Sugar Precursors Research Articles

Variation in the Complex Carbohydrate Biosynthesis Loci of Acinetobacter baumannii Genomes

Extracellular polysaccharides are major immunogenic components of the bacterial cell envelope. However, little is known about their biosynthesis in the genus Acinetobacter, which includes A. baumannii, an important nosocomial pathogen. Whether Acinetobacter sp. produce a capsule or a lipopolysaccharide carrying an O antigen or both is not resolved. To explore these issues, genes involved in the synthesis of complex polysaccharides were located in 10 complete A. baumannii genome sequences, and the function of each of their products was predicted via comparison to enzymes with a known function. The absence of a gene encoding a WaaL ligase, required to link the carbohydrate polymer to the lipid A-core oligosaccharide (lipooligosaccharide) forming lipopolysaccharide, suggests that only a capsule is produced. Nine distinct arrangements of a large capsule biosynthesis locus, designated KL1 to KL9, were found in the genomes. Three forms of a second, smaller variable locus, likely to be required for synthesis of the outer core of the lipid A-core moiety, were designated OCL1 to OCL3 and also annotated. Each K locus includes genes for capsule export as well as genes for synthesis of activated sugar precursors, and for glycosyltransfer, glycan modification and oligosaccharide repeat-unit processing. The K loci all include the export genes at one end and genes for synthesis of common sugar precursors at the other, with a highly variable region that includes the remaining genes in between. Five different capsule loci, KL2, KL6, KL7, KL8 and KL9 were detected in multiply antibiotic resistant isolates belonging to global clone 2, and two other loci, KL1 and KL4, in global clone 1. This indicates that this region is being substituted repeatedly in multiply antibiotic resistant isolates from these clones.

Metabolic engineering of Bacillus subtilis for the efficient biosynthesis of uniform hyaluronic acid with controlled molecular weights

Bacillus subtilis was engineered into an efficient hyaluronic acid (HA) producer by introducing two inducible artificial operons carrying HA synthase gene from Pasteurella multocida and precursor genes encoding enzymes involved in synthesis of the sugar precursors. A two-stage induction strategy was established for metabolic engineering of recombinant B. subtilis to efficiently produce uniform HA with controlled molecular weights. Strain TPG223 produced larger HA molecules (yield=6.8g/L; molecular weight=4.5MDa) than strain PG6181 (yield=2.4g/L; molecular weight=13KDa), indicating that the enzymes involved in the synthesis of UDP-glucuronic acid are essential for HA biosynthesis. Strain TPG223 was able to synthesize HA molecules ranging in molecular weight from 8KDa to 5.4MDa indicating that size control is achievable in vivo through appropriate tools. The work reported here not only advanced mechanisms research of size control in vivo, but also could be an attractive alternative for commercial preparation of uniform size-defined HA.

LC‐MS‐based metabolic characterization of high monoclonal antibody‐producing Chinese hamster ovary cells

The selection of suitable mammalian cell lines with high specific productivities is a crucial aspect of large-scale recombinant protein production. This study utilizes a metabolomics approach to elucidate the key characteristics of Chinese hamster ovary (CHO) cells with high monoclonal antibody productivities (q(mAb)). Liquid chromatography-mass spectrometry (LC-MS)-based intracellular metabolite profiles of eight single cell clones with high and low q(mAb) were obtained at the mid-exponential phase during shake flask batch cultures. Orthogonal projection to latent structures discriminant analysis (OPLS-DA) subsequently revealed key differences between the high and low q(mAb) clones, as indicated by the variable importance for projection (VIP) scores. The mass peaks were further examined for their potential association with q(mAb) across all clones using Pearson's correlation analysis. Lastly, the identities of metabolites with high VIP and correlation scores were confirmed by comparison with standards through LC-MS-MS. A total of seven metabolites were identified-NADH, FAD, reduced and oxidized glutathione, and three activated sugar precursors. These metabolites are involved in key cellular pathways of citric acid cycle, oxidative phosphorylation, glutathione metabolism, and protein glycosylation. To our knowledge, this is the first study to identify metabolites that are associated closely with q(mAb). The results suggest that the high producers had elevated levels of specific metabolites to better regulate their redox status. This is likely to facilitate the generation of energy and activated sugar precursors to meet the demands of producing more glycosylated recombinant monoclonal antibodies.

Synthesis of multiple Pseudomonas aeruginosa biofilm matrix exopolysaccharides is post‐transcriptionally regulated

Exopolysaccharide is a critical biofilm matrix component, yet little is known about how the synthesis of multiple exopolysaccharides is regulated. Pseudomonas aeruginosa can produce several biofilm matrix exopolysaccharides that include alginate, Psl and Pel. Here we demonstrated that AlgC, a key enzyme that provides sugar precursors for the synthesis of alginate and lipopolysaccharides (LPS) is also required for both Psl and Pel production. We showed that forced-synthesis of Psl in alginate-producing mucoid bacteria reduced alginate production but this was not due to transcription of the alginate biosynthesis-operon. Likewise, when either alginate or Psl were overproduced, levels of B-band LPS decreased. Induction of Pel resulted in a reduction of Psl levels. Because the effects of reduced exopolysaccharide synthesis when another is overproduced didn't appear to be regulated at the transcriptional level, this suggests that the biosynthesis pathways of Psl, Pel, alginate, and LPS compete for common sugar precursors. As AlgC is the only enzyme that provides precursors for each of these exopolysaccharides, we propose that AlgC is a key checkpoint enzyme that coordinates the total amount of exopolysaccharide biosynthesis by controlling sugar precursor pool. Our data also provide a plausible strategy that P.aeruginosa utilizes to modulate its biofilm matrix exopolysaccharides.

Glycosaminoglycan polysaccharide biosynthesis and production: today and tomorrow

Glycosaminoglycans [GAGs] are essential heteropolysaccharides in vertebrate tissues that are also, in certain cases, employed as virulence factors by microbes. Hyaluronan [HA], heparin, and chondroitin sulfate [CS] are GAGs currently used in various medical applications and together are multi-billion dollar products thus targets for production by animal-free manufacture. By using bacteria as the source of GAGs, the pathogen's sword may be converted into a plowshare to help avoid potential liabilities springing from the use of animal-derived GAGs including adventitious agents (e.g., prions, pathogens), antigenicity, degradation of the environment, and depletion of endangered species. HA from microbes, which have a chemical structure identical to human HA, has already been commercialized and sold at the ton-scale. Substantial progress towards microbial heparin and CS has been made, but these vertebrate polymers are more complicated structurally than the unsulfated bacterial polysaccharide precursors thus require additional processing steps. This review provides an overview of GAG structure, medical applications, microbial biosynthesis, and the state of bacterial GAG production systems. Representatives of all glycosyltransferase enzymes that polymerize the sugar chains of the three main GAGs have been identified and serve as the core technology to harness, but the proteins involved in sugar precursor formation and chain export steps of biosynthesis are also essential to the GAG production process. In addition, this review discusses future directions and potential important issues. Overall, this area is poised to make great headway to produce safer (both increased purity and more secure supply chains) non-animal GAG-based therapeutics.

Etherification and reductive etherification of 5-(hydroxymethyl)furfural: 5-(alkoxymethyl)furfurals and 2,5-bis(alkoxymethyl)furans as potential bio-diesel candidates

A low energy intensive process for the production of diesel fuel has been delineated from both 5-(hydroxymethyl)furfural (HMF) and its sugar precursor D-(–)-fructose. Alcoholic solutions of the above produced a mixture of potential bio-diesel candidates namely, 5-(alkoxymethyl)furfural, 5-(alkoxymethyl)furfural dialkylacetal, and alkyl levulinate, in the presence of solid acid catalysts. Sulfonic acid functionalized resins, Amberlyst-15 and Dowex DR2030 showed exceptional reactivity and selectivity for these reactions. Production of another potential diesel candidate 2,5-bis(alkoxymethyl)furan has been optimized through both sequential reduction/etherification and one-pot reductive etherification processes. During the metal catalyzed hydrogenation of HMF, platinum showed an exclusive selectivity for the reduction of the carbonyl functionality of HMF. Both Pt and Pt/Sn supported on Al2O3 catalysts have been optimized for the production of 2,5-bis(alkoxymethyl)furan from HMF. The reaction mechanisms of etherification and reductive etherification have been discussed in detail on the basis of intermediates observed during these processes.

Synthesis, Improved Antisense Activity and Structural Rationale for the Divergent RNA Affinities of 3′-Fluoro Hexitol Nucleic Acid (FHNA and Ara-FHNA) Modified Oligonucleotides

The synthesis, biophysical, structural, and biological properties of both isomers of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides are reported. Synthesis of the FHNA and Ara-FHNA thymine phosphoramidites was efficiently accomplished starting from known sugar precursors. Optimal RNA affinities were observed with a 3'-fluorine atom and nucleobase in a trans-diaxial orientation. The Ara-FHNA analog with an equatorial fluorine was found to be destabilizing. However, the magnitude of destabilization was sequence-dependent. Thus, the loss of stability is sharply reduced when Ara-FHNA residues were inserted at pyrimidine-purine (Py-Pu) steps compared to placement within a stretch of pyrimidines (Py-Py). Crystal structures of A-type DNA duplexes modified with either monomer provide a rationalization for the opposing stability effects and point to a steric origin of the destabilization caused by the Ara-FHNA analog. The sequence dependent effect can be explained by the formation of an internucleotide C-F···H-C pseudo hydrogen bond between F3' of Ara-FHNA and C8-H of the nucleobase from the 3'-adjacent adenosine that is absent at Py-Py steps. In animal experiments, FHNA-modified antisense oligonucleotides formulated in saline showed a potent downregulation of gene expression in liver tissue without producing hepatotoxicity. Our data establish FHNA as a useful modification for antisense therapeutics and also confirm the stabilizing influence of F(Py)···H-C(Pu) pseudo hydrogen bonds in nucleic acid structures.

Identification of new protein complexes of Escherichia coli inorganic pyrophosphatase using pull-down assay

Inorganic pyrophosphatase (PPase) is a conserved and essential enzyme catalyzing the hydrolysis of pyrophosphate PP i. Its activity is required to promote a lot of thermodynamically unfavorable reactions including biosynthesis of activated precursors of sugars and amino acids. Several protein partners of PPase were found so far in Escherichia coli by large-scale approaches. Functional role of these interactions was not studied. In this paper we report the identification of three protein partners of E. coli PPase not found earlier. Pull-down assay on the Ni 2+-chelating column using 6His-tagged PPase as bait was used to isolate PPase complexes from stationary-phase cells. Of several isolated protein components, five were identified by MALDI–TOF mass-spectrometry: two chaperones (DnaK and GroEL) and three enzymes of carbohydrate and amino acid metabolism (FbaB, fructose-1,6-bisphosphate aldolase, class I; GadA, l-glutamate decarboxylase; and KduI, 5-keto-4-deoxyuronate isomerase). These three proteins were cloned, expressed and purified in 6His-tagged and/or tag-free forms. Their binary interactions with PPase were verified by independent approaches. Initial characterization of the complexes indicates that PPase may stabilize its protein partners against unfolding or degradation. Comparative analysis of the PPase protein partners allowed an insight into its possible involvement in the cell metabolic regulation.

Low-temperature calcination at 800 °C of alumina–zirconia nanocomposites using sugar as a gelling agent

Alumina–zirconia nanocomposite powder was synthesized at a room temperature and calcined at a lower temperature than 1000 °C of about 800 °C by sugar precursor process. In the synthesis process of the nanocomposite powder, sugar was used as a gelling agent. The synthesized powder was calcined at temperatures of 800 °C, 900 °C, 1000 °C, 1100 °C, and 1200 °C for 5 h. The calcined powder was characterized by X-ray diffraction (XRD), a SYMPATEC NIMBUS particle size analysis, and transmission electron microscopy (TEM). Alumina–zirconia nanocomposites were obtained at 800 °C, where the phases formed were γ-Al 2O 3, m-ZrO 2, and t-ZrO 2. The TEM results showed that the average grain sizes of the nanocomposite were less than 25 nm in diameter. At 800 °C, a particle size analyzer measured the largest nanocomposite particles as having a size of about 40 nm, accounting for 3.49% of the total, while the most commonly sized partical was about 10 nm for 39.87%. A further phase transformation of alumina–zirconia nanocomposites was obtained at 1100 °C, where the phases formed were α-Al 2O 3, m-ZrO 2, and t-ZrO 2; the TEM results show that the average grain sizes of the nanocomposite powder were below 50 nm in diameter.

Catalytic effects of Murchison material: prebiotic synthesis and degradation of RNA precursors.

Mineral components of the Murchison meteorite were investigated in terms of potential catalytic effects on synthetic and hydrolytic reactions related to ribonucleic acid. We found that the mineral surfaces catalyzed condensation reactions of formamide to form carboxylic acids, amino acids, nucleobases and sugar precursors. These results suggest that formamide condensation reactions in the parent bodies of carbonaceous meteorites could give rise to multiple organic compounds thought to be required for the emergence of life. Previous studies have demonstrated similar catalytic effects for mineral assemblies likely to have been present in the early Earth environment. The minerals had little or no effect in promoting hydrolysis of RNA (24mer of polyadenylic acid) at 80°C over a pH range from 4.2 to 9.3. RNA was most stable in the neutral pH range, with a half-life ~5h, but at higher and lower pH ranges the half-life decreased to ~1h. These results suggest that if RNA was somehow incorporated into a primitive form of RNA-based thermophilic life, either it must be protected from random hydrolytic events, or the rate of synthesis must exceed the rate of hydrolysis.

Properties of recombinant Strep-tagged and untagged hyperthermophilic D-arabitol dehydrogenase from Thermotoga maritima

The first hyperthermophilic D-arabitol dehydrogenase from Thermotoga maritima was heterologously purified from Escherichia coli. The protein was purified with and without a Strep-tag. The enzyme exclusively catalyzed the NAD(H)-dependent oxidoreduction of D-arabitol, D-xylitol, D-ribulose, or D-xylulose. A twofold increase of catalytic rates was observed upon addition of Mg(2+) or K(+). Interestingly, only the tag-less protein was thermostable, retaining 90% of its activity after 90 min at 85 °C. However, the tag-less form of D-arabitol dehydrogenase had similar kinetic parameters compared to the tagged enzyme, demonstrating that the Strep-tag was not deleterious to protein function but decreased protein stability. A single band at 27.6 kDa was observed on SDS-PAGE and native PAGE revealed that the protein formed a homohexamer and a homododecamer. The enzyme catalyzed oxidation of D-arabitol to D: -ribulose and therefore belongs to the class of D-arabitol 2-dehydrogenases, which are typically observed in yeast and not bacteria. The product D-ribulose is a rare ketopentose sugar that has numerous industrially applications. Given its thermostability and specificity, D-arabitol 2-dehydrogenase is a desirable biocatalyst for the production of rare sugar precursors.

Overcoming the Recalcitrance for the Conversion of Kenaf Pulp to Glucose via Microwave-Assisted Pre-Treatment Processes

This study evaluates the pre-treatment of cellulose from kenaf plant to yield sugar precursors for the production of ethanol or butanol for use as biofuel additives. In order to convert the crystalline cellulosic form to the amorphous form that can undergo enzymatic hydrolysis of the glycosidic bond to yield sugars, kenaf pulp samples were subjected to two different pre-treatment processes. In the acid pre-treatment, the pulp samples were treated with 37.5% hydrochloric acid in the presence of FeCl3 at 50 °C or 90 °C whereas in the alkaline method, the pulp samples were treated with 25% sodium hydroxide at room temperature and with 2% or 5% sodium hydroxide at 50 °C. Microwave-assisted NaOH-treatment of the cellulose was also investigated and demonstrated to be capable of producing high glucose yield without adverse environmental impact by circumventing the use of large amounts of concentrated acids i.e., 83–85% phosphoric acid employed in most digestion processes. The treated samples were digested with the cellulase enzyme from Trichoderma reesei. The amount of glucose produced was quantified using the Quantichrom™ glucose bioassay for assessing the efficiency of glucose production for each of the treatment processes. The microwave-assisted alkaline pre-treatment processes conducted at 50 °C were found to be the most effective in the conversion of the crystalline cellulose to the amorphous form based on the significantly higher yields of sugar produced by enzymatic hydrolysis compared to the untreated sample.

L‐arabinitol‐based functional polyurethanes

AbstractThree new polymerizable diols, based on mono‐, di‐, and tri‐O‐allyl‐L‐arabinitol derivatives, were prepared from L‐arabinitol as versatile materials for the preparation of tailor‐made polyurethanes with varied degrees of functionalization. Their allyl functional groups can take part in thiol‐ene reactions, to obtain greatly diverse materials. This “click” reaction with 2‐mercaptoethanol was firstly studied on the highly hindered sugar precursor 2,3,4‐tri‐O‐allyl‐1,5‐di‐O‐trityl‐L‐arabinitol, to apply it later to macromolecules. A polyurethane with multiple pendant allyl groups was synthesized by polyaddition reaction of 2,3,4‐tri‐O‐allyl‐L‐arabinitol with 1,6‐hexamethylene diisocyanate, and then functionalized by thiol‐ene reaction. The coupling reaction took place in every allyl group, as confirmed by standard techniques. The thermal stability of the novel polyurethanes was investigated by thermogravimetric analysis and differential scanning calorimetry (DSC). This strategy provides a simple and versatile platform for the design of new materials whose functionality can be easily modified. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011

Development of Grilled-type Shrimp Flavor by Maillard Reaction and Sensory Evaluation

A grilled-type shrimp flavor was developed through the Maillard reaction to reduce or mask fish odor or off-flavor in seafood. Model systems were created by using enzymatic hydrolysate of shrimp and adding precursors to increase flavor quality and stability. Amino acid precursors such as cysteine and methionine, sugar precursors such as glucose, xylose, ribose, and sucrose, and one particular compound of glucosamine were tried and their flavor qualities were tested by sensory evaluation. Also, the optimum reaction condition was investigated using the pH values of pH 5, 6, 7, and 8 with reaction times of 1 hr, 2 hr and 3 hr after the best precursors were determined. The best condition of the precursors for grilled-type shrimp flavor was the mixtures of methionine, threonine, xylose, and glucosamine. The optimum reaction condition was at pH 8.0 and 2 hr reaction time.

Development of Boiled-type Shrimp Flavor by Maillard Reaction and Sensory Evaluation

Boiled-type shrimp flavor was developed using Maillard reaction to reduce or mask fish odor or off-flavor in seafood. Model systems were created using enzymatic hydrolysate of shrimp and adding precursor compounds to increase flavor quality and stability. Amino acid precursors of cysteine and methionine and sugar precursors such as glucose, xylose, ribose and sucrose were tried and their flavor qualities were tested by sensory evaluation. After the optimal precursors were determined, the optimum reaction condition was investigated using pHs of 5, 6, 7, and 8 and reaction times of 1, 2 and 3 hours. The best precursors for boiled-type shrimp flavor were methionine and sucrose. The optimum reaction condition was pH 8.0 and a one hour reaction time.

Internally Protected Amino Sugar Equivalents from Enantiopure 1,2‐Oxazines: Synthesis of Variably Configured Carbohydrates with C‐Branched Amino Sugar Units

A stereodivergent synthesis of differently configured C2-branched 4-amino sugar derivatives was accomplished. The Lewis acid mediated rearrangement of phenylthio-substituted 1,2-oxazines delivered glycosyl donor equivalents that can directly be employed in glycosidation reactions. Treatment with methanol provided internally protected amino sugar equivalents that have been transformed into the stereoisomeric methyl glycosides 28, ent-28, 29, ent-29 and 34 in two simple reductive steps. Reaction with natural carbohydrates or bicyclic amino sugar precursors allowed the synthesis of homo-oligomeric di- and trisaccharides 44, 46 and 47 or a hybrid trisaccharide 51 with natural carbohydrates. Access to a bivalent amino sugar derivative 54 was accomplished by reaction of rearrangement product 10 with 1,5-pentanediol. Alternatively, when a protected L-serine derivative was employed as glycosyl acceptor, the glycosylated amino acid 60 was efficiently prepared in few steps. In this report we describe the synthesis of unusual amino sugar building blocks from enantiopure 1,2-oxazines that can be attached to natural carbohydrates or natural product aglycons to produce new natural product analogues with potential applications in medicinal chemistry.

Synthesis of 3-fluoro-6- S-(2- S-pyridyl) nucleosides as potential lead cytostatic agents

The 3-deoxy-3-fluoro-6- S-(2- S-pyridyl)-6-thio- β- d-glucopyranosyl nucleoside analogs 7 were prepared via two facile synthetic routes. Their precursors, 3-fluoro-6-thio-glucopyranosyl nucleosides 5a- e, were obtained by the sequence of deacetylation of 3-deoxy-3-fluoro- β- d-glucopyranosyl nucleosides 2a- e, selective tosylation of the primary OH of 3 and finally treatment with potassium thioacetate. The desired thiolpyridine protected analogs 7a- c, f, g were obtained by the sequence of deacetylation of 5a- c followed by thiopyridinylation and/or condensation of the corresponding heterocyclic bases with the newly synthesized peracetylated 6- S-(2- S-pyridyl) sugar precursor 13, which was obtained via a novel synthetic route from glycosyl donor 12. None of the compounds 6 and 7 showed antiviral activity, but the 5-fluorouracil derivative 7c and particularly the uracil derivative 7b were endowed with an interesting and selective cytostatic action against a variety of murine and human tumor cell cultures.

An investigation of intracellular glycosylation activities in CHO cells: Effects of nucleotide sugar precursor feeding

Controlling glycosylation of recombinant proteins produced by CHO cells is highly desired as it can be directed towards maintaining or increasing product quality. To further our understanding of the different factors influencing glycosylation, a glycosylation sub-array of 79 genes and a capillary electrophoresis method which simultaneously analyzes 12 nucleotides and 7 nucleotide sugars; were used to generate intracellular N-glycosylation profiles. Specifically, the effects of nucleotide sugar precursor feeding on intracellular glycosylation activities were analyzed in CHO cells producing recombinant human interferon-gamma (IFN-gamma). Galactose (+/-uridine), glucosamine (+/-uridine), and N-acetylmannosamine (ManNAc) (+/-cytidine) feeding resulted in 12%, 28%, and 32% increase in IFN-gamma sialylation as compared to the untreated control cultures. This could be directly attributed to increases in nucleotide sugar substrates, UDP-Hex ( approximately 20-fold), UDP-HexNAc (6- to 15-fold) and CMP-sialic acid (30- to 120-fold), respectively. Up-regulation of B4gal and St3gal could also have enhanced glycan addition onto the proteins, leading to more complete glycosylation (sialylation). Combined feeding of glucosamine + uridine and ManNAc + cytidine increased UDP-HexNAc and CMP-sialic acid by another two- to fourfold as compared to feeding sugar precursors alone. However, it did not lead to a synergistic increase in IFN-gamma sialylation. Other factors such as glycosyltransferase or glycan substrate levels could have become limiting. In addition, uridine feeding increased the levels of uridine- and cytidine-activated nucleotide sugars simultaneously, which could imply that uridine is one of the limiting substrates for nucleotide sugar synthesis in the study. Hence, the characterization of intracellular glycosylation activities has increased our understanding of how nucleotide sugar precursor feeding influence glycosylation of recombinant proteins produced in CHO cells. It has also led to the optimization of more effective strategies for manipulating glycan quality.

ChemInform Abstract: Synthesis of a Novel Sialyl Lewis X Analogue Containing a Pyrrolidine in Place of N-Acetyl Glucosamine.

ABSTRACT The synthesis of the new sialyl Lewis X analogue, 4-O-(α-L-fucopyranosyl)-3-O-(3-O-sodium sulfonato-β-D-galactopyranosyl)-(2S,3R, 4R)-2-ethyl-3,4-dihydroxypyrrolidine 2 has been achieved. The N-acetyl glucosamine unit of natural Lewis X has been replaced by a rigid 3R/4R-dihydroxylated pyrrolidine 12. This one has been synthezised from the methyl 4-O-benzoyl-2,3-di-O-benzyl-6-deoxy-6-iodo-α-D-altropyranoside sugar precursor 10 using the Ganem/Bernotas one-pot elimination-reductive amination ring contraction reaction. The (2S, 3R, 4R)-1-benzyloxycarbonyl-3,4-dihydroxy-2-ethylpyrrolidine 12 obtained was subsequently regioselectively glycosylated, using 2,3,4-tri-O-benzyl-α-L-fucopyranosyl fluoride and 2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl bromide as glycosyl donors. Disaccharide containing pyrrolidine 21 was finally transformed into the target O-sulfated analog 2, after regioselective sulfation and usual deprotection.

Structural and Kinetic Characterization of the LPS Biosynthetic Enzyme d-α,β-d-Heptose-1,7-bisphosphate Phosphatase (GmhB) from Escherichia coli,

Lipopolysaccharide is a major component of the outer membrane of gram-negative bacteria and provides a permeability barrier to many commonly used antibiotics. ADP-heptose residues are an integral part of the LPS inner core, and mutants deficient in heptose biosynthesis demonstrate increased membrane permeability. The heptose biosynthesis pathway involves phosphorylation and dephosphorylation steps not found in other pathways for the synthesis of nucleotide sugar precursors. Consequently, the heptose biosynthetic pathway has been marked as a novel target for antibiotic adjuvants, which are compounds that facilitate and potentiate antibiotic activity. D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) catalyzes the third essential step of LPS heptose biosynthesis. This study describes the first crystal structure of GmhB and enzymatic analysis of the protein. Structure-guided mutations followed by steady state kinetic analysis, together with established precedent for HAD phosphatases, suggest that GmhB functions through a phosphoaspartate intermediate. This study provides insight into the structure-function relationship of GmhB, a new target for combatting gram-negative bacterial infection.