Comprehensive Structural and Substrate Specificity Classification of the Saccharomyces cerevisiae Methyltransferome

Methylation is one of the most common chemical modifications of biologically active molecules and it occurs in all life forms. Its functional role is very diverse and involves many essential cellular processes, such as signal transduction, transcriptional control, biosynthesis, and metabolism. Here, we provide further insight into the enzymatic methylation in S. cerevisiae by conducting a comprehensive structural and functional survey of all the methyltransferases encoded in its genome. Using distant homology detection and fold recognition, we found that the S. cerevisiae methyltransferome comprises 86 MTases (53 well-known and 33 putative with unknown substrate specificity). Structural classification of their catalytic domains shows that these enzymes may adopt nine different folds, the most common being the Rossmann-like. We also analyzed the domain architecture of these proteins and identified several new domain contexts. Interestingly, we found that the majority of MTase genes are periodically expressed during yeast metabolic cycle. This finding, together with calculated isoelectric point, fold assignment and cellular localization, was used to develop a novel approach for predicting substrate specificity. Using this approach, we predicted the general substrates for 24 of 33 putative MTases and confirmed these predictions experimentally in both cases tested. Finally, we show that, in S. cerevisiae, methylation is carried out by 34 RNA MTases, 32 protein MTases, eight small molecule MTases, three lipid MTases, and nine MTases with still unknown substrate specificity

Influence of Soil-Cement Composition on its Selected Properties

The paper discusses the results of mechanical and technological tests of soil-cement composites made with cohesive soil. The compositions of analysed soil-cement mixtures differed in terms of their cement paste volume fractions and water-cement ratios. Limiting values of these technological parameters that enable the application of the soil-cement mixtures obtained in real life conditions for the purposes of the Deep Soil Mixing (DSM) method were determined. Based on the test results obtained, it was found that mechanical properties of the materials analysed were very sensitive to changes in their compositions. Variations in the volume fraction of cement paste within the range analysed caused mechanical properties to change even by an order of magnitude

Domain architecture of <i>S. cerevisiae</i> MTases.

<p>The MTases were grouped according to their common substrate specificities (e.g., protein, RNA, small molecule or lipid) and the fold of catalytic domain. Known MTases with experimentally determined substrate specificity are shown in a regular font, putative MTases in italics, and newly detected MTase in bold. Non-periodic MTases are underlined. The new domains identified in this study are marked with a red asterisk. Sandwich, beta sandwich; Xyl TIM, TIM beta/alpha-barrel belonging to the Xylose isomerase-like superfamily; Alpha, α-helical domain; Ankyrin, Ankyrin repeats; ZnF, zinc finger; Spb1C, Spb1 C-terminal domain; Defensin, defensin-like fold; iSET, SET-inserted domain; Rubisco, Rubisco LSMT C-terminal-like domain; SRI, SET2 Rpb1 interacting domain; PHD, PHD zinc finger; DNA/RNA, DNA/RNA-binding 3-helical bundle; RNase H, RNase H-like domain; ARM, ARM repeat; RNA_rb, RNA ribose binding domain; SirohemeN, Siroheme synthase N-terminal domain-like; SirohemeM, Siroheme synthase middle domain-like; Cobal_N, Cobalamin-independent synthase N-terminal domain; CoCoA_N, Calcium binding and coiled-coil domain-like (N-terminal); OB, OB-fold domain; RNAb, RNA binding domain.</p

YBR271W and YLR285W (NNT1) are protein MTases.

<p>Recombinant proteins (MTases) were incubated with native yeast extracts from the respective knockout strains (ΔMTase ext) and [³H] AdoMet (lane 1). Reaction products were resolved on SDS-PAGE gel and exposed to tritium screen. To test the specificity of these reactions, analyzed proteins were also incubated with yeast extract from the wild-type strain (wt ext) and [³H] AdoMet (lane 2). As a control, yeast extracts from knockout and wild-type strains were incubated with [³H] AdoMet only (lanes 3 and 4). HMT1 (a protein MTase) and TRM4 (an RNA MTase) were used as positive and negative controls, respectively.</p

Hierarchical clustering tree for all <i>S. cerevisiae</i> periodic MTases.

<p>Seventy-two periodic MTases were divided into five clusters, each containing MTases with similar expression profiles during the Yeast Metabolic Cycle (YMC). Branch lengths correspond to correlation coefficients of gene expression profiles during the YMC obtained from SCEPTRANS.</p

Comprehensive picture of the <i>S. cerevisiae</i> methyltransferome.

<p>(A) Structural (fold) and substrate specificity classifications. Eighty-six MTases (known MTases with experimentally verified activity and putative MTases identified in previous bioinformatic studies, including one newly detected here) were divided into several groups based on similarity of their structure (within catalytic domain) and substrate. (B) Detailed structural vs. substrate specificity classification.</p

Putative MTase YIL096C binds AdoMet.

<p>Purified YIL096C (with HIStagSUMO), HTM1 and TEV protease were exposed to UV light in the presence of [³H] AdoMet. Both Coomassie stained proteins (left panel) and the autoradiography of crosslink products (right panel) are shown. HMT1 (known MTase) and TEV protease were used as positive and negative controls, respectively.</p

The <i>S. cerevisiae</i> methyltransferome.

<p>MTases are grouped according to the structural similarity of their catalytic domains (Fold) and substrate specificity (Substrate). Known MTases with experimentally determined substrate specificity are shown in regular font, putative MTases are italicized, and the newly detected MTase is highlighted in bold. Non-periodic MTases are underlined. With the exception of ^aDNA, containing 6-O-methylguanine, ^b5-methyltetrahydropteroyltri-L-glutamate, and ^c5,10-methylenetetrahydrofolate, AdoMet is the methyl group donor.</p