Structural studies of the phosphoribosyltransferase involved in cobamide biosynthesis in methanogenic archaea and cyanobacteria.

Cobamides (Cbas) are coenzymes used by cells across all domains of life, but de novo synthesis is only found in some bacteria and archaea. Five enzymes assemble the nucleotide loop in the alpha phase of the corrin ring. Condensation of the activated ring and nucleobase yields adenosyl-Cba 5'-phosphate, which upon dephosphorylation yields the biologically active coenzyme (AdoCba). Base activation is catalyzed by a phosphoribosyltransferase (PRTase). The structure of the Salmonella enterica PRTase enzyme (i.e., SeCobT) is well-characterized, but archaeal PRTases are not. To gain insights into the mechanism of base activation by the PRTase from Methanocaldococcus jannaschii (MjCobT), we solved crystal structures of the enzyme in complex with substrate and products. We determined several structures: (i) a 2.2 Å structure of MjCobT in the absence of ligand (apo), (ii) structures of MjCobT bound to nicotinate mononucleotide (NaMN) and α-ribazole 5'-phosphate (α-RP) or α-adenylyl-5'-phosphate (α-AMP) at 2.3 and 1.4 Å, respectively. In MjCobT the general base that triggers the reaction is an aspartate residue (Asp 52) rather than a glutamate residue (E317) as in SeCobT. Notably, the dimer interface in MjCobT is completely different from that observed in SeCobT. Finally, entry PDB 3L0Z does not reflect the correct structure of MjCobT.

Yeast pericentrin/Spc110 contains multiple domains required for tethering the γ-tubulin complex to the centrosome.

The Saccharomyces cerevisiae spindle pole body (SPB) serves as the sole microtubule-organizing center of the cell, nucleating both cytoplasmic and nuclear microtubules. Yeast pericentrin, Spc110, binds to and activates the γ-tubulin complex via its N terminus, allowing nuclear microtubule polymerization to occur. The Spc110 C terminus links the γ-tubulin complex to the central plaque of the SPB by binding to Spc42, Spc29, and calmodulin (Cmd1). Here, we show that overexpression of the C terminus of Spc110 is toxic to cells and correlates with its localization to the SPB. Spc110 domains that are required for SPB localization and toxicity include its Spc42-, Spc29-, and Cmd1-binding sites. Overexpression of the Spc110 C terminus induces SPB defects and disrupts microtubule organization in both cycling and G2/M arrested cells. Notably, the two mitotic SPBs are affected in an asymmetric manner such that one SPB appears to be pulled away from the nucleus toward the cortex but remains attached via a thread of nuclear envelope. This SPB also contains relatively fewer microtubules and less endogenous Spc110. Our data suggest that overexpression of the Spc110 C terminus acts as a dominant-negative mutant that titrates endogenous Spc110 from the SPB causing spindle defects.

Structural and functional analysis of an l-serine O-phosphate decarboxylase involved in norcobamide biosynthesis.

Structural diversity of natural cobamides (Cbas, B12 vitamers) is limited to the nucleotide loop. The loop is connected to the cobalt-containing corrin ring via an (R)-1-aminopropan-2-ol O-2-phosphate (AP-P) linker moiety. AP-P is produced by the l-threonine O-3-phosphate (l-Thr-P) decarboxylase CobD. Here, the CobD homolog SMUL_1544 of the organohalide-respiring epsilonproteobacterium Sulfurospirillum multivorans was characterized as a decarboxylase that produces ethanolamine O-phosphate (EA-P) from l-serine O-phosphate (l-Ser-P). EA-P is assumed to serve as precursor of the linker moiety of norcobamides that function as cofactors in the respiratory reductive dehalogenase. SMUL_1544 (SmCobD) is a pyridoxal-5'-phosphate (PLP)-containing enzyme. The structural analysis of the SmCobD apoprotein combined with the characterization of truncated mutant proteins uncovered a role of the SmCobD N-terminus in efficient l-Ser-P conversion.

Structure and function of Spc42 coiled-coils in yeast centrosome assembly and duplication.

Centrosomes and spindle pole bodies (SPBs) are membraneless organelles whose duplication and assembly is necessary for bipolar mitotic spindle formation. The structural organization and functional roles of major proteins in these organelles can provide critical insights into cell division control. Spc42, a phosphoregulated protein with an N-terminal dimeric coiled-coil (DCC), assembles into a hexameric array at the budding yeast SPB core, where it functions as a scaffold for SPB assembly. Here, we present in vitro and in vivo data to elucidate the structural arrangement and biological roles of Spc42 elements. Crystal structures reveal details of two additional coiled-coils in Spc42: a central trimeric coiled-coil and a C-terminal antiparallel DCC. Contributions of the three Spc42 coiled-coils and adjacent undetermined regions to the formation of an ∼145 Šhexameric lattice in an in vitro lipid monolayer assay and to SPB duplication and assembly in vivo reveal structural and functional redundancy in Spc42 assembly. We propose an updated model that incorporates the inherent symmetry of these Spc42 elements into a lattice, and thereby establishes the observed sixfold symmetry. The implications of this model for the organization of the central SPB core layer are discussed.

Kinesin-2 motors: Kinetics and biophysics.

Kinesin-2s are major transporters of cellular cargoes. This subfamily contains both homodimeric kinesins whose catalytic domains result from the same gene product and heterodimeric kinesins with motor domains derived from two different gene products. In this Minireview, we focus on the progress to define the biochemical and biophysical properties of the kinesin-2 family members. Our understanding of their mechanochemical capabilities has been advanced by the ability to identify the kinesin-2 genes in multiple species, expression and purification of these motors for single-molecule and ensemble assays, and development of new technologies enabling quantitative measurements of kinesin activity with greater sensitivity.

Determinants and Expansion of Specificity in a Trichothecene UDP-Glucosyltransferase from Oryza sativa.

Family 1 UDP-glycosyltransferases (UGTs) in plants primarily form glucose conjugates of small molecules and, besides other functions, play a role in detoxification of xenobiotics. Indeed, overexpression of a barley UGT in wheat has been shown to control Fusarium head blight, which is a plant disease of global significance that leads to reduced crop yields and contamination with trichothecene mycotoxins such as deoxynivalenol (DON), T-2 toxin, and many other structural variants. The UGT Os79 from rice has emerged as a promising candidate for inactivation of mycotoxins because of its ability to glycosylate DON, nivalenol, and hydrolyzed T-2 toxin (HT-2). However, Os79 is unable to modify T-2 toxin (T-2), produced by pathogens such as Fusarium sporotrichioides and Fusarium langsethii. Activity toward T-2 is desirable because it would allow a single UGT to inactivate co-occurring mycotoxins. Here, the structure of Os79 in complex with the products UDP and deoxynivalenol 3-O-glucoside is reported together with a kinetic analysis of a broad range of trichothecene mycotoxins. Residues associated with the trichothecene binding pocket were examined by site-directed mutagenesis that revealed that trichothecenes substituted at the C4 position, which are not glycosylated by wild-type Os79, can be accommodated in the binding pocket by increasing its volume. The H122A/L123A/Q202L triple mutation, which increases the volume of the active site and attenuates polar contacts, led to strong and equivalent activity toward trichothecenes with C4 acetyl groups. This mutant enzyme provides the broad specificity required to control multiple toxins produced by different Fusarium species and chemotypes.

The PrpF protein of Shewanella oneidensis MR-1 catalyzes the isomerization of 2-methyl-cis-aconitate during the catabolism of propionate via the AcnD-dependent 2-methylcitric acid cycle.

The 2-methylcitric acid cycle (2-MCC) is a common route of propionate catabolism in microorganisms. In Salmonella enterica, the prpBCDE operon encodes most of the 2-MCC enzymes. In other organisms, e.g., Shewanella oneidensis MR-1, two genes, acnD and prpF replace prpD, which encodes 2-methylcitrate dehydratase. We showed that together, S. oneidensis AcnD and PrpF (SoAcnD, SoPrpF) compensated for the absence of PrpD in a S. enterica prpD strain. We also showed that SoAcnD had 2-methylcitrate dehydratase activity and that PrpF has aconitate isomerase activity. Here we report in vitro evidence that the product of the SoAcnD reaction is an isomer of 2-methyl-cis-aconitate (2-MCA], the product of the SePrpD reaction. We show that the SoPrpF protein isomerizes the product of the AcnD reaction into the PrpD product (2-MCA], a known substrate of the housekeeping aconitase (AcnB]. Given that SoPrpF is an isomerase, that SoAcnD is a dehydratase, and the results from in vivo and in vitro experiments reported here, it is likely that 4-methylaconitate is the product of the AcnD enzyme. Results from in vivo studies using a S. enterica prpD strain show that SoPrpF variants with substitutions of residues K73 or C107 failed to support growth with propionate as the sole source of carbon and energy. High-resolution (1.22 Å) three-dimensional crystal structures of PrpFK73E in complex with trans-aconitate or malonate provide insights into the mechanism of catalysis of the wild-type protein.

Homodimeric Kinesin-2 KIF3CC Promotes Microtubule Dynamics.

KIF3C is one subunit of the functional microtubule-based kinesin-2 KIF3AC motor, an anterograde cargo transporter in neurons. However, KIF3C has also been implicated as an injury-specific kinesin that is a key regulator of axonal growth and regeneration by promoting microtubule dynamics for reorganization at the neuronal growth cone. To test its potential role as a modulator of microtubule dynamics in vitro, an engineered homodimeric KIF3CC was incorporated into a dynamic microtubule assay and examined by total internal reflection fluorescence microscopy. The results reveal that KIF3CC is targeted to the microtubule plus-end, acts as a potent catastrophe factor through an increase in microtubule catastrophe frequency, and does so by elimination of the dependence of the catastrophe rate on microtubule lifetime. Moreover, KIF3CC accelerates the catastrophe rate without altering the microtubule growth rate. Therefore, the ATP-promoted KIF3CC mechanism of catastrophe is different from the well-described catastrophe factors kinesin-13 MCAK and kinesin-8 Kip3/KIF18A. The properties of KIF3CC were not shared by heterodimeric KIF3AC and required the unique KIF3C-specific sequence extension in loop L11 at the microtubule interface. At the microtubule plus-end, the presence of KIF3CC resulted in modulation of the tapered structure typically seen in growing dynamic microtubules to microtubule blunt plus-ends. Overall our results implicate homodimeric KIF3CC as a unique promoter of microtubule catastrophe and substantiate its physiological role in cytoskeletal remodeling.

The molecular architecture of the yeast spindle pole body core determined by Bayesian integrative modeling.

Microtubule-organizing centers (MTOCs) form, anchor, and stabilize the polarized network of microtubules in a cell. The central MTOC is the centrosome that duplicates during the cell cycle and assembles a bipolar spindle during mitosis to capture and segregate sister chromatids. Yet, despite their importance in cell biology, the physical structure of MTOCs is poorly understood. Here we determine the molecular architecture of the core of the yeast spindle pole body (SPB) by Bayesian integrative structure modeling based on in vivo fluorescence resonance energy transfer (FRET), small-angle x-ray scattering (SAXS), x-ray crystallography, electron microscopy, and two-hybrid analysis. The model is validated by several methods that include a genetic analysis of the conserved PACT domain that recruits Spc110, a protein related to pericentrin, to the SPB. The model suggests that calmodulin can act as a protein cross-linker and Spc29 is an extended, flexible protein. The model led to the identification of a single, essential heptad in the coiled-coil of Spc110 and a minimal PACT domain. It also led to a proposed pathway for the integration of Spc110 into the SPB.

Design considerations in coiled-coil fusion constructs for the structural determination of a problematic region of the human cardiac myosin rod.

X-ray structural determination of segments of the myosin rod has proved difficult because of the strong salt-dependent aggregation properties and repeating pattern of charges on the surface of the coiled-coil that lead to the formation of paracrystals. This problem has been resolved in part through the use of globular assembly domains that improve protein folding and prevent aggregation. The primary consideration now in designing coiled-coil fusion constructs for myosin is deciding where to truncate the coiled-coil and which amino acid residues to include from the folding domain. This is especially important for myosin that contains numerous regions of low predicted coiled-coil propensity. Here we describe the strategy adopted to determine the structure of the region that extends from Arg1677 - Leu1797 that included two areas that do not show a strong sequence signature of a conventional left-handed coiled coil or canonical heptad repeat. This demonstrates again that, with careful choice of fusion constructs, overlapping structures exhibit very similar conformations for the myosin rod fragments in the canonical regions. However, conformational variability is seen around Leu1706 which is a hot spot for cardiomyopathy mutations suggesting that this might be important for function.

Functional characterization of a soluble NADPH-cytochrome P450 reductase from Fusarium graminearum.

Fusarium head blight is a devastating disease in wheat caused by some fungal pathogens of the Fusarium genus mainly F. graminearum, due to accumulation of toxic trichothecenes. Most of the trichothecene biosynthetic pathway has been mapped, although some proteins of the pathway remain uncharacterized, including an NADPH-cytochrome P450 reductase. We subcloned a F. graminearum cytochrome P450 reductase that might be involved in the trichothecene biosynthesis. It was expressed heterologously in E. coli as N-terminal truncated form with an octahistidine tag for purification. The construct yielded a soluble apoprotein and its incubation with flavins yielded the corresponding monomeric holoprotein. It was characterized for activity in the pH range 5.5-9.5, using thiazolyl blue tetrazolium bromide (MTT) or cytochrome c as substrates. Binding of the small molecule MTT was weaker than for cytochrome c, however, the rate of MTT reduction was faster. Contrary to other studies of cytochrome reductase proteins, MTT reduction proceeded in a cooperative manner in our studies. Optimum kinetic activity was found at pH 7.5-8.5 for bothMTT and cytochrome c. This is the first paper presenting characterization of a cytochrome P450 reductase from F. graminearum which most likely is involved in mycotoxin biosynthesis or some primary metabolic pathway such as sterol biosynthesis in F. graminearum.

Reversible swelling of SBMV is associated with reversible disordering.

The structures of the compact and swollen southern bean mosaic virus (SBMV) particles have been compared by X-ray diffraction and proton magnetic resonance (PMR). Small-angle X-ray scattering showed that removal of divalent cations at alkaline pH causes the particle diameter to increase from 289Å in the native SBMV by 12% in solution and by 9% in microcrystals. The swelling is fully reversible upon re-addition of Ca2+ and Mg2+ ions, as shown by the X-ray patterns at 6Å resolution and by the 270MHz PMR spectra. Beyond 30Å resolution, X-ray patterns from the compact SBMV in solution and in microcrystals show fine fringes of ∼1/225Å-1 width extending to 6Å resolution, whereas patterns from the swollen SBMV in solution and in microcrystals show only broader fringes of ∼1/90Å-1 width, Model calculations demonstrate that the fine fringes from compact SBMV arise from regular packing of the protein subunits on the icosahedral surface lattice; the smearing of fine fringes in the swollen virus pattern can be simulated by uncorrelated displacements of pentamers and hexamers of protein subunits, with a standard deviation of 6Å from their mean locations. The PMR spectrum of compact SBMV is poorly resolved, whereas PMR spectrum of swollen SBMV shows sharp resonances in the methyl proton region. The line-narrowing for a fraction of the aliphatic protons upon swelling cannot be accounted for by rotational relaxation of the particle of 6×106MW, but must be attributed to internal motion in small regions of the protein subunits.

Crystal Structure of Os79 (Os04g0206600) from Oryza sativa: A UDP-glucosyltransferase Involved in the Detoxification of Deoxynivalenol.

Fusarium head blight is a plant disease with significant agricultural and health impact which affects cereal crops such as wheat, barley, and maize and is characterized by reduced grain yield and the accumulation of trichothecene mycotoxins such as deoxynivalenol (DON). Studies have identified trichothecene production as a virulence factor in Fusarium graminearum and have linked DON resistance to the ability to form DON-3-O-glucoside in wheat. Here, the structures of a deoxynivalenol:UDP-glucosyltransferase (Os79) from Oryza sativa are reported in complex with UDP in an open conformation, in complex with UDP in a closed conformation, and in complex with UDP-2-fluoro-2-deoxy-d-glucose and trichothecene at 1.8, 2.3, and 2.2 Å resolution, respectively. The active site of Os79 lies in a groove between the N-terminal acceptor and the C-terminal donor-binding domains. Structural alignments reveal that Os79 likely utilizes a catalytic mechanism similar to those of other plant UGTs, with His 27 activating the trichothecene O3 hydroxyl for nucleophilic attack at C1' of the UDP-glucose donor. Kinetic analysis of mutant Os79 revealed that Thr 291 plays a critical role in catalysis as a catalytic acid or to position the UDP moiety during the nucleophilic attack. Steady-state kinetic analysis demonstrated that Os79 conjugates multiple trichothecene substrates such as DON, nivalenol, isotrichodermol, and HT-2 toxin, but not T-2 toxin. These data establish a foundation for understanding substrate specificity and activity in this enzyme and can be used to guide future efforts to increase DON resistance in cereal crops.

Heterodimerization of Kinesin-2 KIF3AB Modulates Entry into the Processive Run.

Mammalian KIF3AB is an N-terminal processive kinesin-2 that is best known for its roles in intracellular transport. There has been significant interest in KIF3AB to define the key principles that underlie its processivity but also to define the mechanistic basis of its sensitivity to force. In this study, the kinetics for entry into the processive run were quantified. The results show for KIF3AB that the kinetics of microtubule association at 7 µm-1 s-1 is less than the rates observed for KIF3AA at 13 µm-1 s-1 or KIF3BB at 11.9 µm-1 s-1 ADP release after microtubule association for KIF3AB is 33 s-1 and is significantly slower than ADP release from homodimeric KIF3AA and KIF3BB, which reach 80-90 s-1 To explore the interhead communication implied by the rate differences at these first steps, we compared the kinetics of KIF3AB microtubule association followed by ADP release with the kinetics for mixtures of KIF3AA plus KIF3BB. Surprisingly, the kinetics of KIF3AB are not equivalent to any of the mixtures of KIF3AA + KIF3BB. In fact, the transients for each of the mixtures overlay the transients for KIF3AA and KIF3BB. These results reveal that intermolecular communication within the KIF3AB heterodimer modulates entry into the processive run, and the results suggest that it is the high rate of microtubule association that drives rebinding to the microtubule after force-dependent motor detachment.

Family-specific Kinesin Structures Reveal Neck-linker Length Based on Initiation of the Coiled-coil.

Kinesin-1, -2, -5, and -7 generate processive hand-over-hand 8-nm steps to transport intracellular cargoes toward the microtubule plus end. This processive motility requires gating mechanisms to coordinate the mechanochemical cycles of the two motor heads to sustain the processive run. A key structural element believed to regulate the degree of processivity is the neck-linker, a short peptide of 12-18 residues, which connects the motor domain to its coiled-coil stalk. Although a shorter neck-linker has been correlated with longer run lengths, the structural data to support this hypothesis have been lacking. To test this hypothesis, seven kinesin structures were determined by x-ray crystallography. Each included the neck-linker motif, followed by helix α7 that constitutes the start of the coiled-coil stalk. In the majority of the structures, the neck-linker length differed from predictions because helix α7, which initiates the coiled-coil, started earlier in the sequence than predicted. A further examination of structures in the Protein Data Bank reveals that there is a great disparity between the predicted and observed starting residues. This suggests that an accurate prediction of the start of a coiled-coil is currently difficult to achieve. These results are significant because they now exclude simple comparisons between members of the kinesin superfamily and add a further layer of complexity when interpreting the results of mutagenesis or protein fusion. They also re-emphasize the need to consider factors beyond the kinesin neck-linker motif when attempting to understand how inter-head communication is tuned to achieve the degree of processivity required for cellular function.

Fast or Slow, Either Head Can Start the Processive Run of Kinesin-2 KIF3AC.

Mammalian KIF3AC contains two distinct motor polypeptides and is best known for its role in organelle transport in neurons. Our recent studies showed that KIF3AC is as processive as conventional kinesin-1, suggesting that their ATPase mechanochemistry may be similar. However, the presence of two different motor polypeptides in KIF3AC implies that there must be a cellular advantage for the KIF3AC heterodimer. The hypothesis tested was whether there is an intrinsic bias within KIF3AC such that either KIF3A or KIF3C initiates the processive run. To pursue these experiments, a mechanistic approach was used to compare the pre-steady-state kinetics of KIF3AC to the kinetics of homodimeric KIF3AA and KIF3CC. The results indicate that microtubule collision at 11.4 µM(-1) s(-1) coupled with ADP release at 78 s(-1) are fast steps for homodimeric KIF3AA. In contrast, KIF3CC exhibits much slower microtubule association at 2.1 µM(-1) s(-1) and ADP release at 8 s(-1). For KIF3AC, microtubule association at 6.6 µM(-1) s(-1) and ADP release at 51 s(-1) are intermediate between the constants for KIF3AA and KIF3CC. These results indicate that either KIF3A or KIF3C can initiate the processive run. Surprisingly, the kinetics of the initial event of microtubule collision followed by ADP release for KIF3AC is not equivalent to 1:1 mixtures of KIF3AA plus KIF3CC homodimers at the same motor concentration. These results reveal that the intermolecular communication within the KIF3AC heterodimer modulates entry into the processive run regardless of whether the run is initiated by the KIF3A or KIF3C motor domain.

A composite approach towards a complete model of the myosin rod.

Sarcomeric myosins have the remarkable ability to form regular bipolar thick filaments that, together with actin thin filaments, constitute the fundamental contractile unit of skeletal and cardiac muscle. This has been established for over 50 years and yet a molecular model for the thick filament has not been attained. In part this is due to the lack of a detailed molecular model for the coiled-coil that constitutes the myosin rod. The ability to self-assemble resides in the C-terminal section of myosin known as light meromyosin (LMM) which exhibits strong salt-dependent aggregation that has inhibited structural studies. Here we evaluate the feasibility of generating a complete model for the myosin rod by combining overlapping structures of five sections of coiled-coil covering 164 amino acid residues which constitute 20% of LMM. Each section contains ∼ 7-9 heptads of myosin. The problem of aggregation was overcome by incorporating the globular folding domains, Gp7 and Xrcc4 which enhance crystallization. The effect of these domains on the stability and conformation of the myosin rod was examined through biophysical studies and overlapping structures. In addition, a computational approach was developed to combine the sections into a contiguous model. The structures were aligned, trimmed to form a contiguous model, and simulated for >700 ns to remove the discontinuities and achieve an equilibrated conformation that represents the native state. This experimental and computational strategy lays the foundation for building a model for the entire myosin rod.

Kinesin-2 KIF3AC and KIF3AB Can Drive Long-Range Transport along Microtubules.

Mammalian KIF3AC is classified as a heterotrimeric kinesin-2 that is best known for organelle transport in neurons, yet in vitro studies to characterize its single molecule behavior are lacking. The results presented show that a KIF3AC motor that includes the native helix α7 sequence for coiled-coil formation is highly processive with run lengths of ∼1.23 µm and matching those exhibited by conventional kinesin-1. This result was unexpected because KIF3AC exhibits the canonical kinesin-2 neck-linker sequence that has been reported to be responsible for shorter run lengths observed for another heterotrimeric kinesin-2, KIF3AB. However, KIF3AB with its native neck linker and helix α7 is also highly processive with run lengths of ∼1.62 µm and exceeding those of KIF3AC and kinesin-1. Loop L11, a component of the microtubule-motor interface and implicated in activating ADP release upon microtubule collision, is significantly extended in KIF3C as compared with other kinesins. A KIF3AC encoding a truncation in KIF3C loop L11 (KIF3ACΔL11) exhibited longer run lengths at ∼1.55 µm than wild-type KIF3AC and were more similar to KIF3AB run lengths, suggesting that L11 also contributes to tuning motor processivity. The steady-state ATPase results show that shortening L11 does not alter kcat, consistent with the observation that single molecule velocities are not affected by this truncation. However, shortening loop L11 of KIF3C significantly increases the microtubule affinity of KIF3ACΔL11, revealing another structural and mechanistic property that can modulate processivity. The results presented provide new, to our knowledge, insights to understand structure-function relationships governing processivity and a better understanding of the potential of KIF3AC for long-distance transport in neurons.

Biochemical Characterization of a Recombinant UDP-glucosyltransferase from Rice and Enzymatic Production of Deoxynivalenol-3-O-ß-D-glucoside.

Glycosylation is an important plant defense mechanism and conjugates of Fusarium mycotoxins often co-occur with their parent compounds in cereal-based food and feed. In case of deoxynivalenol (DON), deoxynivalenol-3-O-ß-D-glucoside (D3G) is the most important masked mycotoxin. The toxicological significance of D3G is not yet fully understood so that it is crucial to obtain this compound in pure and sufficient quantities for toxicological risk assessment and for use as an analytical standard. The aim of this study was the biochemical characterization of a DON-inactivating UDP-glucosyltransferase from rice (OsUGT79) and to investigate its suitability for preparative D3G synthesis. Apparent Michaelis constants (Km) of recombinant OsUGT79 were 0.23 mM DON and 2.2 mM UDP-glucose. Substrate inhibition occurred at DON concentrations above 2 mM (Ki = 24 mM DON), and UDP strongly inhibited the enzyme. Cu2+ and Zn2+ (1 mM) inhibited the enzyme completely. Sucrose synthase AtSUS1 was employed to regenerate UDP-glucose during the glucosylation reaction. With this approach, optimal conversion rates can be obtained at limited concentrations of the costly co-factor UDP-glucose. D3G can now be synthesized in sufficient quantity and purity. Similar strategies may be of interest to produce ß-glucosides of other toxins.