Spinning sugars in antigen biosynthesis: characterization of the Coxiella burnetii and Streptomyces griseus TDP-sugar epimerases.

The sugars streptose and dihydrohydroxystreptose (DHHS) are unique to the bacteria Streptomyces griseus and Coxiella burnetii, respectively. Streptose forms the central moiety of the antibiotic streptomycin, while DHHS is found in the O-antigen of the zoonotic pathogen C. burnetii. Biosynthesis of these sugars has been proposed to follow a similar path to that of TDP-rhamnose, catalyzed by the enzymes RmlA, RmlB, RmlC, and RmlD, but the exact mechanism is unclear. Streptose and DHHS biosynthesis unusually requires a ring contraction step that could be performed by orthologs of RmlC or RmlD. Genome sequencing of S. griseus and C. burnetii has identified StrM and CBU1838 proteins as RmlC orthologs in these respective species. Here, we demonstrate that both enzymes can perform the RmlC 3'',5'' double epimerization activity necessary to support TDP-rhamnose biosynthesis in vivo. This is consistent with the ring contraction step being performed on a double epimerized substrate. We further demonstrate that proton exchange is faster at the 3''-position than the 5''-position, in contrast to a previously studied ortholog. We additionally solved the crystal structures of CBU1838 and StrM in complex with TDP and show that they form an active site highly similar to those of the previously characterized enzymes RmlC, EvaD, and ChmJ. These results support the hypothesis that streptose and DHHS are biosynthesized using the TDP pathway and that an RmlD paralog most likely performs ring contraction following double epimerization. This work will support the elucidation of the full pathways for biosynthesis of these unique sugars.

Crystallographic analysis of TarI and TarJ, a cytidylyltransferase and reductase pair for CDP-ribitol synthesis in Staphylococcus aureus wall teichoic acid biogenesis.

The cell wall of many pathogenic Gram-positive bacteria contains ribitol-phosphate wall teichoic acid (WTA), a polymer that is linked to virulence and regulation of essential physiological processes including cell division. CDP-ribitol, the activated precursor for ribitol-phosphate polymerization, is synthesized by a cytidylyltransferase and reductase pair known as TarI and TarJ, respectively. In this study, we present crystal structures of Staphylococcus aureus TarI and TarJ in their apo forms and in complex with substrates and products. The TarI structures illustrate the mechanism of CDP-ribitol synthesis from CTP and ribitol-phosphate and reveal structural changes required for substrate binding and catalysis. Insights into the upstream step of ribulose-phosphate reduction to ribitol-phosphate is provided by the structures of TarJ. Furthermore, we propose a general topology of the enzymes in a heterotetrameric form built using restraints from crosslinking mass spectrometry analysis. Together, our data present molecular details of CDP-ribitol production that may aid in the design of inhibitors against WTA biosynthesis.

Streptococcal dTDP-L-rhamnose biosynthesis enzymes: functional characterization and lead compound identification.

Biosynthesis of the nucleotide sugar precursor dTDP-L-rhamnose is critical for the viability and virulence of many human pathogenic bacteria, including Streptococcus pyogenes (Group A Streptococcus; GAS), Streptococcus mutans and Mycobacterium tuberculosis. Streptococcal pathogens require dTDP-L-rhamnose for the production of structurally similar rhamnose polysaccharides in their cell wall. Via heterologous expression in S. mutans, we confirmed that GAS RmlB and RmlC are critical for dTDP-L-rhamnose biosynthesis through their action as dTDP-glucose-4,6-dehydratase and dTDP-4-keto-6-deoxyglucose-3,5-epimerase enzymes respectively. Complementation with GAS RmlB and RmlC containing specific point mutations corroborated the conservation of previous identified catalytic residues. Bio-layer interferometry was used to identify and confirm inhibitory lead compounds that bind to GAS dTDP-rhamnose biosynthesis enzymes RmlB, RmlC and GacA. One of the identified compounds, Ri03, inhibited growth of GAS, other rhamnose-dependent streptococcal pathogens as well as M. tuberculosis with an IC50 of 120-410 µM. Importantly, we confirmed that Ri03 inhibited dTDP-L-rhamnose formation in a concentration-dependent manner through a biochemical assay with recombinant rhamnose biosynthesis enzymes. We therefore conclude that inhibitors of dTDP-L-rhamnose biosynthesis, such as Ri03, affect streptococcal and mycobacterial viability and can serve as lead compounds for the development of a new class of antibiotics that targets dTDP-rhamnose biosynthesis in pathogenic bacteria.

Multiplexed Capillary Electrophoresis as Analytical Tool for Fast Optimization of Multi-Enzyme Cascade Reactions - Synthesis of Nucleotide Sugars: Dedicated to Prof. Dr. Vladimir Kren on the occasion of his 60th birthday.

Nucleotide sugars are considered as bottleneck and expensive substrates for enzymatic glycan synthesis using Leloir-glycosyltransferases. Synthesis from cheap substrates such as monosaccharides is accomplished by multi-enzyme cascade reactions. Optimization of product yields in such enzyme modules is dependent on the interplay of multiple parameters of the individual enzymes and governed by a considerable time effort when convential analytic methods like capillary electrophoresis (CE) or HPLC are applied. We here demonstrate for the first time multiplexed CE (MP-CE) as fast analytical tool for the optimization of nucleotide sugar synthesis with multi-enzyme cascade reactions. We introduce a universal separation method for nucleotides and nucleotide sugars enabling us to analyze the composition of six different enzyme modules in a high-throughput format. Optimization of parameters (T, pH, inhibitors, kinetics, cofactors and enzyme amount) employing MP-CE analysis is demonstrated for enzyme modules for the synthesis of UDP-α-D-glucuronic acid (UDP-GlcA) and UDP-α-D-galactose (UDP-Gal). In this way we achieve high space-time-yields: 1.8 g/L⋆h for UDP-GlcA and 17 g/L⋆h for UDP-Gal. The presented MP-CE methodology has the impact to be used as general analytical tool for fast optimization of multi-enzyme cascade reactions.

ISPD produces CDP-ribitol used by FKTN and FKRP to transfer ribitol phosphate onto α-dystroglycan.

Mutations in genes required for the glycosylation of α-dystroglycan lead to muscle and brain diseases known as dystroglycanopathies. However, the precise structure and biogenesis of the assembled glycan are not completely understood. Here we report that three enzymes mutated in dystroglycanopathies can collaborate to attach ribitol phosphate onto α-dystroglycan. Specifically, we demonstrate that isoprenoid synthase domain-containing protein (ISPD) synthesizes CDP-ribitol, present in muscle, and that both recombinant fukutin (FKTN) and fukutin-related protein (FKRP) can transfer a ribitol phosphate group from CDP-ribitol to α-dystroglycan. We also show that ISPD and FKTN are essential for the incorporation of ribitol into α-dystroglycan in HEK293 cells. Glycosylation of α-dystroglycan in fibroblasts from patients with hypomorphic ISPD mutations is reduced. We observe that in some cases glycosylation can be partially restored by addition of ribitol to the culture medium, suggesting that dietary supplementation with ribitol should be evaluated as a therapy for patients with ISPD mutations.

Characterization of Early Enzymes Involved in TDP-Aminodideoxypentose Biosynthesis en Route to Indolocarbazole AT2433.

The characterization of TDP-α-D-glucose dehydrogenase (AtmS8), TDP-α-D-glucuronic acid decarboxylase (AtmS9), and TDP-4-keto-α-D-xylose 2,3-dehydratase (AtmS14), involved in Actinomadura melliaura AT2433 aminodideoxypentose biosynthesis, is reported. This study provides the first biochemical evidence that both deoxypentose and deoxyhexose biosynthetic pathways share common strategies for sugar 2,3-dehydration/reduction and implicates the sugar nucleotide base specificity of AtmS14 as a potential mechanism for sugar nucleotide commitment to secondary metabolism. In addition, a re-evaluation of the AtmS9 homologue involved in calicheamicin aminodeoxypentose biosynthesis (CalS9) reveals that CalS9 catalyzes UDP-4-keto-α-D-xylose as the predominant product, rather than UDP-α-D-xylose as previously reported. Cumulatively, this work provides additional fundamental insights regarding the biosynthesis of novel pentoses attached to complex bacterial secondary metabolites.

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP-4-dehydrorhamnose reductases (RmlD).

The sugar nucleotide dTDP-L-rhamnose is critical for the biosynthesis of the Group A Carbohydrate, the molecular signature and virulence determinant of the human pathogen Group A Streptococcus (GAS). The final step of the four-step dTDP-L-rhamnose biosynthesis pathway is catalyzed by dTDP-4-dehydrorhamnose reductases (RmlD). RmlD from the Gram-negative bacterium Salmonella is the only structurally characterized family member and requires metal-dependent homo-dimerization for enzymatic activity. Using a biochemical and structural biology approach, we demonstrate that the only RmlD homologue from GAS, previously renamed GacA, functions in a novel monomeric manner. Sequence analysis of 213 Gram-negative and Gram-positive RmlD homologues predicts that enzymes from all Gram-positive species lack a dimerization motif and function as monomers. The enzymatic function of GacA was confirmed through heterologous expression of gacA in a S. mutans rmlD knockout, which restored attenuated growth and aberrant cell division. Finally, analysis of a saturated mutant GAS library using Tn-sequencing and generation of a conditional-expression mutant identified gacA as an essential gene for GAS. In conclusion, GacA is an essential monomeric enzyme in GAS and representative of monomeric RmlD enzymes in Gram-positive bacteria and a subset of Gram-negative bacteria. These results will help future screens for novel inhibitors of dTDP-L-rhamnose biosynthesis.

Production of a novel N-monomethylated dideoxysugar.

The importance of unusual deoxysugars in biology has become increasingly apparent over the past decade. Some, for example, play key roles in the physiological activities of the natural products to which they are attached. Here we describe a study of TylM1, a dimethyltransferase from Streptomyces fradiae involved in the production of dTDP-mycaminose. From this investigation, the manner in which the enzyme binds its dimethylated product has been revealed. More significantly, by providing the enzyme with an alternative substrate, it was possible to produce a monomethylated product not observed in nature. This has important ramifications for the production of unique carbohydrates that may prove useful in drug design.

Synthesis of nucleotide sugars and α-galacto-oligosaccharides by recombinant Escherichia coli cells with trehalose substrate.

Useful nucleoside diphosphate (NDP)-sugars and α-galacto-oligosaccharides were synthesized by recombinant Escherichia coli whole cells and compared to those produced by enzyme-coupling. Production yields of NDP-glucoses (Glcs) by whole cells harboring trehalose synthase (TS) were 60% for ADP-Glc, 82% for GDP-Glc, and 27% for UDP-Glc, based on NDP used. Yield of UDP-galactose (Gal) by the whole-cell harboring a UDP-Gal 4-epimerase (pGALE) was 26% of the quantity of UDP-Glc. α-Galacto-oligosaccharides, α-Gal epitope (Galα-3Galß-4Glu) and globotriose (Galα-4Galß-4Glu), were produced by the combination of three recombinant whole cells harboring TS, pGALE, and α-galactosyltransferase, with production yields of 48% and 54%, based on UDP, respectively. Production yields of NDP-sugars and α-galacto-oligosaccharides by recombinant whole-cell reactions were approximately 1.5 times greater than those of enzyme-coupled reactions. These results suggest that a recombinant whole-cell system using cells harboring TS with trehalose as a substrate may be used as an alternative and practical method for the production of NDP-sugars and α-galacto-oligosaccharides.

Structural and functional studies on a 3'-epimerase involved in the biosynthesis of dTDP-6-deoxy-D-allose.

Unusual deoxy sugars are often attached to natural products such as antibiotics, antifungals, and chemotherapeutic agents. One such sugar is mycinose, which has been found on the antibiotics chalcomycin and tylosin. An intermediate in the biosynthesis of mycinose is dTDP-6-deoxy-D-allose. Four enzymes are required for the production of dTDP-6-deoxy-D-allose in Streptomyces bikiniensis, a soil-dwelling microbe first isolated from the Bikini and Rongelap atolls. Here we describe a combined structural and functional study of the enzyme ChmJ, which reportedly catalyzes the third step in the pathway leading to dTDP-6-deoxy-D-allose formation. Specifically, it has been proposed that ChmJ is a 3'-epimerase that converts dTDP-4-keto-6-deoxyglucose to dTDP-4-keto-6-deoxyallose. This activity, however, has never been verified in vitro. As reported here, we demonstrate using (1)H nuclear magnetic resonance that ChmJ, indeed, functions as a 3'-epimerase. In addition, we determined the structure of ChmJ complexed with dTDP-quinovose to 2.0 Å resolution. The structure of ChmJ shows that it belongs to the well-characterized "cupin" superfamily. Two active site residues, His 60 and Tyr 130, were subsequently targeted for study via site-directed mutagenesis and kinetic analyses, and the three-dimensional architecture of the H60N/Y130F mutant protein was determined to 1.6 Å resolution. Finally, the structure of the apoenzyme was determined to 2.2 Å resolution. It has been previously suggested that the position of a conserved tyrosine, Tyr 130 in the case of ChmJ, determines whether an enzyme in this superfamily functions as a mono- or diepimerase. Our results indicate that the orientation of the tyrosine residue in ChmJ is a function of the ligand occupying the active site cleft.

The Pseudomonas aeruginosa rmlBDAC operon, encoding dTDP-L-rhamnose biosynthetic enzymes, is regulated by the quorum-sensing transcriptional regulator RhlR and the alternative sigma factor σS.

Pseudomonas aeruginosa produces as biosurfactants rhamnolipids, containing one (mono-rhamnolipid) or two (di-rhamnolipid) l-rhamnose molecules. The rhamnosyltransferase RhlB catalyses the synthesis of mono-rhamnolipid using as precursors dTDP-l-rhamnose and 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) produced by RhlA, while the rhamnosyltransferase RhlC synthesizes di-rhamnolipid using mono-rhamnolipid and dTDP-l-rhamnose as substrates. The Las and Rhl quorum-sensing systems coordinately regulate the production of these surfactants, as well as that of other exoproducts involved in bacterial virulence, at the transcriptional level in a cell density-dependent manner. In this work we study the transcriptional regulation of the rmlBDAC operon, encoding the enzymes involved in the production of dTDP-l-rhamnose, the substrate of both rhamnosyltransferases, RhlB and RhlC, and also a component of P. aeruginosa lipopolysaccharide. Here we show that the rmlBDAC operon possesses three promoters. One of these transcriptional start sites (P2) is responsible for most of its expression and is dependent on the stationary phase sigma factor σ(S) and on RhlR/C(4)-HSL through its binding to an atypical 'las box'.

Investigating Mithramycin deoxysugar biosynthesis: enzymatic total synthesis of TDP-D-olivose.

Mix'n'match: Enzymatic total synthesis of TDP-D-olivose was achieved, starting from TDP-4-keto-6-deoxy-D-glucose, by combining three pathway enzymes with one cofactor-regenerating enzyme. The results also revealed that MtmC is a bifunctional enzyme that can perform a 4-ketoreduction necessary for D-olivose biosynthesis besides the previously found C-methyltransfer for D-mycarose biosynthesis.

Using simple donors to drive the equilibria of glycosyltransferase-catalyzed reactions.

We report that simple glycoside donors can drastically shift the equilibria of glycosyltransferase-catalyzed reactions, transforming NDP-sugar formation from an endothermic to an exothermic process. To demonstrate the utility of this thermodynamic adaptability, we highlight the glycosyltransferase-catalyzed synthesis of 22 sugar nucleotides from simple aromatic sugar donors, as well as the corresponding in situ formation of sugar nucleotides as a driving force in the context of glycosyltransferase-catalyzed reactions for small-molecule glycodiversification. These simple aromatic donors also enabled a general colorimetric assay for glycosyltransfer, applicable to drug discovery, protein engineering and other fundamental sugar nucleotide-dependent investigations. This study directly challenges the general notion that NDP-sugars are 'high-energy' sugar donors when taken out of their traditional biological context.

Combined structural and functional investigation of a C-3''-ketoreductase involved in the biosynthesis of dTDP-L-digitoxose.

l-Digitoxose is an unusual dideoxysugar found attached to various pharmacologically active natural products, including the antitumor antibiotic tetrocarcin A and the antibiotics kijanimicin and jadomycin B. Six enzymes are required for its production starting from glucose 1-phosphate. Here we describe a combined structural and functional investigation of KijD10, an NADPH-dependent C-3''-ketoreductase that catalyzes the third step of l-digitoxose biosynthesis in the African soil-dwelling bacterium Actinomadura kijaniata. KijD10 belongs to the glucose-fructose oxidoreductase superfamily. For this investigation, both binary and ternary complexes of KijD10 were crystallized, and their structures were determined to 2.0 Å resolution or better. On the basis of these high-resolution structures, two potential active site acids were identified, Lys 102 and Tyr 186. These residues were individually mutated and the resultant proteins investigated both kinetically and structurally. The Y186F mutant protein demonstrated significant catalytic activity, and its structure was virtually identical to that of the wild-type enzyme except for the positioning of the nicotinamide ring. All lysine mutations, on the other hand, resulted in proteins with either abolished or drastically reduced catalytic activities. Structures for the K102A and K102E mutant proteins were determined and showed that the abrogation of catalytic activity was not a result of large conformational changes. Taken together, these data suggest that Lys 102 donates a proton to the C-3'' keto group during the reaction and that Tyr 186 serves only an auxiliary role. This is in contrast to that proposed for glucose-fructose oxidoreductase and other family members in which the tyrosines, or in some cases similarly positioned histidines, are thought to play major catalytic roles.

Characterization of the CDP-2-glycerol biosynthetic pathway in Streptococcus pneumoniae.

Capsule polysaccharide (CPS) plays an important role in the virulence of Streptococcus pneumoniae and is usually used as the pneumococcal vaccine target. Glycerol-2-phosphate is found in the CPS of S. pneumoniae types 15A and 23F and is rarely found in the polysaccharides of other bacteria. The biosynthetic pathway of the nucleotide-activated form of glycerol-2-phosphate (NDP-2-glycerol) has never been identified. In this study, three genes (gtp1, gtp2, and gtp3) from S. pneumoniae 23F that have been proposed to be involved in the synthesis of NDP-2-glycerol were cloned and the enzyme products were expressed, purified, and assayed for their respective activities. Capillary electrophoresis was used to detect novel products from the enzyme-substrate reactions, and the structure of the product was elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. Gtp1 was identified as a reductase that catalyzes the conversion of 1,3-dihydroxyacetone to glycerol, Gtp3 was identified as a glycerol-2-phosphotransferase that catalyzes the conversion of glycerol to glycerol-2-phosphate, and Gtp2 was identified as a cytidylyltransferase that transfers CTP to glycerol-2-phosphate to form CDP-2-glycerol as the final product. The kinetic parameters of Gtp1 and Gtp2 were characterized in depth, and the effects of temperature, pH, and cations on these two enzymes were analyzed. This is the first time that the biosynthetic pathway of CDP-2-glycerol has been identified biochemically; this pathway provides a method to enzymatically synthesize this compound.

Characterization and mechanistic studies of DesII: a radical S-adenosyl-L-methionine enzyme involved in the biosynthesis of TDP-D-desosamine.

D-desosamine (1) is a 3-(N,N-dimethylamino)-3,4,6-trideoxyhexose found in a number of macrolide antibiotics including methymycin (2), neomethymycin (3), pikromycin (4), and narbomycin (5) produced by Streptomyces venezuelae . It plays an essential role in conferring biological activities to its parent aglycones. Previous genetic and biochemical studies of the biosynthesis of desosamine in S. venezuelae showed that the conversion of TDP-4-amino-4,6-dideoxy-D-glucose (8) to TDP-3-keto-4,6-dideoxy-D-glucose (9) is catalyzed by DesII, which is a member of the radical S-adenosyl-L-methionine (SAM) enzyme superfamily. Here, we report the purification and reconstitution of His(6)-tagged DesII, characterization of its [4Fe-4S] cluster using UV-vis and EPR spectroscopies, and the capability of flavodoxin, flavodoxin reductase, and NADPH to reduce the [4Fe-4S](2+) cluster. Also included are a steady-state kinetic analysis of DesII-catalyzed reaction and an investigation of the substrate flexibility of DesII. Studies of deuterium incorporation into SAM using TDP-[3-(2)H]-4-amino-4,6-dideoxy-D-glucose as the substrate provides strong evidence for direct hydrogen atom transfer to a 5'-deoxyadenosyl radical in the catalytic cycle. The fact that hydrogen atom abstraction occurs at C-3 also sheds light on the mechanism of this intriguing deamination reaction.

Functional characterization and substrate specificity of spinosyn rhamnosyltransferase by in vitro reconstitution of spinosyn biosynthetic enzymes.

Spinosyn, a potent insecticide, is a novel tetracyclic polyketide decorated with d-forosamine and tri-O-methyl-L-rhamnose. Spinosyn rhamnosyltransferase (SpnG) is a key biocatalyst with unique sequence identity and controls the biosynthetic maturation of spinosyn. The rhamnose is critical for the spinosyn insecticidal activity and cell wall biosynthesis of the spinosyn producer, Saccharopolyspora spinosa. In this study, we have functionally expressed and characterized SpnG and the three enzymes, Gdh, Epi, and Kre, responsible for dTDP-L-rhamnose biosynthesis in S. spinosa by purified enzymes from Escherichia coli. Most notably, the substrate specificity of SpnG was thoroughly characterized by kinetic and inhibition experiments using various NDP sugar analogs made by an in situ combination of NDP-sugar-modifying enzymes. SpnG was found to exhibit striking substrate promiscuity, yielding corresponding glycosylated variants. Moreover, the critical residues presumably involved in catalytic mechanism of Gdh and SpnG were functionally evaluated by site-directed mutagenesis. The information gained from this study has provided important insight into molecular recognition and mechanism of the enzymes, especially SpnG. The results have made possible the structure-activity characterization of SpnG, as well as the use of SpnG or its engineered form to serve as a combinatorial tool to make spinosyn analogs with altered biological activities and potency.

Characterization of the dTDP-D-fucofuranose biosynthetic pathway in Escherichia coli O52.

D-fucofuranose (D-Fucf) is a component of Escherichia coli O52 O antigen. This uncommon sugar is also the sugar moiety of the anticancer drug--gilvocarcin V produced by many streptomycetes. In E. coli O52, rmlA, rmlB, fcf1 and fcf2 were proposed in a previous study by our group to encode the enzymes of the dTDP-D-Fucf (the nucleotide-activated form of D-Fucf) biosynthetic pathway. In this study, Fcf1 and Fcf2 from E. coli O52 were expressed, purified and assayed for their respective activities. Novel product peaks from enzyme-substrate reactions were detected by capillary electrophoresis and the structures of the product compounds were elucidated by electro-spray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. Fcf1 was confirmed to be a dTDP-6-deoxy-D-xylo-hex-4-ulopyranose reductase for the conversion of dTDP-6-deoxy-D-xylo-hex-4-ulopyranose to dTDP-D-fucopyranose (dTDP-D-Fucp), and Fcf2 a dTDP-D-Fucp mutase for the conversion of dTDP-D-Fucp to dTDP-D-Fucf. The K(m) of Fcf1 for dTDP-6-deoxy-D-xylo-hex-4-ulopyranose was determined to be 0.38 mM, and of Fcf2 for dTDP-D-Fucp to be 3.43 mM. The functional role of fcf1 and fcf2 in the biosynthesis of E. coli O52 O antigen were confirmed by mutation and complementation tests. This is the first time that the biosynthetic pathway of dTDP-D-Fucf has been fully characterized.

In vitro characterization of the enzymes involved in TDP-D-forosamine biosynthesis in the spinosyn pathway of Saccharopolyspora spinosa.

Forosamine (4-dimethylamino)-2,3,4,6-tetradeoxy-beta-D-threo-hexopyranose) is a highly deoxygenated sugar component of several important natural products, including the potent yet environmentally benign insecticide spinosyns. To study D-forosamine biosynthesis, the five genes (spnO, N, Q, R, and S) from the spinosyn gene cluster thought to be involved in the conversion of TDP-4-keto-6-deoxy-D-glucose to TDP-D-forosamine were cloned and heterologously expressed, and the corresponding proteins were purified and their activities examined in vitro. Previous work demonstrated that SpnQ functions as a pyridoxamine 5'-monophosphate (PMP)-dependent 3-dehydrase which, in the presence of the cellular reductase pairs ferredoxin/ferredoxin reductase or flavodoxin/flavodoxin reductase, catalyzes C-3 deoxygenation of TDP-4-keto-2,6-dideoxy-D-glucose. It was also established that SpnR functions as a transaminase which converts the SpnQ product, TDP-4-keto-2,3,6-trideoxy-D-glucose, to TDP-4-amino-2,3,4,6-tetradeoxy-D-glucose. The results presented here provide a full account of the characterization of SpnR and SpnQ and reveal that SpnO and SpnN functions as a 2,3-dehydrase and a 3-ketoreductase, respectively. These two enzymes act sequentially to catalyze C-2 deoxygenation of TDP-4-keto-6-deoxy-D-glucose to form the SpnQ substrate, TDP-4-keto-2,6-dideoxy-D-glucose. Evidence has also been obtained to show that SpnS functions as the 4-dimethyltransferase that converts the SpnR product to TDP-D-forosamine. Thus, the biochemical functions of the five enzymes involved in TDP-D-forosamine formation have now been fully elucidated. The steady-state kinetic parameters for the SpnQ-catalyzed reaction have been determined, and the substrate specificities of SpnQ and SpnR have been explored. The implications of this work for natural product glycodiversification and comparative mechanistic analysis of SpnQ and related NDP-sugar 3-dehydrases E1 and ColD are discussed.