% of non-group O donors, p < 0.0001). The higher rate of positivity was primarily due to Class I reactivity. This difference in antibody frequency was not observed at centers using the flow-based assay. Centers using the ELISA may have a higher rate of permanent deferral from plateletpheresis donation among group O female donors. Although the reasons for the higher rate of reactivity on Class I ELISA testing are unknown, this could result from test system characteristics or differences in group O donor antibody strength or specificity.

Identification of a residue responsible for UDP-sugar donor selectivity of a dihydroxybenzoic acid glycosyltransferase from Arabidopsis natural accessions.

UDP-glycosyltransferase (UGT) plays a major role in the diversity and reactivity of plant specialized metabolites by catalyzing the transfer of the sugar moiety from activated UDP-sugars to various acceptors. Arabidopsis UGT89A2 was previously identified from a genome-wide association study as a key factor that affects the differential accumulation of dihydroxybenzoic acid (DHBA) glycosides in distinct Arabidopsis natural accessions, including Col-0 and C24. The invitro enzyme assays indicate that these distinct metabolic phenotypes reflect the divergence of UGT89A2 enzyme properties in the Col-0 and C24 accessions. UGT89A2 from Col-0 is highly selective toward UDP-xylose as the sugar donor, and the isoform from C24 can utilize both UDP-glucose and UDP-xylose but with a higher affinity to the glucose donor. The sequences of the two isozymes only differ at six amino acid residues. Examination of these amino acid residues in more natural accessions revealed a strong correlation between the amino acid polymorphism at position 153 and the DHBA glycoside accumulation pattern. Site-directed mutagenesis that swapped residue 153 between UGT89A2 from Col-0 and C24 reversed the UDP-sugar preferences, indicating that residue 153 plays an important role in determining sugar donor specificity of UGT89A2. This study provides insight into the key amino acid changes that confer sugar donor selectivity on UGTs, and demonstrates the usefulness of natural variation in understanding the structure-function relationship of enzymes involved in specialized metabolism.

Acceptor Specificity of Amylosucrase from Deinococcus radiopugnans and Its Application for Synthesis of Rutin Derivatives.

The transglycosylation activity of amylosucrase (ASase) has received significant attention owing to its use of an inexpensive donor, sucrose, and broad acceptor specificity, including glycone and aglycone compounds. The transglycosylation reaction of recombinant ASase from Deinococcus radiopugnans (DRpAS) was investigated using various phenolic compounds, and quercetin-3-O-rutinoside (rutin) was found to be the most suitable acceptor molecule used by DRpAS. Two amino acid residues in DRpAS variants (DRpAS Q299K and DRpAS Q299R), assumed to be involved in acceptor binding, were constructed by site-directed mutagenesis. Intriguingly, DRpAS Q299K and DRpAS Q299R produced 10-fold and 4-fold higher levels of rutin transglycosylation product than did the wild-type (WT) DRpAS, respectively. According to in silico molecular docking analysis, the lysine residue at position 299 in the mutants enables rutin to more easily position inside the active pocket of the mutant enzyme than in that of the WT, due to conformational changes in loop 4.

Donor specificity of YjiC glycosyltransferase determines the conjugation of cytosolic NDP-sugar in in vivo glycosylation reactions

Escherichia coli BL21 (DE3) was engineered by blocking glucose-1-phosphate utilizing glucose phosphate isomerase (pgi), glucose-6-phosphate dehydrogenase (zwf) and uridylyltransferase (galU) genes to produce pool of four different rare dTDP-sugars. The cytosolic pool of dTDP-l-rhamnose, dTDP-d-viosamine, dTDP-4-amino 4,6-dideoxy-d-galactose, and dTDP-3-amino 3,6-dideoxy-d-galactose was generated by overexpressing respective dTDP-sugars biosynthesis genes from various microbial sources. A flexible glycosyltransferase YjiC, from Bacillus licheniformis DSM 13 was also overexpressed to transfer sugar moieties to 3-hydroxyl group of 3-hydroxyflavone, a core unit of flavonoids. Among four rare dTDP-sugars generated in cytosol of engineered strains, YjiC solely transferred l-rhamnose from dTDP-l-rhamnose and tuned to rhamnosyltransferase.

Minimalist Protein Engineering of an Aldolase Provokes Unprecedented Substrate Promiscuity

Application of aldolases for the asymmetric synthesis of multifunctional chiral products is hampered by their reputed strict nucleophile (=aldol donor) specificity owing to a mechanistic requirement for creating a carbanion nucleophile in aqueous medium. Here we report that a minimalist engineering can extensively broaden the substrate scope of native d-fructose-6-phosphate aldolase (FSA) from Escherichia coli, for which hydroxyacetone is the most proficient substrate, to accept an unprecedented wide variety of alternative nucleophiles. By single- or double-space-generating mutations using simple conservative Leu to Ala replacement of active site residues, we found enzyme variants to efficiently convert larger ketols and bioisosteric ether components with up to seven skeletal atoms, including linear and branched-chain structures. All reactions occurred with full retention of the natural d-threo diastereospecificity. These FSA variants open new avenues toward the synthesis of novel product families that hitherto were inaccessible by biological catalysis.

Human acetyl-CoA:glucosamine-6-phosphate N-acetyltransferase 1 has a relaxed donor specificity and transfers acyl groups up to four carbons in length

Glucosamine-6-phosphate N-acetyltransferase1 (GNA1) catalyses the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to glucosamine-6-phosphate (GlcN6P) to form N-acetylglucosamine-6-phosphate (GlcNAc6P), which is an essential intermediate in UDP-GlcNAc biosynthesis. An analog of GlcNAc, N-butyrylglucosamine (GlcNBu) has shown healing properties for bone and articular cartilage in animal models of arthritis. The goal of this work was to examine whether GNA1 has the ability to transfer a butyryl group from butyryl-CoA to GlcN6P to form GlcNBu6P, which can then be converted to GlcNBu. We developed fluorescent and radioactive assays and examined the donor specificity of human GNA1. Acetyl, propionyl, n-butyryl, and isobutyryl groups were all transferred to GlcN6P, but isovaleryl-CoA and decanoyl-CoA did not serve as donor substrates. Site-specific mutants were produced to examine the role of amino acids potentially affecting the size and properties of the AcCoA binding pocket. All of the wild type and mutant enzymes showed activities of both acetyl and butyryl transfer and can therefore be used for the enzymatic synthesis of GlcNBu for biomedical applications.

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.

Tuning the Phosphoryl Donor Specificity of Dihydroxyacetone Kinase from ATP to Inorganic Polyphosphate. An Insight from Computational Studies.

Dihydroxyacetone (DHA) kinase from Citrobacter freundii provides an easy entry for the preparation of DHA phosphate; a very important C3 building block in nature. To modify the phosphoryl donor specificity of this enzyme from ATP to inorganic polyphosphate (poly-P); a directed evolution program has been initiated. In the first cycle of evolution, the native enzyme was subjected to one round of error-prone PCR (EP-PCR) followed directly (without selection) by a round of DNA shuffling. Although the wild-type DHAK did not show activity with poly-P, after screening, sixteen mutant clones showed an activity with poly-phosphate as phosphoryl donor statistically significant. The most active mutant presented a single mutation (Glu526Lys) located in a flexible loop near of the active center. Interestingly, our theoretical studies, based on molecular dynamics simulations and hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) optimizations, suggest that this mutation has an effect on the binding of the poly-P favoring a more adequate position in the active center for the reaction to take place.

Evolutionary Analyses and Natural Selection of Betaine-Homocysteine S-Methyltransferase (BHMT) and BHMT2 Genes.

Betaine-homocysteine S-methyltransferase (BHMT) and BHMT2 convert homocysteine to methionine using betaine and S-methylmethionine, respectively, as methyl donor substrates. Increased levels of homocysteine in blood are associated with cardiovascular disease. Given their role in human health and nutrition, we identified BHMT and BHMT2 genes and proteins from 38 species of deuterostomes including human and non-human primates. We aligned the genes to look for signatures of selection, to infer evolutionary rates and events across lineages, and to identify the evolutionary timing of a gene duplication event that gave rise to two genes, BHMT and BHMT2. We found that BHMT was present in the genomes of the sea urchin, amphibians, reptiles, birds and mammals; BHMT2 was present only across mammals. BHMT and BHMT2 were present in tandem in the genomes of all monotreme, marsupial and placental species examined. Evolutionary rates were accelerated for BHMT2 relative to BHMT. Selective pressure varied across lineages, with the highest dN/dS ratios for BHMT and BHMT2 occurring immediately following the gene duplication event, as determined using GA Branch analysis. Nine codons were found to display signatures suggestive of positive selection; these contribute to the enzymatic or oligomerization domains, suggesting involvement in enzyme function. Gene duplication likely occurred after the divergence of mammals from other vertebrates but prior to the divergence of extant mammalian subclasses, followed by two deletions in BHMT2 that affect oligomerization and methyl donor specificity. The faster evolutionary rate of BHMT2 overall suggests that selective constraints were reduced relative to BHMT. The dN/dS ratios in both BHMT and BHMT2 was highest following the gene duplication, suggesting that purifying selection played a lesser role as the two paralogs diverged in function.

Expanding the threonine aldolase toolbox for the asymmetric synthesis of tertiary α-amino acids.

The direct biochemical synthesis of tertiary α-amino acids with a wide range of diversity was recently reported using natural threonine aldolases LTA from Aeromonas jandei and DTA from Pseudomonas sp. Here, we describe the identification of five novel threonine aldolases which accept alanine and serine as amino acid donors. The enzymes were found by sequence database analysis using known aldolases as template. All enzymes were overexpressed in Escherichia coli and purified, and their biochemical properties were characterized. The new enantiocomplementary L- and D-threonine aldolases catalyze the asymmetric synthesis of β-hydroxy α-methyl- and α-hydroxymethyl-α-amino acids with good conversion and perfect enantioselectivity at α-carbon of the products (e.e. >99 %). The structural basis for the broad donor specificity of these threonine aldolases is analyzed based on crystal structure alignments and amino acid sequences comparison.

Recent Developments in Enzymatic Synthesis of Modified Sialic Acid Derivatives

AbstractSialic acid‐containing glycans play important roles in biology, but their synthesis by standard chemical approaches is challenging. Enzymatic assembly is generally a better approach. In the last two decades, a number of bacterial sialyltransferases (STs) and trans‐sialidases have been identified and a number of them found to exhibit broad substrate specificity. In this review, we will focus on the donor and acceptor substrate specificities of these enzymes along with the enzymatic routes employed to prepare sialosides bearing modifications at specific positions on the sialic acid carbon skeleton. Understanding the substrate tolerance of these enzymes will help researchers to choose the best biocatalyst for the assembly of specific sialosides, which can be valuable tools in the study of or inhibition of sialidases and STs and sialic acid‐binding proteins. In addition, the ability to study these processes using synthetic sialylated glycans will lead to a greater understanding of their biology and will lead to new therapeutic targets.magnified image

Endogenous graft-specific Tregs expand and differentiate into effector Tregs during the induction of tolerance to allografts (TRAN1P.949)

Abstract Peripheral transplantation tolerance is donor-specific and requires the presence of Tregs. We hypothesized that allospecific Tregs are critical for the donor specificity of tolerance. To date, studies of Foxp3+ regulatory T cells (Tregs) in transplantation tolerance have examined endogenous Tregs without defining their antigen specificity, or used seeded populations of TCR-transgenic T cells that only allow the study of induced Tregs. To study endogenous alloreactive Tregs in tolerance, we used a novel transgenic donor mouse that expresses the model antigen 2W and 2W:I-Ab tetramers to track 2W-specific (2W+) Tregs and Tconv cells. We used hearts from F1 (BALB/cxC57Bl/6.Act-2W) mice and C57Bl/6 recipients. Tolerance was induced with anti-CD154 (D0,7&amp;14) + donor splenocytes (D0) or with CTLA-4Ig (D0&amp;2). In both models, 2W+ Tconv increased 3-fold whereas the 2W+ Tregs increased 9-fold over naïve mice. 2W+ Tregs differentiated from a naïve, CD44loCD62Lhi to an effector CD44hiCD62Llo phenotype but 2W- Tregs largely did not. Without treatment, 2W+ Tconv cells increased 17-fold at D30 post-transplant whereas the 2W+ Tregs increased 6-fold. These observations unexpectedly show that graft-specific Tregs expand in tolerance and rejection, and that Tconv cells are uniquely constrained during tolerance induction. The ability to track the fate of endogenous donor-reactive Tregs is a powerful approach to gain insight into their fate in tolerance induction and maintenance.

Volatile Glycosylation in Tea Plants: Sequential Glycosylations for the Biosynthesis of Aroma β-Primeverosides Are Catalyzed by Two Camellia sinensis Glycosyltransferases.

Tea plants (Camellia sinensis) store volatile organic compounds (VOCs; monoterpene, aromatic, and aliphatic alcohols) in the leaves in the form of water-soluble diglycosides, primarily as β-primeverosides (6-O-β-D-xylopyranosyl-β-D-glucopyranosides). These VOCs play a critical role in plant defenses and tea aroma quality, yet little is known about their biosynthesis and physiological roles in planta. Here, we identified two UDP-glycosyltransferases (UGTs) from C. sinensis, UGT85K11 (CsGT1) and UGT94P1 (CsGT2), converting VOCs into β-primeverosides by sequential glucosylation and xylosylation, respectively. CsGT1 exhibits a broad substrate specificity toward monoterpene, aromatic, and aliphatic alcohols to produce the respective glucosides. On the other hand, CsGT2 specifically catalyzes the xylosylation of the 6'-hydroxy group of the sugar moiety of geranyl β-D-glucopyranoside, producing geranyl β-primeveroside. Homology modeling, followed by site-directed mutagenesis of CsGT2, identified a unique isoleucine-141 residue playing a crucial role in sugar donor specificity toward UDP-xylose. The transcripts of both CsGTs were mainly expressed in young leaves, along with β-primeverosidase encoding a diglycoside-specific glycosidase. In conclusion, our findings reveal the mechanism of aroma β-primeveroside biosynthesis in C. sinensis. This information can be used to preserve tea aroma better during the manufacturing process and to investigate the mechanism of plant chemical defenses.

Cofactor Dependent Cooperation Between Dimeric hSULT1B1 Subunits

The cytosolic sulfotransferases (SULTs) catalyze the transformation of drugs and steroids into sulfate esters, generally “flagging” the molecules for excretion from the cell and body. Despite the variable substrate specificities between hSULT isoforms, all isoforms share a conserved specificity for the sulfonate donor, 3′‐phosphoadenosine 5′‐phosphosulfate (PAPS). Interactions involving the cofactor's 3′‐phosphate ensure the SULTs are not vulnerable to inhibition by other ATP analogues in the cell. However, with this gain in specificity, the SULTs sacrifice catalytic efficiency due to the resulting high affinity for the reaction byproduct, 3′, 5′‐diphosphoadenosine (PAP). We hypothesize the SULTs counteract susceptibility to PAP inhibition by communicating in a cooperative manner between dimer subunits, the result of which is a half‐site reaction mechanism. Molecular dynamic simulations (MDS) of hSULT1B1 dimers indicate the binding of PAP(S) to a single dimer subunit elicits stabilizing effects on its dimeric partner. Coupled with in vitro data showing two distinct PAPS binding affinities for the hSULT1B1 dimer, these data strongly suggest cofactor binding stimulates communication between dimer subunits, altering the cofactor binding affinity of the dimer partner. Further results indicate the presence of PAP actually promotes the binding of PAPS to the SULT dimer, triggering a structural shift that favors the release of PAP from the dimer subunit. Together, the data imply SULT half‐site reactivity results from an oscillating mechanism that promotes PAPS binding and PAP release across the SULT dimerization domain. This oscillatory cycle could be crucial in maintaining SULT catalytic efficiency under physiological PAPS concentrations, which vary throughout the body.

Highlights 2014 on glycoscience; glycosyltransferases and glycobiomarkers.

The field of glycobiology has continued to grow and mature, establishing the role of glycans in a myriad of biological processes. Many new functions of glycans have emerged from studies on different eukaryotic and prokaryotic organisms. The development of new analytical tools and techniques have fuelled the generation of a lot of information on the structure of glycans, factors determining their structure and their derangement in several pathological conditions. One of the major themes of the recently held International Symposium on Carbohydrates in Bangalore, India was glycoconjugates and glycobiology, in which scientists working at the forefront of research in these areas reported their findings on different aspects of glycans in physiology and pathology. Selected contributions from these sessions are presented in this special issue of the journal. These contributions comprise short critical reviews or original research papers focusing on (a) glycosyl transferases in physiology and pathology (b) glycobiomarkers and (c) structural analysis of glycans. Critical information embedded within the oligosaccharides on the glycoconjugates as represented by the structural features of the glycans are important in specific biological processes however alterations in glycan structures occur during development of diseases such as cancer. Glycosyl transferases, which constitute a large family of enzymes displaying exquisite specificity for both glycosyl donor and the acceptor, are largely responsible for glycosyl transfer reactions that generate a diversity of glycan structures on glycoconjugates. Many substances including natural compounds are known to affect the activity of these enzymes, making them potential drug targets. At least six articles in the ensuing pages describe the biological importance and features of glycosyl transferases. These include data relating to (a) resiliency to mutagenicity of glycosyltransferases involved in bloodgroup antigen synthesis (b) role of O-linked glycans and protein –O-glycosylation in protein folding (c) microbial cell wall biosynthesis as well as formation of GPI anchors (d) characterisation of glycosyltransferases from new sources (e) glycosyl transferasebased assays to demonstrate glycan changes in cancer tissues. A number of changes can occur in glycan expression during the development of pathological conditions. Identification and validation of these glycan changes in disease conditions make them useful surrogate markers for the disease or drug response. A strategy for developing glycobiomarkers for disease has been described in one of the contributions while another paper reports the potential of 9-O-acylated sialic acids in differentiating normal haematopoietic stem cells from leukemic stem cells. Further, changes in cell surface proteoglycans in tumor cells and the potential of cell surface molecules such as receptor for advanced glycation end-products in recognising such molecules and development of glycomimetics based on these, have been discussed. P. R. Sudhakaran (*) Department of Computational Biology and Bioinformatics, University of Kerala, Karyavattom, Trivandrum, Kerala, India e-mail: prslab@gmail.com

Flexibility and mutagenic resiliency of glycosyltransferases.

The human blood group A and B antigens are synthesized by two highly homologous enzymes, glycosyltransferase A (GTA) and glycosyltransferase B (GTB), respectively. These enzymes catalyze the transfer of either GalNAc or Gal from their corresponding UDP-donors to αFuc1-2βGal-R terminating acceptors. GTA and GTB differ at only four of 354 amino acids (R176G, G235S, L266M, G268A), which alter the donor specificity from UDP-GalNAc to UDP-Gal. Blood type O individuals synthesize truncated or non-functional enzymes. The cloning, crystallization and X-ray structure elucidations for GTA and GTB have revealed key residues responsible for donor discrimination and acceptor binding. Structural studies suggest that numerous conformational changes occur during the catalytic cycle. Over 300 ABO alleles are tabulated in the blood group antigen mutation database (BGMUT) that provides a framework for structure-function studies. Natural mutations are found in all regions of GTA and GTB from the active site, flexible loops, stem region and surfaces remote from the active site. Our characterizations of natural mutants near a flexible loop (V175M), on a remote surface site (P156L), in the metal binding motif (M212V) and near the acceptor binding site (L232P) demonstrate the resiliency of GTA and GTB to mutagenesis.

Exploring the new threonine aldolases with broad donor specificity

Exploring the new threonine aldolases with broad donor specificity

A fluorescence-based assay to monitor autopalmitoylation of zDHHC proteins applicable to high-throughput screening

Palmitoylation, the posttranslational thioester-linked modification of a 16-carbon saturated fatty acid onto the cysteine residue of a protein, has garnered considerable attention due to its implication in a multitude of disease states. The signature DHHC motif (Asp-His-His-Cys) identifies a family of protein acyltransferases (PATs) that catalyze the S-palmitoylation of target proteins via a two-step mechanism. In the first step, autopalmitoylation, palmitate is transferred from palmitoyl-CoA to the PAT, creating a palmitoyl:PAT intermediate and releasing reduced CoA. The palmitoyl moiety is then transferred to a protein substrate in the second step of the reaction. We have developed an in vitro, single-well, fluorescence-based enzyme assay that monitors the first step of the PAT reaction by coupling the production of reduced CoA to the reduction of NAD+ using the α-ketoglutarate dehydrogenase complex. This assay is suitable for determining PAT kinetic parameters, elucidating lipid donor specificity and measuring PAT inhibition by 2-bromopalmitate. Finally, it can be used for high-throughput screening (HTS) campaigns for modulators of protein palmitoylation.

Structural and mutational analysis of amino acid residues involved in ATP specificity of Escherichia coli acetate kinase

Acetate kinase (AK) generally utilizes ATP as a phosphoryl donor, but AK from Entamoeba histolytica (PPi-ehiAK) uses pyrophosphate (PPi), not ATP, and is PPi-specific. The determinants of the phosphoryl donor specificity are unknown. Here, we inferred 5 candidate amino acid residues associated with this specificity, based on structural information. Each candidate residue in Escherichia coli ATP-specific AK (ATP-ecoAK), which is unable to use PPi, was substituted with the respective PPi-ehiAK amino acid residue. Each variant ATP-ecoAK had an increased Km for ATP, indicating that the 5 residues are the determinants for the specificity to ATP in ATP-ecoAK. Moreover, Asn-337 of ATP-ecoAK was shown to be particularly significant for the specificity to ATP. The 5 residues are highly conserved in 2625 PPi-ehiAK homologs, implying that almost all organisms have ATP-dependent, rather than PPi-dependent, AK.

Identification and functional characterization of a flax UDP-glycosyltransferase glucosylating secoisolariciresinol (SECO) into secoisolariciresinol monoglucoside (SMG) and diglucoside (SDG).

BackgroundLignans are a class of diphenolic nonsteroidal phytoestrogens often found glycosylated in planta. Flax seeds are a rich source of secoisolariciresinol diglucoside (SDG) lignans. Glycosylation is a process by which a glycosyl group is covalently attached to an aglycone substrate and is catalyzed by uridine diphosphate glycosyltransferases (UGTs). Until now, very little information was available on UGT genes that may play a role in flax SDG biosynthesis. Here we report on the identification, structural and functional characterization of 5 putative UGTs potentially involved in secoisolariciresinol (SECO) glucosylation in flax.ResultsFive UGT genes belonging to the glycosyltransferases’ family 1 (EC 2.4.x.y) were cloned and characterized. They fall under four UGT families corresponding to five sub-families referred to as UGT74S1, UGT74T1, UGT89B3, UGT94H1, UGT712B1 that all display the characteristic plant secondary product glycosyltransferase (PSPG) conserved motif. However, diversity was observed within this 44 amino acid sequence, especially in the two peptide sequences WAPQV and HCGWNS known to play a key role in the recognition and binding of diverse aglycone substrates and in the sugar donor specificity. In developing flax seeds, UGT74S1 and UGT94H1 showed a coordinated gene expression with that of pinoresinol-lariciresinol reductase (PLR) and their gene expression patterns correlated with SDG biosynthesis. Enzyme assays of the five heterologously expressed UGTs identified UGT74S1 as the only one using SECO as substrate, forming SECO monoglucoside (SMG) and then SDG in a sequential manner.ConclusionWe have cloned and characterized five flax UGTs and provided evidence that UGT74S1 uses SECO as substrate to form SDG in vitro. This study allowed us to propose a model for the missing step in SDG lignan biosynthesis.