Genome-Scale Identification of Essential Metabolic Processes for Targeting the Plasmodium Liver Stage.

Plasmodium gene functions in mosquito and liver stages remain poorly characterized due to limitations in the throughput of phenotyping at these stages. To fill this gap, we followed more than 1,300 barcoded P. berghei mutants through the life cycle. We discover 461 genes required for efficient parasite transmission to mosquitoes through the liver stage and back into the bloodstream of mice. We analyze the screen in the context of genomic, transcriptomic, and metabolomic data by building a thermodynamic model of P. berghei liver-stage metabolism, which shows a major reprogramming of parasite metabolism to achieve rapid growth in the liver. We identify seven metabolic subsystems that become essential at the liver stages compared with asexual blood stages: type II fatty acid synthesis and elongation (FAE), tricarboxylic acid, amino sugar, heme, lipoate, and shikimate metabolism. Selected predictions from the model are individually validated in single mutants to provide future targets for drug development.

Expedient assembly of Oligo-LacNAcs by a sugar nucleotide regeneration system: Finding the role of tandem LacNAc and sialic acid position towards siglec binding.

Sialosides containing (oligo-)N-acetyllactosamine (LacNAc, Galß(1,4)GlcNAc) as core structure are known to serve as ligands for Siglecs. However, the role of tandem inner epitope for Siglec interaction has never been reported. Herein, we report the effect of internal glycan (by length and type) on the binding affinity and describe a simple and efficient chemo-enzymatic sugar nucleotide regeneration protocol for the preparative-scale synthesis of oligo-LacNAcs by the sequential use of ß1,4-galactosyltransferase (ß4GalT) and ß1,3-N-acetylglucosyl transferase (ß3GlcNAcT). Further modification of these oligo-LacNAcs was performed in one-pot enzymatic synthesis to yield sialylated and/or fucosylated analogs. A glycan library of 23 different sialosides containing various LacNAc lengths or Lac core with natural/unnatural sialylation and/or fucosylation was synthesized. These glycans were used to fabricate a glycan microarray that was utilized to screen glycan binding preferences against five different Siglecs (2, 7, 9, 14 and 15).

Enzymatic synthesis of N-acetyllactosamine from lactose enabled by recombinant ß1,4-galactosyltransferases.

Utilising a fast and sensitive screening method based on imidazolium-tagged probes, we report unprecedented reversible activity of bacterial ß1,4-galactosyltransferases to catalyse the transgalactosylation from lactose to N-acetylglucosamine to form N-acetyllactosamine in the presence of UDP. The process is demonstrated by the preparative scale synthesis of pNP-ß-LacNAc from lactose using ß1,4-galactosyltransferase NmLgtB-B as the only biocatalyst.

Enhanced Production of 6-(N-Hydroxyethyl)-Amino-6-Deoxy-α-L-Sorbofuranose by Immobilized Gluconobacter oxydanson Corn Stover with a pH Control Strategy in a Bubble Column Bioreactor.

6-(N-Hydroxyethyl)-amino-6-deoxy-α-L-sorbofuranose (6NSL) is a key intermediate in the synthesis of miglitol. Biotransformation of N-2-hydroxyethyl glucamine (NHEG) to 6NSL was performed by immobilized Gluconobacter oxydans, which was prepared by cultivating the cells in a home-made bubble column bioreactor where corn stover particles were loaded. The optimal carrier addition and aeration rate for 6NSL production by immobilized cells in the bioreactor were determined to be 25 g/L and 2.5 vvm respectively. The supplementation of NH4Cl was conducive to the biotransformation of NHEG and was performed by adding aqueous ammonia and HCl, which was taken as the pH controlling agents as well. An optimal pH control strategy using the mixture of aqueous ammonia and NaOH was applied, resulting in a 9.9% increased production of 6NSL, while repeated batches of biotransformation increased from three times to four times. Finally, the 6NSL concentration and the conversion rate of NHEG to 6NSLreached 44.2 ± 1.5 g/L and 88.4 ± 2.0%, respectively, in average after four cycles of biotransformation under the optimized condition.

Acinetobacter baumannii isolate BAL_212 from Vietnam produces the K57 capsular polysaccharide containing a rarely occurring amino sugar N-acetylviosamine.

The structures of capsular polysaccharides (CPSs) produced by different Acinetobacter baumannii strains have proven to be invaluable in confirming the role of specific genes in the synthesis of rare sugars through the correlation of genetic content at the CPS biosynthesis locus with sugars found in corresponding CPS structures. A module of four genes (rmlA, rmlB, vioA and vioB) was identified in the KL57 capsule biosynthesis gene cluster of A. baumannii isolate BAL_212 from Vietnam. These genes were predicted to direct the synthesis of 4-acetamido-4,6-dideoxy-d-glucose (N-acetylviosamine, d-Qui4NAc) and the K57 CPS was found to contain this monosaccharide. The K57 structure was determined and, in addition to d-Qui4NAc, included three N-acetylgalactosamine residues in the main chain, with a single glucose side branch. The KL57 gene cluster has not been found in any other A. baumannii genomes, but the rmlA-rmlB-vioA-vioB module is present in the KL119 gene cluster that would likely produce a d-Qui4NAc-containing CPS.

Characterisation of a Bacterial Galactokinase with High Activity and Broad Substrate Tolerance for Chemoenzymatic Synthesis of 6-Aminogalactose-1-Phosphate and Analogues.

Glycosyl phosphates are important intermediates in many metabolic pathways and are substrates for diverse carbohydrate-active enzymes. Thus, there is a need to develop libraries of structurally similar analogues that can be used as selective chemical probes in glycomics. Here, we explore chemoenzymatic cascades for the fast generation of glycosyl phosphate libraries without protecting-group strategies. The key enzyme is a new bacterial galactokinase (LgGalK) cloned from Leminorella grimontii, which was produced in Escherichia coli and shown to catalyse 1-phosphorylation of galactose. LgGalK displayed a broad substrate tolerance, being able to catalyse the 1-phosphorylation of a number of galactose analogues, including 3-deoxy-3-fluorogalactose and 4-deoxy-4-fluorogalactose, which were first reported to be substrates for wild-type galactokinase. LgGalK and galactose oxidase variant M1 were combined in a one-pot, two-step system to synthesise 6-oxogalactose-1-phosphate and 6-oxo-2-fluorogalactose-1-phosphate, which were subsequently used to produce a panel of 30 substituted 6-aminogalactose-1-phosphate derivatives by chemical reductive amination in a one-pot, three-step chemoenzymatic process.

Biosynthesis of three N-acetylaminosugar-conjugated flavonoids using engineered Escherichia coli.

BACKGROUND: Nucleotide sugars serve as sugar donors for the synthesis of various glycones. The biological and chemical properties of glycones can be altered depending which sugar is attached. Bacteria synthesize unusual nucleotide sugars. A novel nucleotide sugar can be synthesized in Escherichia coli by introducing nucleotide biosynthetic genes from other microorganisms into E. coli. The engineered E. coli strains can be used as a platform for the synthesis of novel glycones. RESULTS: Four genes, Pdeg (UDP-N-acetylglucosamine C4,6-dehydratase), Preq (UDP-4-reductase), UDP-GlcNAc 6-DH (UDP-N-acetylglucosamine 6-dehydrogenase), and UXNAcS (UDP-N-acetylxylosamine synthase), were employed to synthesize UDP-quinovosamine, UDP-N-acetylglucosaminuronic acid, and UDP-N-acetylxylosamine in E. coli. We engineered an E. coli nucleotide sugar biosynthetic pathway to increase the pool of substrate for the target nucleotide sugars. Uridine diphosphate dependent glycosyltransferase (UGT) was also selected and introduced into E. coli. Using engineered E. coli, high levels of three novel flavonoid glycosides were obtained; 158.3 mg/L quercetin 3-O-(N-acetyl)quinovosamine, 172.5 mg/L luteolin 7-O-(N-acetyl)glucosaminuronic acid, and 160.8 mg/L quercetin 3-O-(N-acetyl)xylosamine. CONCLUSIONS: We reconstructed an E. coli nucleotide pathway for the synthesis of UDP-quinovosamine, UDP-N-acetylglucosaminuronic acid and UDP-N-acetylxylosamine in an E. coli galU (UDP-glucose 1-phosphate uridylyltransferase) or pgm (phosphoglucomutase) deletion mutant. Using engineered E. coli strains harboring a specific UGT, three novel flavonoids glycones were synthesized. The E. coli strains used in this study can be used for the synthesis of diverse glycones.

Biosynthetic and synthetic access to amino sugars.

Amino sugars are important constituents of a number of biomacromolecules and products of microbial secondary metabolism, including antibiotics. For most of them, the amino group is located at the positions C1, C2 or C3 of the hexose or pentose ring. In biological systems, amino sugars are formed due to the catalytic activity of specific aminotransferases or amidotransferases by introducing an amino functionality derived from L-glutamate or L-glutamine to the keto forms of sugar phosphates or sugar nucleotides. The synthetic introduction of amino functionalities in a regio- and stereoselective manner onto sugar scaffolds represents a substantial challenge. Most of the modern methods of for the preparation of 1-, 2- and 3-amino sugars are those starting from "an active ester" of carbohydrate derivatives, glycals, alcohols, carbonyl compounds and amino acids. A substantial progress in the development of region- and stereoselective methods of amino sugar synthesis has been made in the recent years, due to the application of metal-based catalysts and tethered approaches. A comprehensive review on the current state of knowledge on biosynthesis and chemical synthesis of amino sugars is presented.

Chemo-enzymatic synthesis of imidazolium-tagged sialyllactosamine probes.

Two novel α-linked sialyltrisaccharide imidazolium-type probes (ITags) based on the structures of biologically relevant 6'-sialyllactosamine and 3'-sialyllactosamine were efficiently and stereoselectively prepared using a chemo-enzymatic approach. The apparent kinetic parameters for the enzyme catalyzed transformations with α-2,3-sialyltransferase (α-2,3-ST) and α-2,6-sialyltransferase (α-2,6-ST) were measured by LC-MS using the ionic probes. This strategy demonstrates the suitability of the ITags to probe glycosyltransferase activity and their versatility in the preparation of sialylated epitopes for glycobiology research.

Differential accumulation and function of proinflammatory 6-sulfo LacNAc dendritic cells in lymph node and colon of Crohn's versus ulcerative colitis patients.

Human Slan DCs have been studied in patients with psoriasis, rheumatoid arthritis, cancer, and autoimmune diseases. In this study, we investigated the frequency, phenotype, and function of Slan DCs in blood, colon, as well as mLNs of patients with IBD. We first show that the frequency of circulating CD14(dull)Slan DCs was reduced in CD patients refractory to immunosuppressive drugs or TNF-α blockers relative to untreated CD, UC, and healthy subjects. In blood of CD patients, Slan DCs expressed CD172a, as detected by CD47 fusion protein binding, when compared with its lack of expression in control subjects. Next, we demonstrate that CD172a(+)Slan DCs that produced IL-1ß and TNF-α accumulated in mLNs and colons of CD patients. The CD172a(+)Slan DCs up-regulated their expression of CD14 in CD tissues and the proinflammatory cytokines were produced in CD14(bright)CD172a(+)Slan DCs. By contrast, no difference was noted in the frequency of Slan DCs between inflamed, noninflamed colonic mucosa of UC patients and control, non-IBD donors. Finally, the percentage of cytokine-producing Slan DCs also augmented in response to TLR2 and NOD2 in in vitro stimulation in PBMCs of CD, but not UC, patients. In conclusion, we propose that proinflammatory CD14(bright)CD172a(+)Slan DCs are a distinguishing feature between CD and UC, as these cells accumulate uniquely in mLNs and colonic mucosa of CD patients. Thus, Slan DCs may contribute to CD immunopathogenesis.

3'-Azidothymidine may potently inhibit the biosynthesis of polylactosamine chains on highly glycosylated-CD147 and reduce matrix metalloproteinase-2 expression in SGC-7901 and U251 cells.

Alterations to N­linked glycans are closely associated with cancer progression. Of particular importance in tumor growth and invasion, is the synthesis of complex N­linked oligosaccharides containing poly­N­acetyllactosamine (polylactosamine) chains, which have previously been reported to inhibit 3'­azidothymidine (AZT). Cluster of differentiation 147 (CD147) is a glycoprotein that carries ß1,6­branched polylactosamine sugars on its N­glycans. The present study aimed to explore the mechanism by which AZT may affect matrix metalloproteinase­2 (MMP2) expression and the cell cycle via regulation of the N­glycans on CD147 in SGC­7901 and U251 cell lines. Subsequent to treatment with various concentrations of AZT, the N­glycans of highly glycosylated (HG)­CD147 were observed to decrease in the two cell lines, and the expression of MMP2 was also significantly decreased. In addition, cell cycle analysis demonstrated that the percentage of the cells in the G1 phase increased in a dose­dependent manner with AZT treatment, indicating that AZT may inhibit cell proliferation in SGC­7901 cells. It was suggested that AZT may reduce the biosynthesis of polylactosamine chains on CD147 and reduce MMP2 expression to inhibit cell proliferation in SGC­7901 and U251 cells. Thus, AZT is suggested to be an antineoplastic drug, which may be effective therapeutically for certain types of cancer through acting on the N­glycans of HG­CD147.

Knockdown of ß3GnT8 reverses 5-fluorouracil resistance in human colorectal cancer cells via inhibition the biosynthesis of polylactosamine-type N-glycans.

Aberrant glycosylation is known to be associated with cancer chemoresistance. ß-1,3-N-acetyl-glucosaminyltransferase (ß3GnT)8, which synthesizes polylactosamine on ß1-6 branched N-glycans, is dramatically upregulated in colorectal cancer (CRC). 5-Fluorouracil (5-FU) resistance remains a major obstacle to the chemotherapy of CRC. However, little is known with regard to the correlation between 5­FU resistance and the expression of ß3GnT8 in CRC. In this study, a 5-FU­resistant cell line (SW620/5-FU) was generated, and 50% inhibition concentration (IC50) of 5-FU was determined by MTT assay. Flow cytometry and lectin blot analysis were performed to detect the alteration of polylactosamine structures. Quantitative RT-­PCR and western blot analysis were used to identify and evaluate candidate genes involved in the synthesis of polylactosamine in SW620/5-FU cells. We found polylactosamine chains were significantly increased in SW620/5-FU cells. Inhibition of the biosynthesis of polylactosamine by 3'-azidothymidine (AZT) was able to reduce 5-FU tolerance. Further studies showed that ß3GnT8 expression was also upregulated in 5-FU­resistant cancer cells, and knockdown of ß3GnT8 by RNA interference reversed 5-FU resistance through, at least partly, by suppressing the formation of polylactosamine. In conclusion, the alteration of ß3GnT8 in CRC cells correlates with tumor sensitivity to the chemotherapeutic drug and has significant implication for the development of new treatment strategies.

Rational design of a glycosynthase by the crystal structure of ß-galactosidase from Bacillus circulans (BgaC) and its use for the synthesis of N-acetyllactosamine type 1 glycan structures.

The crystal structure of ß-galactosidase from Bacillus circulans (BgaC) was determined at 1.8Å resolution. The overall structure of BgaC consists of three distinct domains, which are the catalytic domain with a TIM-barrel structure and two all-ß domains (ABDs). The main-chain fold and steric configurations of the acidic and aromatic residues at the active site were very similar to those of Streptococcus pneumoniae ß(1,3)-galactosidase BgaC in complex with galactose. The structure of BgaC was used for the rational design of a glycosynthase. BgaC belongs to the glycoside hydrolase family 35. The essential nucleophilic amino acid residue has been identified as glutamic acid at position 233 by site-directed mutagenesis. Construction of the active site mutant BgaC-Glu233Gly gave rise to a galactosynthase transferring the sugar moiety from α-d-galactopyranosyl fluoride (αGalF) to different ß-linked N-acetylglucosamine acceptor substrates in good yield (40-90%) with a remarkably stable product formation. Enzymatic syntheses with BgaC-Glu233Gly afforded the stereo- and regioselective synthesis of ß1-3-linked key galactosides like galacto-N-biose or lacto-N-biose.

Differential expression patterns of N-acetylglucosaminyl transferases and polylactosamines in uterine lesions.

Polylactosamine (polyLacNAc) is a fundamental structure in glycoconjugates and it is expressed in specific cells/tissues associated with the development and carcinogenesis. ß1,3-N-acetylglucosaminyl transferases (ß3GnTs) play an important role in polyLacNAc synthesis, however the roles of these glycosyltransferases and their products in cancer progression are still unclear. In this sense, this work aimed to evaluate differential expression pattern of the N-acetylglucosaminyl transferases and polylactosamines in invasive and premalignant lesions of the uterus cervix. The expression of ß3GnT2 and ß3GnT3 were evaluated in normal (n=10) and uterine cervix lesions (n= 120) malignant (squamous carcinoma - SC) and premalignant (cervical intraepithelial neoplasia - CIN - grades 1, 2 and 3) using immunohistochemistry. Besides, lectin histochemistry with Phytolacca americana lectin (PWM) and Wheat germ agglutinin (WGA) was also carried out to observe the presence of polyLacNAc chains and N-acetylglucosamine (GlcNAc), respectively. The ß3GnT3 was expressed in almost all samples (99%) and ß3GnT2 was higher expressed in disease samples mainly in CIN 3, when compared with normal (P=0.002), CIN 1 (P=0.009) and CIN 2 (P=0.03). The expression of polyLacNAc was higher is SC samples, when compared with normal (P=0.03), CIN 1 (P=0.02) and CIN 3 (P=0.004), and was observed only nuclear expression in nearly 50% of the SC samples, showing a statistically significant when compared with normal (P=0.01), CIN 1 (P=0.002), CIN 2 (P=0.007) and CIN 3 (P=0.04). Deferring from transferases and polyLacNAc chains, GlcNAc (WGA ligand) reveals a gradual staining pattern decrease with the increase of the lesion degree, being more expressed in CIN 1 lesions when compared with normal (P<0.0001), CIN 2 (P<0.0001), SC (P<0.0001) and CIN 3 (P=0.0003). Our data reveals ß3GnT2 and polyLacNAc may be involved in the progression of the pre-malignant lesions of human the uterine cervix. In addition, polyLacNAc expression only in the nucleus can be associated a poor prognostic in uterine lesions.

Medium-chain dehydrogenases with new specificity: amino mannitol dehydrogenases on the azasugar biosynthetic pathway.

Azasugar biosynthesis involves a key dehydrogenase that oxidizes 2-amino-2-deoxy-D-mannitol to the 6-oxo compound. The genes encoding homologous NAD-dependent dehydrogenases from Bacillus amyloliquefaciens FZB42, B. atrophaeus 1942, and Paenibacillus polymyxa SC2 were codon-optimized and expressed in BL21(DE3) Escherichia coli. Relative to the two Bacillus enzymes, the enzyme from P. polymyxa proved to have superior catalytic properties with a Vmax of 0.095 ± 0.002 µmol/min/mg, 59-fold higher than the B. amyloliquefaciens enzyme. The preferred substrate is 2- amino-2-deoxy-D-mannitol, though mannitol is accepted as a poor substrate at 3% of the relative rate. Simple amino alcohols were also accepted as substrates at lower rates. Sequence alignment suggested D283 was involved in the enzyme's specificity for aminopolyols. Point mutant D283N lost its amino specificity, accepting mannitol at 45% the rate observed for 2-amino-2-deoxy-D-mannitol. These results provide the first characterization of this class of zinc-dependent medium chain dehydrogenases that utilize aminopolyol substrates.

Helicobacter pylori ß1,3-N-acetylglucosaminyltransferase for versatile synthesis of type 1 and type 2 poly-LacNAcs on N-linked, O-linked and I-antigen glycans.

Poly-N-acetyllactosamine extensions on N- and O-linked glycans are increasingly recognized as biologically important structural features, but access to these structures has not been widely available. Here, we report a detailed substrate specificity and catalytic efficiency of the bacterial ß3-N-acetylglucosaminyltransferase (ß3GlcNAcT) from Helicobacter pylori that can be adapted to the synthesis of a rich diversity of glycans with poly-LacNAc extensions. This glycosyltransferase has surprisingly broad acceptor specificity toward type-1, -2, -3 and -4 galactoside motifs on both linear and branched glycans, found commonly on N-linked, O-linked and I-antigen glycans. This finding enables the production of complex ligands for glycan-binding studies. Although the enzyme shows preferential activity for type 2 (Galß1-4GlcNAc) acceptors, it is capable of transferring N-acetylglucosamine (GlcNAc) in ß1-3 linkage to type-1 (Galß1-3GlcNAc) or type-3/4 (Galß1-3GalNAcα/ß) sequences. Thus, by alternating the use of the H. pylori ß3GlcNAcT with galactosyltransferases that make the ß1-4 or ß1-3 linkages, various N-linked, O-linked and I-antigen acceptors could be elongated with type-2 and type-1 LacNAc repeats. Finally, one-pot incubation of di-LacNAc biantennary N-glycopeptide with the ß3GlcNAcT and GalT-1 in the presence of uridine diphosphate (UDP)-GlcNAc and UDP-Gal, yielded products with 15 additional LacNAc units on the precursor, which was seen as a series of sequential ion peaks representing alternative additions of GlcNAc and Gal residues, on matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. Overall, our data demonstrate a broader substrate specificity for the H. pylori ß3GlcNAcT than previously recognized and demonstrate its ability as a potent resource for preparative chemo-enzymatic synthesis of complex glycans.

Radical SAM enzymes in the biosynthesis of sugar-containing natural products.

Carbohydrates play a key role in the biological activity of numerous natural products. In many instances their biosynthesis requires radical mediated rearrangements, some of which are catalyzed by radical SAM enzymes. BtrN is one such enzyme responsible for the dehydrogenation of a secondary alcohol in the biosynthesis of 2-deoxystreptamine. DesII is another example that catalyzes a deamination reaction necessary for the net C4 deoxygenation of a glucose derivative en route to desosamine formation. BtrN and DesII represent the two most extensively characterized radical SAM enzymes involved in carbohydrate biosynthesis. In this review, we summarize the biosynthetic roles of these two enzymes, their mechanisms of catalysis, the questions that have arisen during these investigations and the insight they can offer for furthering our understanding of radical SAM enzymology. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.

Biosynthesis of glycosylated derivatives of tylosin in Streptomyces venezuelae.

Streptomyces venezuelae YJ028, bearing a deletion of the entire biosynthetic gene cluster encoding the pikromycin polyketide synthases and desosamine biosynthetic enzymes, was used as a bioconversion system for combinatorial biosynthesis of glycosylated derivatives of tylosin. Two engineered deoxysugar biosynthetic pathways for the biosynthesis of TDP-3-O-demethyl-D-chalcose or TDP-Lrhamnose in conjunction with the glycosyltransferaseauxiliary protein pair DesVII/DesVIII were expressed in a S. venezuelae YJ028 mutant strain. Supplementation of each mutant strain capable of producing TDP-3-O-demethyl- D-chalcose or TDP-L-rhamnose with tylosin aglycone tylactone resulted in the production of the 3-O-demethyl- D-chalcose, D-quinovose, or L-rhamnose-glycosylated tylactone.

A comparative study of the regioselectivity of the ß-galactosidases from Kluyveromyces lactis and Bacillus circulans in the enzymatic synthesis of N-Acetyl-lactosamine in aqueous media.

The enzymatic synthesis of N-acetyl-lactosamine (LacNAc) was studied in aqueous media with high substrate concentrations using the transgalactosylation of N-acetyl-D-glucosamine (GlcNAc), starting from lactose as a galactosyl donor. The efficiency and regioselectivity of the ß-galactosidases from Kluyveromyces lactis (KlßGal) and Bacillus circulans (BcßGal) were compared. The reaction was optimized by varying the experimental conditions (pH, catalytic activity concentration, and mass concentration ratio of the substrates), which enhanced the synthesis yields with both enzymes and especially with BcßGal. BcßGal catalyzed the formation of the maximal LacNAc concentration obtained (101 mM or 39 g L(-1), corresponding to a yield of 11% on the basis of GlcNAc conversion), after 5 h at pH 6.5 and for a substrate mass concentration ratio of 1. This enzyme also gave an optimal synthesis yield of about 17.5%. No change in regioselectivity was observed when using KlßGal, whereas the regioselectivity of BcßGal proved to be subject to variations, the 1-4 and 1-6 linkages being favored under kinetic and thermodynamic control conditions, respectively. Finally, it was demonstrated that the N-acetyl-allolactosamine synthesized during the GlcNAc transgalactosylation catalyzed by BcßGal was a thermodynamic product and did not result from a chemical and/or enzymatic isomerization of LacNAc.

Characterization of the enzymes encoded by the anthrose biosynthetic operon of Bacillus anthracis.

Bacillus anthracis spores, the etiological agents of anthrax, possess a loosely fitting outer layer called the exosporium that is composed of a basal layer and an external hairlike nap. The filaments of the nap are formed by trimers of the collagenlike glycoprotein BclA. Multiple pentasaccharide and trisaccharide side chains are O linked to BclA. The nonreducing terminal residue of the pentasaccharide side chain is the unusual sugar anthrose. A plausible biosynthetic pathway for anthrose biosynthesis has been proposed, and an antABCD operon encoding four putative anthrose biosynthetic enzymes has been identified. In this study, we genetically and biochemically characterized the activities of these enzymes. We also used mutant B. anthracis strains to determine the effects on BclA glycosylation of individually inactivating the genes of the anthrose operon. The inactivation of antA resulted in the appearance of BclA pentasaccharides containing anthrose analogs possessing shorter side chains linked to the amino group of the sugar. The inactivation of antB resulted in BclA being replaced with only trisaccharides, suggesting that the enzyme encoded by the gene is a dTDP-ß-L-rhamnose α-1,3-L-rhamnosyl transferase that attaches the fourth residue of the pentasaccharide side chain. The inactivation of antC and antD resulted in the disappearance of BclA pentasaccharides and the appearance of a tetrasaccharide lacking anthrose. These phenotypes are entirely consistent with the proposed roles for the antABCD-encoded enzymes in anthrose biosynthesis. Purified AntA was then shown to exhibit ß-methylcrotonyl-coenzyme A (CoA) hydratase activity, as we predicted. Similarly, we confirmed that purified AntC had aminotransferase activity and that purified AntD displayed N-acyltransferase activity.