Light-dependent subcellular localization of nucleoside diphosphate kinase-1 in Neurospora crassa

Nucleoside diphosphate kinase (NDK) is a housekeeping enzyme localized in cellular organelles and distributed in various organs in prokaryotes and eukaryotes. In Neurospora crassa, NDK-1 is suggested to control catalases in response to heat, oxidative stress and light. In this study, we identified the presence of NDK-1 during most developmental stages in submerged mycelia, aerial hyphae, asexual conidia and perithecia, and the localization of it in soluble, mitochondrial, nuclear and membrane fractions in the mycelial cell. A light-dependent localization of NDK-1 was shown by Western blotting and immunohistochemical analysis using anti-NDK-1 antibody. In the mycelia, NDK-1 was compartmentalized on the plasma membrane in darkness, while it was relocated in the cytoplasm under light. These results suggest that NDK-1 protein was translocated from the plasma membrane to cytoplasm in response to light, and may interact with catalase.     

The nucleoside diphosphate kinase from mimivirus: a peculiar affinity for deoxypyrimidine nucleotides

The first viral Nucleoside Diphosphate Kinase was recently identified in the giant double-stranded DNA virus Acanthamoeba polyphag a Mimivirus (ApM). Here we report its expression and detailed biochemical characterization. NDK_apm exhibits unique features such as a shorter Kpn-loop, a structural motif previously reported to be part of the active site and involved in oligomer formation. Enzymatic activity measurements on the recombinant NDK_apm revealed its preferential affinity for deoxypyrimidine nucleotides. This property might represent an adaptation of NDK_apm to the production of the limiting TTP deoxynucleotide required for the replication of the large A+T rich (72%) viral genome. The NDK_apm might also assume a role in dUTP detoxification to compensate for the surprising absence of Mimivirus dUTPase (deoxyuridine triphosphate pyrophosphatase) an important enzyme conserved in most viruses. Although the phylogenetic analysis of NDK sequences sampled through organisms from the three domains of life is only partially informative, it favors an ancestral origin for NDK_apm over a recent acquisition from a eukaryotic organism by horizontal gene transfer.     

Theoretical model of interactions between ligand-binding sites in a dimeric protein and its application for the analysis of thiamine diphosphate binding to yeast transketolase

The binding of thiamin diphosphate (ThDP) to yeast dimeric apotransketolase (apoTK) is accompanied by the appearance of a band in the absorption spectrum with maximum at 320 nm. The saturation function has been analyzed using a scheme that involves binding of ThDP to each subunit followed by the conformational transition of this subunit. It is assumed that the binding of ThDP to one subunit may affect the conformational transition of the other subunit. Rigorous mathematical expressions describing the dependence of the optical absorption on the total concentration of ThDP are first developed. Equilibrium constants and corresponding rate constants for the binding of ThDP to apoTK have been estimated. The negative cooperativity in the ThDP binding has been characterized by the function reflecting the dependence of the conformational change on the saturation of apoTK by ThDP.     

Synthesis with good enantiomeric excess of both enantiomers of α-ketols and acetolactates by two thiamin diphosphate-dependent decarboxylases

In addition to the decarboxylation of 2-oxo acids, thiamin diphosphate (ThDP)-dependent decarboxylases/dehydrogenases can also carry out so-called carboligation reactions, where the central ThDP-bound enamine intermediate reacts with electrophilic substrates. For example, the enzyme yeast pyruvate decarboxylase (YPDC, from Saccharomyces cerevisiae) or the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1) can produce acetoin and acetolactate, resulting from the reaction of the central thiamin diphosphate-bound enamine with acetaldehyde and pyruvate, respectively. Earlier, we had shown that some active center variants indeed prefer such a carboligase pathway to the usual one [Sergienko, Jordan, Biochemistry 40 (2001) 7369–7381; Nemeria et al., J. Biol. Chem. 280 (2005) 21,473–21,482]. Herein is reported detailed analysis of the stereoselectivity for forming the carboligase products acetoin, acetolactate, and phenylacetylcarbinol by the E477Q and D28A YPDC, and the E636A and E636Q PDHc-E1 active-center variants. Both pyruvate and β-hydroxypyruvate were used as substrates and the enantiomeric excess was analyzed by a combination of NMR, circular dichroism and chiral-column gas chromatographic methods. Remarkably, the two enzymes produced a high enantiomeric excess of the opposite enantiomer of both acetoin-derived and acetolactate-derived products, strongly suggesting that the facial selectivity for the electrophile in the carboligation is different in the two enzymes. The different stereoselectivities exhibited by the two enzymes could be utilized in the chiral synthesis of important intermediates.     

Covalent Binding of 1,N 6-ethenoadenosine diphosphate to catalytic and noncatalytic sites of chloroplast ATP synthase

Catalytic and noncatalytic sites of the chloroplast coupling factor (CF₁) were selectively modified by incubation with the dialdehyde derivative of fluorescent adenosine diphosphate analog 1,N⁶-ethenoadenosine diphosphate. The modified CF₁ was reconstituted with EDTA-treated thylakoid membranes of chloroplasts. The effects of light-induced transmembrane proton gradient and phosphate ions on the fluorescence of 1,N⁶-ethenoadenosine diphosphate, covalently bound to the catalytic sites of ATP synthase, were studied. Quenching of fluorescence of covalently bound 1,N⁶-ethenoadenosine diphosphate was observed under illumination of thylakoid membranes with saturating white light. Addition of inorganic phosphate to the reaction mixture in the dark increased the fluorescence of the label. Quenching reappeared under repeated illumination; however, addition of phosphate ions had no effect on the fluorescence yield in this case. When 1,N⁶-ethenoadenosine diphosphate was covalently bound to noncatalytic sites of ATP synthase, no similar fluorescence changes were observed. The relation between the observed changes of 1,N⁶-ethenoadenosine diphosphate fluorescence and the mechanism of energy-dependent structural changes in the catalytic site of ATP synthase is discussed.     

Structure-based phylogeny of polyene macrolide antibiotic glycosyltransferases

Antibiotic glycosyltransferases (AGts) attach unusual deoxy-sugars to aglycons so antibiotics can exert function. It has been reported that polyene macrolide (PEM) AGts have different evolutionary origin when compared with other polyketide AGts, and our previous analysis have suggested that they could be results of horizontal gene transfer (HGT) from eukaryotes. In this paper, we compared the structures of PEM AGts with structures of eukaryotes and other AGts, and then built models of the representative PEM AGts and GT-1 glycosyltransferases. We also constructed the Neighbor-Joining (NJ) trees based on the normalized Root Mean Square (RMS) distance, the Bayesian tree guided by structural alignments, and carried out analysis on several key conserved residues in PEM AGts. The NJ tree showed a close relationship between PEM AGts and eukaryotic glycosyltransferases, and Bayesian tree further supported their affinity with UDP-glucuronosyltransferases (UGTs). Analysis on key conserved residues showed that PEM AGts may have similar interaction mechanism such as in the formation of hydrogen bonds as eukaryotic glycosyltransferases. Using structure-based phylogenetic approaches, this study further supported that PEM AGts were the result of HGT between prokaryotes and eukaryotes.     

Gene network reconstruction identifies the authentic trans-prenyl diphosphate synthase that makes the solanesyl moiety of ubiquinone-9 in Arabidopsis

Ubiquinone (coenzyme Q) is the generic name of a class of lipid-soluble electron carriers formed of a redox active benzoquinone ring attached to a prenyl side chain. The length of the latter varies among species, and depends upon the product specificity of a trans-long-chain prenyl diphosphate synthase that elongates an allylic diphosphate precursor. In Arabidopsis, this enzyme is assumed to correspond to an endoplasmic reticulum-located solanesyl diphosphate synthase, although direct genetic evidence was lacking. In this study, the reconstruction of the functional network of Arabidopsis genes linked to ubiquinone biosynthesis singled out an unsuspected solanesyl diphosphate synthase candidate--product of gene At2g34630--that, extraordinarily, had been shown previously to be targeted to plastids and to contribute to the biosynthesis of gibberellins. Green fluorescent protein (GFP) fusion experiments in tobacco and Arabidopsis, and complementation of a yeast coq1 knockout lacking mitochondrial hexaprenyl diphosphate synthase demonstrated that At2g34630 is also targeted to mitochondria. At2g34630 is the main--if not sole--contributor to solanesyl diphosphate synthase activity required for the biosynthesis of ubiquinone, as demonstrated by the dramatic (75-80%) reduction of the ubiquinone pool size in corresponding RNAi lines. Overexpression of At2g34630 gave up to a 40% increase in ubiquinone content compared to wild-type plants. None of the silenced or overexpressing lines, in contrast, displayed altered levels of plastoquinone. Phylogenetic analyses revealed that At2g34630 is the only Arabidopsis trans-long-chain prenyl diphosphate synthase that clusters with the Coq1 orthologs involved in the biosynthesis of ubiquinone in other eukaryotes.     

NMR for direct determination of Km and Vmax of enzyme reactions based on the Lambert W function-analysis of progress curves

¹H NMR spectroscopy was used to follow the cleavage of sucrose by invertase. The parameters of the enzyme's kinetics, K_m and V_max, were directly determined from progress curves at only one concentration of the substrate. For comparison with the classical Michaelis-Menten analysis, the reaction progress was also monitored at various initial concentrations of 3.5 to 41.8 mM. Using the Lambert W function the parameters K_m and V_max were fitted to obtain the experimental progress curve and resulted in K_m = 28 mM and V_max = 13 μM/s. The result is almost identical to an initial rate analysis that, however, costs much more time and experimental effort. The effect of product inhibition was also investigated. Furthermore, we analyzed a much more complex reaction, the conversion of farnesyl diphosphate into (+)-germacrene D by the enzyme germacrene D synthase, yielding K_m = 379 μM and k_cat = 0.04 s^− 1. The reaction involves an amphiphilic substrate forming micelles and a water insoluble product; using proper controls, the conversion can well be analyzed by the progress curve approach using the Lambert W function.     

The structural basis of chain length control in Rv1086

In Mycobacterium tuberculosis, two related Z-prenyl diphosphate synthases, E,Z-farnesyl diphosphate synthase (Rv1086) and decaprenyl diphosphate synthase (Rv2361c), work in series to synthesize decaprenyl phosphate (C₅₀) from isopentenyl diphosphate and E-geranyl diphosphate. Decaprenyl phosphate plays a central role in the biosynthesis of essential mycobacterial cell wall components, such as the mycolyl-arabinogalactan-peptidoglycan complex and lipoarabinomannan; thus, its synthesis has attracted considerable interest as a potential therapeutic target. Rv1086 is a unique prenyl diphosphate synthase in that it adds only one isoprene unit to geranyl diphosphate, generating the 15-carbon product (E,Z-farnesyl diphosphate). Rv2361c then adds a further seven isoprene units to E,Z-farnesyl diphosphate in a processive manner to generate the 50-carbon prenyl diphosphate, which is then dephosphorylated to generate a carrier for activated sugars. The molecular basis for chain-length discrimination by Rv1086 during synthesis is unknown. We also report the structure of apo Rv1086 with citronellyl diphosphate bound and with the product mimic E,E-farnesyl diphosphate bound. We report the structures of Rv2361c in the apo form, with isopentenyl diphosphate bound and with a substrate analogue, citronellyl diphosphate. The structures confirm the enzymes are very closely related. Detailed comparison reveals structural differences that account for chain-length control in Rv1086. We have tested this hypothesis and have identified a double mutant of Rv1086 that makes a range of longer lipid chains.