Preliminary X-ray analysis of a new crystal form of the<i>Escherichia coli</i>KDO8P synthase

Radaev, Sergei; Dastidar, Parthasarathi; Patel, M.; Woodard, Ronald W.; Gatti, Domenico L.

doi:10.1107/s0907444900002389

Cited by 17 publications

(17 citation statements)

References 13 publications

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“…Two crystal forms of E. coli KDO8PS previously reported by this laboratory contain instead a homotetramer of the enzyme with 222 local symmetry (9,11). A third crystal form reported by Wagner et al (10) contains only one chain in the asymmetric unit, but application of crystal symmetry generates a tetramer essentially identical to that present in the other two crystal forms.…”

Section: Resultsmentioning

confidence: 97%

Substrate and Metal Complexes of 3-Deoxy-d-manno-octulosonate-8-phosphate Synthase from Aquifex aeolicus at 1.9-Å Resolution

Duewel

Radaev

Wang

et al. 2001

Journal of Biological Chemistry

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157

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3-Deoxy-D-manno-octulosonate-8-phosphate synthase (KDO8PS) from the hyperthermophilic bacteriumAquifex aeolicus differs from its Escherichia coli counterpart in the requirement of a divalent metal for activity (Duewel, H. S., and Woodard, R. W. (2000) J. Biol. Chem. 275, 22824 -22831). Here we report the crystal structure of the A. aeolicus enzyme, which was determined by molecular replacement using E. coli KDO8PS as a model. The structures of the metal-free and Cd 2؉forms of the enzyme were determined in the uncomplexed state and in complex with various combinations of phosphoenolpyruvate (PEP), arabinose 5-phosphate (A5P), and erythrose 4-phosphate (E4P). Like the E. coli enzyme, A. aeolicus KDO8PS is a homotetramer containing four distinct active sites at the interface between subunits. The active site cavity is open in the substratefree enzyme or when either A5P alone or PEP alone binds, and becomes isolated from the aqueous phase when both PEP and A5P (or E4P) bind together. In the presence of metal, the enzyme is asymmetric and appears to alternate catalysis between the active sites located on one face of the tetramer and those located on the other face. In the absence of metal, the asymmetry is lost. Details of the active site that may be important for catalysis are visible at the high resolution achieved in these structures. Most notably, the shape of the PEPbinding pocket forces PEP to assume a distorted geometry at C-2, which might anticipate the conversion from sp 2 to sp 3 hybridization occurring during intermediate formation and which may modulate PEP reactivity toward A5P. Two water molecules are located in van der Waals contact with the si and re sides of C-2 PEP , respectively. Abstraction of a proton from either of these water molecules by a protein group is expected to elicit a nucleophilic attack of the resulting hydroxide ion on the nearby C-2 PEP , thus triggering the beginning of the catalytic cycle.3-Deoxy-D-manno-octulosonate-8-phosphate synthase (KDO8PS 1 ; phospho-2-dehydro-3-deoxyoctonate aldolase, EC 4.1.2.16) catalyzes the aldol-type condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P and inorganic phosphate ( Fig. 1) (1). This reaction is the first step in the biosynthesis of 3-deoxy-Dmanno-octulosonate, an essential component of the lipopolysaccharide of all Gram-negative bacteria (2). A reaction very similar to KDO8P synthesis is the formation of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) from erythrose 4-phosphate (E4P) and PEP catalyzed by DAH7P synthase (DAH7PS; phospho-2-dehydro-3-deoxyheptonate aldolase, EC 4.1.2.15), which constitutes the first step of the shikimate pathway (3). Earlier studies have established that KDO8PS and DAH7PS share several mechanistic characteristics. For example, in both enzymes, the condensation step of the reaction is stereospecific, involving the addition of the si face of C-3 PEP to the re face of the A5P/E4P carbonyl (4 -7). In particular, KDO8PS and DAH7PS are two of only four known PEP-utilizing e...

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Section: Resultsmentioning

confidence: 97%

Substrate and Metal Complexes of 3-Deoxy-d-manno-octulosonate-8-phosphate Synthase from Aquifex aeolicus at 1.9-Å Resolution

Duewel

Radaev

Wang

et al. 2001

Journal of Biological Chemistry

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157

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“…To a first approximation, binding of 0.25 (manganese), 0.5 (zinc, copper, cadmium), or 1 (iron) mol eq of metal per subunit is observed in the complexes. Several observations provide convincing evidence that A. aeolicus KDO8PS exists as a tetramer in solution: the structure of E. coli KDO8PS determined for two crystal forms reveals a tightly packed homotetramer with four individual active sites (66,67); the oligomeric structure of A. aeolicus KDO8PS is equivalent to the E. coli enzyme in solution (48); and the sequence identity between the two enzymes (46%; see below) is significant. Therefore, it is reasonable that each subunit of the A. aeolicus KDO8PS tetramer will itself contain a lone active site that binds a single metal ion.…”

Section: Discussionmentioning

confidence: 95%

A Metal Bridge between Two Enzyme Families

Duewel

Woodard

2000

Journal of Biological Chemistry

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The enzymes 3-deoxy-D-manno-octulosonic acid-8-phosphate synthase (KDO8PS) and 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthase (DAHPS) catalyze analogous condensation reactions between phosphoenolpyruvate and D-arabinose 5-phosphate or D-erythrose 4-phosphate, respectively. While several similarities exist between the two enzymatic reactions, classic studies on the Escherichia coli enzymes have established that DAHPS is a metalloenzyme, whereas KDO8PS has no metal requirement. Here, we demonstrate that KDO8PS from Aquifex aeolicus, representing only the second member of the KDO8PS family to be characterized in detail, is a metalloenzyme. , and Zn 2؉ and is reversibly inhibited by higher concentrations (>1 mM) of certain metals. Analysis of several metal forms of the enzyme by plasma mass spectrometry suggests that the enzyme preferentially binds one, two, or four metal ions per tetramer. These observations strongly suggest that A. aeolicus KDO8PS is a metalloenzyme in vivo and point to a previously unrecognized relationship between the KDO8PS and DAHPS families.Over the past several years, our understanding of a unique class of enzymes responsible for the incorporation of the 3-carbon skeleton of phosphoenolpyruvate (PEP) 1 into pivotal biosynthetic intermediates has dramatically increased. Common to this particular enzyme class, which can be divided into two broad groups, is cleavage of the C-O bond of PEP concurrent with catalysis, as opposed to the more conventional cleavage of the P-O bond (1-7). Members of the first group, 5-enolpyruvylshikimate-3-phosphate synthase and UDP-N-acetylglucosamine enolpyruvate transferase, promote transfer of the intact carboxyvinyl portion of PEP to a cosubstrate alcohol with the formation of an enolic ether linkage to the C-2 of PEP. The mechanisms of enolpyruvylshikimate-3-phosphate synthase (8 -12) and UDP-N-acetylglucosamine enolpyruvate transferase (13-15) have been thoroughly characterized.The second group of enzymes associated with C-O bond cleavage reactions presently includes 3-deoxy-D-manno-octulosonic acid-8-phosphate (KDO8P) synthase (KDO8PS) (16, 17) and 3-deoxy-D-arabino-heptulosonic acid-7-phosphate (DAHP) synthase (DAHPS) (18,19). Both enzymes catalyze the condensation of PEP with a phosphorylated monosaccharide via coupling of the C-3 of PEP to the C-1 of an aldose cosubstrate to produce a 3-deoxy-2-keto sugar acid three carbons longer. KDO8PS occupies an essential position in the biosynthesis of lipopolysaccharide in Gram-negative microorganisms (20, 21), using D-arabinose 5-phosphate (A5P) in its condensation reaction to yield KDO8P and inorganic phosphate. KDO8P is the precursor to 3-deoxy-D-manno-octulosonic acid (KDO), an unusual octulose found in the inner core of lipopolysaccharide. KDO assumes an important role in the overall assembly of lipopolysaccharide, and its production and utilization have proven central to cellular viability and homeostasis (22)(23)(24)(25). In the reaction catalyzed by DAHPS, D-erythrose 4-phosphate (E4P) serves as the...

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“…Bacterial KDOPSs have been extensively investigated [12][13][14][15][16][17][18][19][20][21][22][23][24]; however, little information is available for plant KDOPSs. In the present study, the kdsA from A. thaliana is overexpressed, purified and characterized.…”

Section: Discussionmentioning

confidence: 99%

“…The microbial non-metallo-KDOPSs from E. coli [12], S. typhimurium [13] and Neisseria gonorrhoeae [14], as well as microbial metallo-KDOPSs from Aquifex aeolicus [15], Helicobacter pylori [16] and Chlamydia psittaci [17] have been characterized. The structure and mechanism of the KDOPSs from E. coli (nonmetallo) [18][19][20][21][22] and A. aeolicus (metallo) [23,24] have been investigated in great detail. Although the KDOPSs from microbial Abbreviations used: A5P, D-arabinose 5-phosphate; BTP, 1,3-bis[tris(hydroxymethyl)-methylamino]propane; DAHPS, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase; DPA, dipicolinic acid; 2dR5P, 2-deoxyribose 5-phosphate; E4P, D-erythrose 4-phosphate; KDO, 3-deoxy-D-manno-octulosonate; KDOPS, 3-deoxy-D-manno-octulosonate 8-phosphate synthase; MALDI-MS, matrix-assisted laser-desorption ionization-mass spectrometry; PEP, phosphoenolpyruvate; R5P, D-ribose 5-phosphate.…”

Section: Introductionmentioning

confidence: 99%

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

Patel

Sundaram

et al. 2004

Biochemical Journal

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An open reading frame, encoding for KDOPS (3-deoxy-D-manno-octulosonate 8-phosphate synthase), from Arabidopsis thaliana was cloned into a T7-driven expression vector. The protein was overexpressed in Escherichia coli and purified to homogeneity. Recombinant A. thaliana KDOPS, in solution, displays an apparent molecular mass of 76 kDa and a subunit molecular mass of 31.519 kDa. Unlike previously studied bacterial KDOPSs, which are tetrameric, A. thaliana KDOPS appears to be a dimer in solution. The optimum temperature of the enzyme is 65 degrees C and the optimum pH is 7.5, with a broad peak between pH 6.5 and 9.5 showing 90% of maximum activity. The enzyme cannot be inactivated by EDTA or dipicolinic acid treatment, nor it can be activated by a series of bivalent metal ions, suggesting that it is a non-metallo-enzyme, as opposed to the initial prediction that it would be a metallo-enzyme. Kinetic studies showed that the enzyme follows a sequential mechanism with K(m)=3.6 microM for phosphoenolpyruvate and 3.8 microM for D-arabinose 5-phosphate and kcat=5.9 s(-1) at 37 degrees C. On the basis of the characterization of A. thaliana KDOPS and phylogenetic analysis, plant KDOPSs may represent a new, distinct class of KDOPSs.

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Preliminary X-ray analysis of a new crystal form of theEscherichia coliKDO8P synthase

Cited by 17 publications

References 13 publications

Substrate and Metal Complexes of 3-Deoxy-d-manno-octulosonate-8-phosphate Synthase from Aquifex aeolicus at 1.9-Å Resolution

Substrate and Metal Complexes of 3-Deoxy-d-manno-octulosonate-8-phosphate Synthase from Aquifex aeolicus at 1.9-Å Resolution

A Metal Bridge between Two Enzyme Families

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

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