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The Escherichia coli TrmB protein and its Saccharomyces cerevisiae ortholog Trm8p catalyze the S-adenosyl-L-methionine-dependent formation of 7-methylguanosine at position 46 (m7G46) in tRNA. To learn more about the sequence-structure-function relationships of these enzymes we carried out a thorough bioinformatics analysis of the tRNA:m7G methyltransferase (MTase) family to predict sequence regions and individual amino acid residues that may be important for the interactions between the MTase and the tRNA substrate, in particular the target guanosine 46. We used site-directed mutagenesis to construct a series of alanine substitutions and tested the activity of the mutants to elucidate the catalytic and tRNA-recognition mechanism of TrmB. The functional analysis of the mutants, together with the homology model of the TrmB structure and the results of the phylogenetic analysis, revealed the crucial residues for the formation of the substrate-binding site and the catalytic center in tRNA:m7G MTases.

Forty-four sequences of ornithine carbamoyltransferases (OTCases) and 33 sequences of aspartate carbamoyltransferases (ATCases) representing the three domains of life were multiply aligned and a phylogenetic tree was inferred from this multiple alignment. The global topology of the composite rooted tree (each enzyme family being used as an outgroup to root the other one) suggests that present-day genes are derived from paralogous ancestral genes which were already of the same size and argues against a mechanism of fusion of independent modules. A closer observation of the detailed topology shows that this tree could not be used to assess the actual order of organismal descent. Indeed, this tree displays a complex topology for many prokaryotic sequences, with polyphyly for Bacteria in both enzyme trees and for the Archaea in the OTCase tree. Moreover, representatives of the two prokaryotic Domains are found to be interspersed in various combinations in both enzyme trees. This complexity may be explained by assuming the occurrence of two subfamilies in the OTCase tree (OTC alpha and OTC beta) and two other ones in the ATCase tree (ATC I and ATC II). These subfamilies could have arisen from duplication and selective losses of some differentiated copies during the successive speciations. We suggest that Archaea and Eukaryotes share a common ancestor in which the ancestral copies giving the present-day ATC II/OTC beta combinations were present, whereas Bacteria comprise two classes: one containing the ATC II/OTC alpha combination and the other harboring the ATC I/OTC beta combination. Moreover, multiple horizontal gene transfers could have occurred rather recently amongst prokaryotes. Whichever the actual history of carbamoyltransferases, our data suggest that the last common ancestor to all extant life possessed differentiated copies of genes coding for both carbamoyltransferases, indicating it as a rather sophisticated organism.

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