Condensation Of Phosphoenolpyruvate Research Articles

Campylobacter jejuni KDO8P Synthase, Its Inhibition by KDO8P Oxime, and Control of the Residence Time of Slow-Binding Inhibition.

3-Deoxy-d- manno-2-octulosonate-8-phosphate (KDO8P) synthase catalyzes the first step of lipopolysaccharide biosynthesis, namely condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P), to produce KDO8P. We have characterized Campylobacter jejuni KDO8P synthase and its inhibition by KDO8P oxime. It was metal-dependent and homotetrameric and followed a rapid equilibrium sequential ordered ter ter kinetic mechanism in which Mn2+ bound first, followed by PEP and then A5P. It was inhibited by KDO8P oxime, an analogue of 3-deoxy-d- arabino-heptulosonate 7-phosphate (DAHP) oxime, a transition-state mimic of DAHP synthase. Inhibition was uncompetitive-like with respect to Mn2+ and competitive with respect to PEP and A5P. It displayed both fast-binding inhibition ( Ki = 10 μM) and slow-binding inhibition ( Ki* = 0.57 μM). The residence times on the enzyme ( tR) ranged from 27 min in the absence of free inhibitor to 69 h with excess inhibitor. The dependence of tR on the free inhibitor concentration suggested intersubunit communication within the homotetramer between high- and low-affinity sites. This confirms the generality of the oxime functional group, a small, neutral phosphate bioisostere, as an α-carboxyketose synthase inhibitor and highlights the challenge that intersubunit communication poses to effective inhibition.

Probing the determinants of phosphorylated sugar-substrate binding for human sialic acid synthase

N-acetylneuraminic acid (NeuNAc), the most naturally abundant sialic acid, is incorporated as the terminal residue of mammalian cell surface glycoconjugates and acts as a key facilitator of cellular recognition, adhesion and signalling. Several pathogenic bacteria similarly express NeuNAc on their cell surfaces, allowing evasion of their host's immune system. Prokaryotic NeuNAc biosynthesis proceeds via condensation of phosphoenolpyruvate (PEP) with N-acetylmannosamine (ManNAc), a reaction catalysed by the domain-swapped homodimeric enzyme, N-acetylneuraminic acid synthase (NeuNAcS). Conversely, the mammalian orthologue, N-acetylneuraminic acid 9-phosphate synthase (NeuNAc 9-PS) utilises the phosphorylated substrate N-acetylmannosamine 6-phosphate (ManNAc 6-P) in catalysis. Here we report an investigation into the determinants of substrate specificity of human NeuNAc 9-PS, using model-guided mutagenesis to delineate binding interactions with ManNAc 6-P. Modelling predicts the formation of a domain-swapped homodimer as observed for bacterial variants, which was supported by experimental small angle X-ray scattering. A number of conserved residues which may play key roles in the selection of ManNAc 6-P were identified and substituted for alanine to assess their function. Lys290 and Thr80 were identified as a putative phosphate binding pair, with the cationic lysine residue extending into the active site from the adjacent chain of the dimeric enzyme. Substitution of these residues results in a significant loss of activity and reduced affinity for ManNAc 6-P. These residues, along with the electropositive β2α2 loop, are likely to facilitate the PEP dependent binding and stabilisation of ManNAc 6-P. By utilising a phosphorylated sugar-substrate, the mammalian enzyme gains considerable catalytic affinity advantage over its bacterial counterpart.

PhDAHP1 is required for floral volatile benzenoid/phenylpropanoid biosynthesis in Petunia × hybrida cv ‘Mitchell Diploid’

Floral volatile benzenoid/phenylpropanoid (FVBP) biosynthesis consists of numerous enzymatic and regulatory processes. The initial enzymatic step bridging primary metabolism to secondary metabolism is the condensation of phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) carried out via 3-DEOXY-D-ARABINO-HEPTULOSONATE-7-PHOSPHATE (DAHP) synthase. Here, identified, cloned, localized, and functionally characterized were two DAHP synthases from the model plant species Petunia × hybrida cv ‘Mitchell Diploid’ (MD). Full-length transcript sequences for PhDAHP1 and PhDAHP2 were identified and cloned using cDNA SMART libraries constructed from pooled MD corolla and leaf total RNA. Predicted amino acid sequence of PhDAHP1 and PhDAHP2 proteins were 76% and 80% identical to AtDAHP1 and AtDAHP2 from Arabidopsis, respectively. PhDAHP1 transcript accumulated to relatively highest levels in petal limb and tube tissues, while PhDAHP2 accumulated to highest levels in leaf and stem tissues. Through floral development, PhDAHP1 transcript accumulated to highest levels during open flower stages, and PhDAHP2 transcript remained constitutive throughout. Radiolabeled PhDAHP1 and PhDAHP2 proteins localized to plastids, however, PhDAHP2 localization appeared less efficient. PhDAHP1 RNAi knockdown petunia lines were reduced in total FVBP emission compared to MD, while PhDAHP2 RNAi lines emitted ‘wildtype’ FVBP levels. These results demonstrate that PhDAHP1 is the principal DAHP synthase protein responsible for the coupling of metabolites from primary metabolism to secondary metabolism, and the ultimate biosynthesis of FVBPs in the MD flower.

Characterization of a putative 3-deoxy-D-manno-2-octulosonic acid (Kdo) transferase gene from Arabidopsis thaliana

The structures of the pectic polysaccharide rhamnogalacturonan II (RG-II) pectin constituent are remarkably evolutionary conserved in all plant species. At least 12 different glycosyl residues are present in RG-II. Among them is the seldom eight-carbon sugar 3-deoxy-d-manno-octulosonic acid (Kdo) whose biosynthetic pathway has been shown to be conserved between plants and Gram-negative bacteria. Kdo is formed in the cytosol by the condensation of phosphoenol pyruvate with d-arabinose-5-P and then activated by coupling to cytidine monophosphate (CMP) prior to its incorporation in the Golgi apparatus by a Kdo transferase (KDTA) into the nascent polysaccharide RG-II. To gain new insight into RG-II biosynthesis and function, we isolated and characterized null mutants for the unique putative KDTA (AtKDTA) encoded in the Arabidopsis genome. We provide evidence that, in contrast to mutants affecting the RG-II biosynthesis, the extinction of the AtKDTA gene expression does not result in any developmental phenotype in the AtkdtA plants. Furthermore, the structure of RG-II from the null mutants was not altered and contained wild-type amount of Rha-alpha(1-5)Kdo side chain. The cellular localization of AtKDTA was investigated by using laser scanning confocal imaging of the protein fused to green fluorescent protein. In agreement with its cellular prediction, the fusion protein was demonstrated to be targeted to the mitochondria. These data, together with data deduced from sequence analyses of higher plant genomes, suggest that AtKDTA encodes a putative KDTA involved in the synthesis of a mitochondrial not yet identified lipid A-like molecule rather than in the synthesis of the cell wall RG-II.

Cloning, expression, and characterization of sialic acid synthases

The most commonly occurring sialic acid, N-acetylneuraminic acid, is the repeating unit in polysialic acid chain of human neuronal cell adhesion molecule as well as in capsular polysialic acid of neuroinvasive bacteria, Escherichia coli K1 and Neisseria meningitidis. Sialic acid synthesis and polymerization occur in slightly different pathways in animals and bacteria. N-Acetylneuraminic acid (NeuNAc) is synthesized by the condensation of phosphoenolpyruvate and N-acetylmannosamine by NeuNAc synthase in bacteria. The mammalian homologue N-acetylneuraminic acid-9-phosphate (NeuNAc-9-P) synthase uses N-acetylmannosamine-6-phosphate in the condensation reaction to produce NeuNAc-9-P. Both subfamilies of sialic acid synthases possess N-terminal triosephosphate isomerase barrel domain and C-terminal antifreeze protein domain. We report cloning of the genes, expression, purification, and characterization of human NeuNAc-9-P synthase and N. meningitidis NeuNAc synthase. Stability of the purified enzymes and effects of pH and temperature on their activities were evaluated. Enzyme kinetics and preliminary mutagenesis experiments reveal the importance of C-terminal antifreeze protein domain and a conserved cysteine residue for the enzyme activities.

Crystal Structure of the Reaction Complex of 3-Deoxy-d-arabino-heptulosonate-7-phosphate Synthase from Thermotoga maritima Refines the Catalytic Mechanism and Indicates a New Mechanism of Allosteric Regulation

3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) catalyzes the first reaction of the aromatic biosynthetic pathway in bacteria, fungi, and plants, the condensation of phosphoenolpyruvate (PEP) and d-erythrose-4-phosphate (E4P) with the formation of DAHP. Crystals of DAHPS from Thermotoga maritima (DAHPSTm) were grown in the presence of PEP and metal cofactor, Cd2+, and then soaked with E4P at 4 °C where the catalytic activity of the enzyme is negligible. The crystal structure of the “frozen” reaction complex was determined at 2.2Å resolution. The subunit of the DAHPSTm homotetramer consists of an N-terminal ferredoxin-like (FL) domain and a (β/α)8-barrel domain. The active site located at the C-end of the barrel contains Cd2+, PEP, and E4P, the latter bound in a non-productive conformation. The productive conformation of E4P is suggested and a catalytic mechanism of DAHPS is proposed. The active site of DAHPSTm is nearly identical to the active sites of the other two known DAHPS structures from Escherichia coli (DAHPSEc) and Saccharomyces cerevisiae (DAHPSSc). However, the secondary, tertiary, and quaternary structures of DAHPSTm are more similar to the functionally related enzyme, 3-deoxy-d-manno-octulosonate-8-phosphate synthase (KDOPS) from E.coli and Aquiflex aeolicus, than to DAHPSEc and DAHPSSc. Although DAHPSTm is feedback-regulated by tyrosine and phenylalanine, it lacks the extra barrel segments that are required for feedback inhibition in DAHPSEc and DAHPSSc. A sequence similarity search revealed that DAHPSs of phylogenetic family Iβ possess a FL domain like DAHPSTm while those of family Iα have extra barrel segments similar to those of DAHPSEc and DAHPSSc. This indicates that the mechanism of feedback regulation in DAHPSTm and other family Iβ enzymes is different from that of family Iα enzymes, most likely being mediated by the FL domain.

Substrate deactivation of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase by erythrose 4-phosphate.

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS, EC 4.1.2.15) catalyzes the condensation of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to give DAH7P via an ordered sequential mechanism. In the absence of PEP (the first substrate to bind), E4P binds covalently to the phenylalanine-sensitive DAH7PS of Escherichia coli, DAH7PS(Phe), deactivating the enzyme. Activity is restored on addition of excess PEP but not if deactivation was carried out in the presence of sodium cyanoborohydride. Electrospray mass spectrometry indicates that a single E4P is bound to the protein. These data are consistent with a slow, reversible Schiff base reaction of the aldehydic functionality of E4P with a buried lysine. Molecular modeling indicates that Lys186, a residue at the base of the substrate-binding cavity involved in hydrogen bonding with PEP, is well placed to react with E4P forming an imine linkage that is substantially protected from solvent water.

Steady-state kinetics and inhibitor binding of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (tryptophan sensitive) from Escherichia coli.

The tryptophan-inhibited 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase [DAHPS(Trp)] of Escherichia coli was analyzed with respect to steady-state kinetics and tryptophan binding. DAHPS(Trp) is one of three differentially regulated isoforms that catalyze the first step of aromatic biosynthesis, the condensation of phosphoenolpyruvate and erythrose-4-phosphate to form 3-deoxy-D-arabino-heptulosonate-7-phosphate. The DAHP synthase isozymes are metalloproteins, being activated in vitro by a variety of divalent metals. Both catalytic activity and substrate affinity are dependent on the species of activating metal ion. We report here kinetic and binding studies of metal-homogeneous (Mn2+-activated) DAHPS(Trp). The homodimeric enzyme had an apparent kcat of 21 s-1 and displayed sigmoidal kinetics with respect to both substrates. The S0.5 was 35 microM for erythrose-4-phosphate and 5.3 microM for phosphoenolpyruvate. Equilibrium binding studies with radiolabeled tryptophan demonstrated two independent inhibitor binding sites per enzyme dimer, with KdTrp of 1 microM. L-Tryptophan binding decreased kcat, increased affinity for both substrates, decreased positive homotropic cooperativity for both substrates and activated the enzyme at low concentrations of erythrose-4-phosphate. The results suggest an inhibition mechanism analogous to system C5 hyperbolic mixed-type inhibition with respect to erythrose-4-phosphate and partial noncompetitive inhibition with respect to phosphoenolpyruvate.