Functional and biochemical characterization of a recombinant <i>Arabidopsis thaliana</i> 3-deoxy-<scp>D</scp>-<i>manno</i>-octulosonate 8-phosphate synthase

Wu, Jing; Patel, M.; Sundaram, Appavu K.; Woodard, Ronald W.

doi:10.1042/bj20040207

Cited by 19 publications

(22 citation statements)

References 43 publications

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“…To determine the effect of divalent metal ions on the API activity of Q5LIW1, samples of Q5LIW1 were diluted in buffer containing 108.2 mM Bis-Tris propane, pH 8.5, and 10.82 M EDTA or divalent metal salt and incubated on ice for 30 min. The API activity was assayed using the Aminoff assay (15). Final concentrations in each reaction mixture were 100 nM Q5LIW1, 100 mM Bis-Tris propane (pH 8.5), 0.15 mg/ml KdsA, 10 mM PEP, and 5 mM Ru5P.…”

Section: Methodsmentioning

confidence: 99%

“…Kinetic parameters for the isomerization of Ru5P to A5P were determined, in triplicate, using a modified coupled Aminoff assay utilizing the 3-deoxy-D-manno-octulosonate 8-phosphate synthase (Kdo8PS) from Arabidopsis thaliana (15,16). Reaction mixtures containing final concentrations of 10 mM phosphoenolpyruvate (PEP), 0.15 mg/ml Kdo8PS, 100 mM Bis-Tris propane (pH 8.5), and 1 mM EDTA and final concentrations of Ru5P ranging from 0 to 10 mM were heated separately from mixtures of Q5LIW1 or EcKdsD⌬2CBS (100 nM final concentration) to 37°C for 3 min.…”

Section: Methodsmentioning

confidence: 99%

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Analysis of the Arabinose-5-Phosphate Isomerase of Bacteroides fragilis Provides Insight into Regulation of Single-Domain Arabinose Phosphate Isomerases

et al. 2014

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Arabinose-5-phosphate isomerases (APIs) catalyze the interconversion of D-ribulose-5-phosphate and D-arabinose-5-phosphate, the first step in the biosynthesis of 3-deoxy-D-manno-octulosonic acid (Kdo), an essential component of the lipopolysaccharide in Gram-negative bacteria. Classical APIs, such as Escherichia coli KdsD, contain a sugar isomerase domain and a tandem cystathionine beta-synthase domain. Despite substantial effort, little is known about structure-function relationships in these APIs. We recently reported an API containing only a sugar isomerase domain. This protein, c3406 from E. coli CFT073, has no known physiological function. In this study, we investigated a putative single-domain API from the anaerobic Gram-negative bacterium Bacteroides fragilis. This putative API (UniProt ID Q5LIW1) is the only protein encoded by the B. fragilis genome with significant identity to any known API, suggesting that it is responsible for lipopolysaccharide biosynthesis in B. fragilis. We tested this hypothesis by preparing recombinant Q5LIW1 protein (here referred to by the UniProt ID Q5LIW1), characterizing its API activity in vitro, and demonstrating that the gene encoding Q5LIW1 (GenBank ID YP_209877.1) was able to complement an APIdeficient E. coli strain. We demonstrated that Q5LIW1 is inhibited by cytidine 5=-monophospho-3-deoxy-D-manno-2-octulosonic acid, the final product of the Kdo biosynthesis pathway, with a K i of 1.91 M. These results support the assertion that Q5LIW1 is the API that supports lipopolysaccharide biosynthesis in B. fragilis and is subject to feedback regulation by CMPKdo. The sugar isomerase domain of E. coli KdsD, lacking the two cystathionine beta-synthase domains, demonstrated API activity and was further characterized. These results suggest that Q5LIW1 may be a suitable system to study API structure-function relationships.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Analysis of the Arabinose-5-Phosphate Isomerase of Bacteroides fragilis Provides Insight into Regulation of Single-Domain Arabinose Phosphate Isomerases

et al. 2014

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“…A second, more sensitive coupled assay was developed to determine API activity in crude cell extracts that utilized 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KdsA) from Arabidopsis thaliana (34). This enzyme catalyzes the irreversible stereospecific condensation of A5P and phosphoenolpyruvate to form 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) and inorganic phosphate.…”

Section: Methodsmentioning

confidence: 99%

Identification of GutQ from Escherichia coli as a d -Arabinose 5-Phosphate Isomerase

Meredith¹,

Woodard

2005

J Bacteriol

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The glucitol operon (gutAEBDMRQ) of Escherichia coli encodes a phosphoenolpyruvate:sugar phosphotransferase system that metabolizes the hexitol D-glucitol (sorbitol). The functions for all but the last gene, gutQ, have been previously assigned. The high sequence similarity between GutQ and KdsD, a D-arabinose 5-phosphate isomerase (API) from the 3-deoxy-D-manno-octulosonate (KDO)-lipopolysaccharide (LPS) biosynthetic pathway, suggested a putative activity, but its role within the context of the gut operon remained unclear. Accordingly, the enzyme was cloned, overexpressed, and characterized. Recombinant GutQ was shown to indeed be a second copy of API from the E. coli K-12 genome with biochemical properties similar to those of KdsD, catalyzing the reversible aldol-ketol isomerization between D-ribulose 5-phosphate (Ru5P) and Darabinose 5-phosphate (A5P). Genomic disruptions of each API gene were constructed in E. coli K-12. TCM11[(⌬kdsD)] was capable of sustaining essential LPS synthesis at wild-type levels, indicating that GutQ functions as an API inside the cell. The gut operon remained inducible in TCM7[(⌬gutQ)], suggesting that GutQ is not directly involved in D-glucitol catabolism. The conditional mutant TCM15[(⌬gutQ⌬kdsD)] was dependent on exogenous A5P both for LPS synthesis/growth and for upregulation of the gut operon. The phenotype was suppressed by complementation in trans with a plasmid encoding a functional copy of GutQ or by increasing the amount of A5P in the medium. As there is no obvious obligatory role for GutQ in the metabolism of D-glucitol and there is no readily apparent link between D-glucitol metabolism and LPS biosynthesis, it is suggested that A5P is not only a building block for KDO biosynthesis but also may be a regulatory molecule involved in expression of the gut operon.D-Arabinose 5-phosphate isomerase (API) is the first enzyme in the biosynthetic pathway of 3-deoxy-D-manno-octulosonate (KDO), a unique 8-carbon sugar component of lipopolysaccharides (LPSs). Lipopolysaccharides are amphiphilic macromolecules located in the outer leaflet of the outer membrane of gram-negative bacteria, forming an asymmetric lipid bilayer that surrounds the cell. The LPS layer is anchored in the outer membrane by the highly conserved lipid A domain and is followed by an oligosaccharide core region usually containing at least one KDO molecule that connects lipid A to an extended hypervariable repeating polysaccharide chain (O antigen) (25). In addition to providing a measure of intrinsic antibiotic resistance and defense against host responses, LPS (also referred to as endotoxin) is the main mediator of gramnegative bacterial pathogenesis (13, 33). The minimal LPS structure required for viability of Escherichia coli under laboratory conditions is KDO 2 -lipid A, also referred to as Re endotoxin (11,25,31).API catalyzes the reversible 1,2-keto/aldol isomerization of the pentose pathway intermediate ribulose 5-phosphate (Ru5P) to D-arabinose 5-phosphate (A5P) and is the intracellular source of A5P used f...

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“…In previous studies, we have determined the crystal structure of KDO8PS from the hyperthermophile Aquifex aeolicus (a Class II KDO8PS) in complex with Cd 2+ (2,3), the metal conferring the highest activity in vitro (9). All known KDO8PSs, with the exception of the Arabidopsis thaliana enzyme (10), are tetramers. However, the asymmetric unit of A. aeolicus KDO8PS crystals contains only two subunits, named here A and B.…”

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confidence: 99%

The Catalytic and Conformational Cycle ofAquifex aeolicusKDO8P Synthase: Role of the L7 Loop^,

Kona

Wang

et al. 2005

Biochemistry

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KDO8P synthase catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form the 8-carbon sugar KDO8P and inorganic phosphate (P i ). The X-ray structure of the wild-type enzyme shows that when both PEP and A5P bind, the active site becomes isolated from the environment due to a conformational change of the L7 loop. The structures of the R106G mutant, without substrates, and with PEP and PEP plus A5P bound, were determined and reveal that in R106G closure of the L7 loop is impaired. The structural perturbations originating from the loss of the Arg 106 side chain point to a role of the L2 loop in stabilizing the closed conformation of the L7 loop. Despite the increased exposure of the R106G active site, no abnormal reaction of PEP with water was observed, ruling out the hypothesis that the primary function of the L7 loop is to shield the active site from bulk solvent during the condensation reaction. However, the R106G enzyme displays several kinetic abnormalities on both the substrate side (smaller K m PEP , larger K i A5P and K m A5P ) and the product side (smaller K i Pi and K i KDO8P ) of the reaction. As a consequence, the mutant enzyme is less severely inhibited by A5P and more severely inhibited by P i and KDO8P. Simulations of the flux of KDO8P synthesis under metabolic steady-state conditions (constant concentration of reactants and products over time) suggest that in vivo R106G is expected to perform optimally in a narrower range of substrate and product concentrations than the wild-type enzyme.3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS, EC 2.5.1.55) catalyzes the irreversible condensation of phosphoenolpyruvate (PEP) 1 and arabinose 5-phosphate (A5P) to form KDO8P and inorganic phosphate ( Figure 1) (1)(2)(3)(4)(5). This reaction is the first step in the biosynthesis of 3-deoxy-D-manno-octulosonate (KDO), an essential component of the lipopolysaccharide of all Gram-negative bacteria (6, 7). † This research was supported by U.S. Public Health Service Grants GM69840 to D.L.G. ‡ The structure-factor amplitudes and refined coordinates of substrate-free R106G, R106G with PEP, and PEP plus A5P, were deposited in the Protein Data Bank (entries 1T99, 1T96, 1T8X, respectively). 1 Abbreviations: KDO, 3-deoxy-D-manno-octulosonate; KDO8P, 3-deoxy-D-manno-octulosonate 8-phosphate; KDO8PS. 3-deoxy-Dmanno-octulosonate 8-phosphate synthase; PEP, phosphoenolpyruvate; A5P, arabinose 5-phosphate; 2-PGA, 2-phosphoglycerate; DSS, 2,2-dimethylsilapentane-5-sulfonic acid; DDM, difference-distance matrix; EDDM, error-scaled difference-distance matrix; SSR, sum of squared residuals; rmsd, root-mean-square deviation; H, helix; S, strand; L, loop. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptThere are two types of KDO8PSs, distinguished by their requirement for divalent metals (Class II) or lack thereof (Class I) (8). In previous studies, we have determined the crystal structure of KDO8PS from the hyperthermophile Aquifex aeolicu...

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Functional and biochemical characterization of a recombinant Arabidopsis thaliana 3-deoxy-D-manno-octulosonate 8-phosphate synthase

Cited by 19 publications

References 43 publications

Analysis of the Arabinose-5-Phosphate Isomerase of Bacteroides fragilis Provides Insight into Regulation of Single-Domain Arabinose Phosphate Isomerases

Analysis of the Arabinose-5-Phosphate Isomerase of Bacteroides fragilis Provides Insight into Regulation of Single-Domain Arabinose Phosphate Isomerases

Identification of GutQ from Escherichia coli as a d -Arabinose 5-Phosphate Isomerase

The Catalytic and Conformational Cycle ofAquifex aeolicusKDO8P Synthase: Role of the L7 Loop^,

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Functional and biochemical characterization of a recombinant Arabidopsis thaliana 3-deoxy-D-manno-octulosonate 8-phosphate synthase

Cited by 19 publications

References 43 publications

Analysis of the Arabinose-5-Phosphate Isomerase of Bacteroides fragilis Provides Insight into Regulation of Single-Domain Arabinose Phosphate Isomerases

Analysis of the Arabinose-5-Phosphate Isomerase of Bacteroides fragilis Provides Insight into Regulation of Single-Domain Arabinose Phosphate Isomerases

Identification of GutQ from Escherichia coli as a d -Arabinose 5-Phosphate Isomerase

The Catalytic and Conformational Cycle ofAquifex aeolicusKDO8P Synthase: Role of the L7 Loop,

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The Catalytic and Conformational Cycle ofAquifex aeolicusKDO8P Synthase: Role of the L7 Loop^,