Metabolic Pathfinding Using RPAIR Annotation

Metabolic databases contain information about thousands of small molecules and reactions, which can be represented as networks. In the context of metabolic reconstruction, pathways can be inferred by searching optimal paths in such networks. A recurrent problem is the presence of pool metabolites (e.g., water, energy carriers, and cofactors), which are connected to hundreds of reactions, thus establishing irrelevant shortcuts between nodes of the network. One solution to this problem relies on weighted networks to penalize highly connected compounds. A more refined solution takes the chemical structure of reactants into account in order to differentiate between side and main compounds of a reaction. Thanks to an intensive annotation effort at KEGG, decompositions of reactions into reactant pairs (RPAIR) categorized by their role (main, trans, cofac, ligase, and leave) are now available.The goal of this article is to evaluate the impact of RPAIR data on pathfinding in metabolic networks. To this end, we measure the impact of different parameters concerning the construction of the metabolic network: mapping of reactions and reactant pairs onto a graph, use of selected categories of reactant pairs, weighting schemes for compounds and reactions, removal of highly connected metabolites, and reaction directionality. In total, we tested 104 combinations of parameters and identified their optimal values for pathfinding on the basis of 55 reference pathways from three organisms.The best-performing metabolic network combines the biochemical knowledge encoded by KEGG RPAIR with a weighting scheme penalizing highly connected compounds. With this network, we could recover reference pathways from Escherichia coli with an average accuracy of 93% (32 pathways), from Saccharomyces cerevisiae with an average accuracy of 66% (11 pathways), and from humans with an average accuracy of 70% (12 pathways). Our pathfinding approach is available as part of the Network Analysis Tools.     

New developments in developmental biology

A report on the 15th International Society of Developmental Biologists Congress, Sydney, Australia, 3-7 September 2005.     

Phytanoyl-CoA hydroxylase: recognition of 3-methyl-branched acyl-coAs and requirement for GTP or ATP and Mg(2+) in addition to its known hydroxylation cofactors

Phytanoyl-CoA hydroxylase is a peroxisomal alpha-oxidation enzyme that catalyzes the 2-hydroxylation of 3-methyl-branched acyl-CoAs. A polyhistidine-tagged human phytanoyl-CoA hydroxylase was expressed in E. coli and subsequently purified as an active protein. The recombinant enzyme required GTP or ATP and Mg(2+), in addition to its known cofactors Fe(2+), 2-oxoglutarate, and ascorbate. The enzyme was active towards phytanoyl-CoA and 3-methylhexadecanoyl-CoA, but not towards 3-methylhexadecanoic acid. Racemic, R- and S-3-methylhexadecanoyl-CoA were equally well hydroxylated. Hydroxylation of R- and S-3-methylhexadecanoyl-CoA yielded the (2S, 3R) and (2R,3S) isomers of 2-hydroxy-3-methylhexadecanoyl-CoA, respectively. Human phytanoyl-CoA hydroxylase did not show any activity towards 2-methyl- and 4-methyl-branched acyl-CoAs or towards long and very long straight chain acyl-CoAs, excluding a possible role for the enzyme in the formation of 2-hydroxylated and odd-numbered straight chain fatty acids, which are abundantly present in brain. In conclusion, we report the unexpected requirement for ATP or GTP and Mg(2+) of phytanoyl-CoA hydroxylase in addition to the known hydroxylation cofactors. Due to the fact that straight chain fatty acyl-CoAs are not a substrate for phytanoyl-CoA hydroxylase, 2-hydroxylation of fatty acids in brain can be allocated to a different enzyme/pathway.     

Complete nucleotide sequences of bovine alpha S2- and beta-casein cDNAs: comparisons with related sequences in other species

The nucleotide sequences corresponding to bovine alpha S2- and beta- casein mRNAs have been determined by cDNA analysis. Both sequences appear to be complete at their 5' ends. The nucleotide sequence of alpha S2-casein, when compared with the corresponding cavine A sequence, helps to define the boundaries of a large amino acid repeat (approximately 80 residues) whereas comparisons with the nucleotide sequences of rat gamma- and mouse epsilon-casein mRNAs also reveal extensive sequence similarities. An alignment of these four sequences shows that the divergence of their translated regions has been characterized by the duplication and deletion of discrete segments of sequence that probably correspond to exons. A high degree of nucleotide substitution is also found when the four sequences are compared, except for well-conserved leader-peptide and phosphorylation-site sequences and, to a lesser extent, the 5'-untranslated regions. Similar comparison of the bovine and rat beta-caseins shows that their divergence has involved a high rate of nucleotide substitution but that no major insertions or deletions of sequence have occurred. The several splice sites that have veen defined in the rat beta-casein gene are likely to have been conserved in the bovine. The contrasting evolutionary histories of the alpha- and beta-casein coding sequences correlate with the distinctive functions of these proteins in the casein micelle system in milk.      

Control of thiophene accumulation in calli of two Tagetes species

The relation between root differentiation and accumulation of biocidal thiophenes was studied in stem calli of two Tagetes species. Disorganized tissues of T. erecta were low in thiophene production. A sharp rise in thiophene content coincided with the emergence of roots on the calli. Root regeneration and the amount of thiophenes produced were found to be quantitatively related. Callus tissues of T. minuta did not differentiate into organs. Nevertheless, they accumulated thiophenes throughout the incubation period. Light at 12 W m^- stimulated thiophene production in T. erecta without having an apparent effect on root regeneration. In T. minuta even low irradiance levels (2 W m∼²) strongly inhibited thiophene accumulation. Under favorable conditions thiophene concentrations in calli of both species were comparable to or somewhat lower than the levels in roots on the plants and in excised, cultured roots of T. erecta. We conclude that in calli of T. erecta thiophene accumulation is coupled to root regeneration whereas a different control mechanism allowing for accumulation in disorganized tissues is operative in T. minuta.     

Metabolism of 1-naphthaleneacetic acid in explants of tobacco: Evidence for release of free hormone from conjugates

1-Naphthaleneacetic acid (1-NAA), required for in vitro flower bud formation, was taken up by pedicel explants of tobacco (Nicotiana tabacum L.) in large amounts and rapidly metabolized into various conjugates. These conjugates have been tentatively identified in four thin-layer Chromatographic systems using authentic standards as references. The major metabolite formed during the first hours of culture comigrated with 1-NAA-glucoside (1-NAGlu). From the 6th hour on, most 1-NAA had been converted into a yet unidentified metabolite. 1-NAglu was an intermediate in the formation of this metabolite. After 24 h, 1-NAA-aspartate (1-NAAsp) became the second major metabolite. The increase in 1-NAAsp formation was induced by 1-NAA. The inactive analog 2-naphthaleneacetic acid (2-NAA) was metabolized similar to 1-NAA, but was unable to increase the formation of the aspartate conjugate. When explants were fed labeled 1-NAGlu, 1-NAAsp or the major unidentified metabolite, radioactivity became associated with free 1-NAA and all major conjugates, indicating interconversion of conjugates and breakdown to free 1-NAA. A regulatory role of conjugation in maintaining a particular level of free 1-NAA in the tissue is proposed herein.     

Amino acid uptake and protein synthesis during early meiosis in Saccharomyces cerevisiae

Uptake and incorporation into proteins of an externally supplied amino acid were followed during early meiosis in yeast. Under conditions optimal for development, an insufficient permeability of the cell leads to an incorporation pattern which reflects the changes in the activity of the amino acid transporting system rather than those in protein synthesis. A more correct picture of protein synthesis during early meiosis is obtained by the use of a mutant with an enhanced level of amino acid uptake.Abbreviation SPM Sporulation medium