The pre-steady-state reaction of Dictyostelium nucleoside diphosphate (NDP) kinase with dideoxynucleotide triphosphates (ddNTP) and AZT triphosphate was studied by quenching of protein fluorescence after manual mixing or by stopped flow. The fluorescence signal, which is correlated with the phosphorylation state of the catalytic histidine in the enzyme active site, decreases upon ddNTP addition according to a monoexponential time course. The pseudo-first order rate constant was determined for different concentrations of the various ddNTPs and was found to be saturable. The data are compatible with a two-step reaction scheme, where fast association of the enzyme with the dideoxynucleotide is followed by a rate-limiting phosphorylation step. The rate constants and dissociation equilibrium constants determined for each dideoxynucleotide were correlated with the steady-state kinetic parameters measured in the enzymatic assay in the presence of the two substrates. It is shown that ddNTPs and AZT triphosphate are poor substrates for NDP kinase with a rate of phosphate transfer of 0.02 to 3.5 s ؊1 and a K S of 1-5 mM. The equilibrium dissociation constants for ADP, GDP, ddADP, and ddGDP were also determined by fluorescence titration of a mutant F64W NDP kinase, where the introduction of a tryptophan at the nucleotide binding site provides a direct spectroscopic probe. The lack of the 3-OH in ddNTP causes a 10-fold increase in K D . Contrary to "natural" NTPs, NDP kinase discriminates between various ddNTPs, with ddGTP the more efficient and ddCTP the least efficient substrate within a range of 100 in k cat values.Nucleoside analogues like 3Ј-deoxy-3Ј-azidothymidine (AZT) 1 and dideoxynucleosides (ddN) are widely used as antiviral drugs, particularly in the multitherapy protocols now used in the treatment of AIDS. These drugs are targeted at the HIV reverse transcriptase as the lack of the 3Ј-OH required for the 3Ј-5Ј phosphodiester bond formation during DNA elongation blocks viral DNA synthesis. To exert their antiviral activity, the nucleoside analogues must be phosphorylated into triphosphates derivatives by cellular kinases. Although AZT, ddT, and ddC are structurally related, they show different patterns of intracellular phosphorylation (1). The synthesis of mono-and diphospho-derivatives involves kinases specific for either purines (for example deoxyguanosine kinase) or pyrimidines (for example deoxycytidine kinase or thymidine kinase). In all cases, the last step in the pathway leading to the triphospho-derivative is catalyzed by nucleoside diphosphate (NDP) kinase (EC 2.7.4.6), which has little specificity toward the nucleobase (2).NDP kinase phosphorylates all nucleoside diphosphates into triphosphates using ATP as the major phosphate donor. The reaction involves the formation of a phosphorylated intermediate according to a ping-pong bi-bi mechanism following the scheme below, where E-P is the phosphorylated intermediate on the catalytic histidine (3).EϳP ϩ N 2 DP 7 N 2 TP ϩ E (reaction B) SCHEME IIn recent yea...

Rapid non-empirical methods for estimating binding free energies are reviewed. A novel approach based on the application of the free energy perturbation formula to a biased ensemble is presented. Preliminary results demonstrating the applicability of this approach in protein systems are shown and the potential of this method in structure-based drug design is discussed.

The sulfate group is directed toward the protein surface. PAPS will be useful for the design of high affinity drugs targeted to NDP kinases.

In Gram-negative bacteria, lipid modification of proteins is catalysed in a three-step pathway. Apolipoprotein N-acyl transferase (Lnt) catalyses the third step in this pathway, whereby it transfers an acyl chain from a phospholipid to the amine group of the N-terminal cysteine residue of the apolipoprotein. Here, we report the 2.6-Å crystal structure of Escherichia coli Lnt. This enzyme contains an exo-membrane nitrilase domain fused to a transmembrane (TM) domain. The TM domain of Lnt contains eight TM helices which form a membrane-embedded cavity with a lateral opening and a periplasmic exit. The nitrilase domain is located on the periplasmic side of the membrane, with its catalytic cavity connected to the periplasmic exit of the TM domain. An amphipathic lid loop from the nitrilase domain interacts with the periplasmic lipid leaflet, forming an interfacial entrance from the lipid bilayer to the catalytic centre for both the lipid donor and acceptor substrates.

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Strigolactones are a novel class of plant hormones that interact with multiple signaling molecules, including auxin, abscisic acid, ethylene, and brassinosteroid, to regulate plant growth and development. Recently, researchers have shown that sugars are involved in bud outgrowth control, suggesting a potential interaction between sugars and strigolactone signaling. To better understand the relationship between strigolactones and sugar in plant development, the sugar sensitivity of strigolactone biosynthesis and signaling mutants (max1 and max2) was evaluated in early seedling development with a low-glucose assay. Both max1 and max2 displayed obvious hyposensitivity to glucose repression, as do gin mutants, but they were hypersensitive like the wild type to the high-glucose conditions used for gin mutant screening. The strigolactones acted synergistically with glucose in repressing seedling establishment. A further comparative transcriptomic analysis indicated that the expression of stress-related genes in the max2 mutant is impaired by glucose, and a carbohydrate analysis revealed a reduced hexose content in the max mutants. Our results suggest that the roles of strigolactones in the regulation of early seedling development are probably independent of the HXK1 signaling pathway. Taken together, these findings provide evidence that strigolactones are involved in sugar signaling, thus modulating early seedling development.

3-hydroxyacyl-CoA dehydrogenase (HAD, EC 1.1.1.35) is a homodimeric enzyme localized in the mitochondrial matrix, which catalyzes the third step in fatty acid β-oxidation. The crystal structures of human HAD and subsequent complexes with cofactor/substrate enabled better understanding of HAD catalytic mechanism. However, numerous human diseases were found related to mutations at HAD dimerization interface that is away from the catalytic pocket. The role of HAD dimerization in its catalytic activity needs to be elucidated. Here, we solved the crystal structure of Caenorhabditis elegans HAD (cHAD) that is highly conserved to human HAD. Even though the cHAD mutants (R204A, Y209A and R204A/Y209A) with attenuated interactions on the dimerization interface still maintain a dimerization form, their enzymatic activities significantly decrease compared to that of the wild type. Such reduced activities are in consistency with the reduced ratios of the catalytic intermediate formation. Further molecular dynamics simulations results reveal that the alteration of the dimerization interface will increase the fluctuation of a distal region (a.a. 60–80) that plays an important role in the substrate binding. The increased fluctuation decreases the stability of the catalytic intermediate formation, and therefore the enzymatic activity is attenuated. Our study reveals the molecular mechanism about the essential role of the HAD dimerization interface in its catalytic activity via allosteric effects.

Nucleoside diphosphate (NDP) kinase phosphorylates nucleoside diphosphates with little specificity for the base and the sugar. Although nucleotide analogues used in antiviral therapies are also metabolized to their triphosphate form by NDP kinase, their lack of the 3 H -hydroxyl of the ribose, which allows them to be DNA chain terminators, severely impairs the catalytic efficiency of NDP kinase. We have analyzed the kinetics parameters of several mutant NDP kinases modified on residues (Lys16, Tyr56, Asn119) interacting with the g-phosphate and/or the 3 H -OH of the Mg 21 -ATP substrate. We compared the relative contributions of the active-site residues and the substrate 3 H -OH for point mutations on Lys16, Tyr56 and Asn119. Analysis of additional data from pH profiles identify the ionization state of these residues in the enzyme active form. X-ray structure of K16A mutant NDP kinase shows no detectable rearrangement of the residues of the active site.Keywords: phosphoryl transfer; substrate assisted catalysis; dideoxynucleotides; tyrosyl titration.Nucleoside diphosphate kinase (ATP: nucleoside diphosphate phosphotransferase, EC 2.7.4.6; NDP kinase) catalyses the phosphorylation of nucleoside diphosphates into triphosphates. The reaction is ping-pong according to:where N 1 TP is usually ATP. It involves a covalent intermediate with a histidine residue phosphorylated in the active site. The enzyme shows little specificity for the base and uses ribonucleotides as well as deoxy-ribonucleotides as substrates [1].Genes coding for NDP kinases were first identified in 1990 [2±5], and more than 70 NDP kinase genes have been cloned to date in prokaryotes and eukaryotes. The crystal structure of several NDP kinases has been determined at high resolution showing a characteristic subunit fold with a babbab motif also called the ferredoxin fold [6]. The structure of the active site is highly conserved from prokaryotes to eukaryotes as could be expected from proteins whose sequences are 40% identical or more. The NDP kinase from Dictyostelium discoideum has often served as a reliable model to study the catalytic reaction at the atomic level, not only because it was the first eukaryotic NDP kinase to be solved but because it is particularly easy to crystallize. Generally, the active site appears as a preformed rigid template to which the nuleotide binds with very little, if any, change in protein conformation [7]. A structure of the phosphorylated enzyme where the phosphate is covalently bound to the active site His122 shows no major conformational change relative to the free protein [8]. The structure of several complexes of NDP kinases with nucleoside diphosphates [7±10] provides a precise picture of the binding mode of a nucleotide in the active site. In all cases, the base is near the protein surface while the ribose and phosphate moieties are located deeper inside the active site. Of particular interest is the 3 H -OH group of the sugar, which is involved in a network of hydrogen bonds with conserved residues of the acti...