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(The FASEB Journal. 2000;14:2365-2368.)
© 2000 FASEB

Phylogenomic analysis of 16S rRNA:(guanine-N2) methyltransferases suggests new family members and reveals highly conserved motifs and a domain structure similar to other nucleic acid amino-methyltransferases

JANUSZ M. BUJNICKI1

Bioinformatics Unit, International Institute of Molecular and Cell Biology, 02–109 Warsaw, Poland; and Molecular Biology Research Program, Henry Ford Health System, Detroit, Michigan 48202, USA

1Correspondence: Bioinformatics Unit, International Institute of Molecular and Cell Biology, ul. ks. Trojdena 4, 02–109 Warsaw, Poland. E-mail: iamb{at}bioinfo.pl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 REFERENCES
 
The sequences of known Escherichia coli 16S rRNA:m2G1207 methyltransferase (MTase) RsmC and hypothetical 16S rRNA:m2G966 MTase encoded by the ygjo open reading frame were used to carry out a database search of other putative m2G-generating enzymes in finished and unfinished genomic sequences. Sequence comparison and phylogenetic analysis of 21 close homologs of RsmC and YgjO revealed the presence of the third paralogous lineage in E. coli and other {gamma}-Proteobacteria, which might correspond to the subfamily of MTases specific for G1516 in 16S rRNA. In addition, the comparative sequence analysis supported by sequence/structure threading suggests that rRNA:m2G MTases are very closely related to RNA and DNA:m6A MTases and that these two enzyme families share common architecture of the active site and presumably a similar mechanism of methyl group transfer onto the exocyclic amino group of their target bases.—Bujnicki, J. M. Phylogenomic analysis of 16S rRNA:(guanine-N2) methyltransferases suggests new family members and reveals highly conserved motifs and a domain structure similar to other nucleic acid amino-methyltransferases.


Key Words: RNA modification • sequence alignment • molecular evolution • structure prediction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
REFERENCES
 
WITH THE ACCUMULATION of sequence information generated by various genome projects, it now becomes possible to predict the function of putative genes based on their homology to the ones characterized experimentally. However, this task is far from trivial for the putative members of the large S-adenosylmethionine (AdoMet) -dependent methyltransferase (MTase) superfamily (comprehensively reviewed in ref 1 ). Although the majority of structurally characterized MTases appear to adopt a very similar 3-dimensional fold, the sequence homologies between the individual members of the superfamily are strikingly low (2 3 4) . Several weakly conserved motifs present among these enzymes have been defined (5 6 7) . Nevertheless, even in cases when the sequence similarities help to guess the likely substrate of a given enzyme (e.g., a nucleic acid base, a protein or some low molecular weight molecule), the precise prediction of reaction specificity usually is hardly possible unless a sufficient number of closely related homologs of known function are found.

This problem is particularly evident for MTases modifying nucleic acids, especially RNA. Many distinct enzymes are present in the cell, catalyzing similar reactions but in different classes of RNA or at different locations in an RNA molecule (8) . The methylated nucleotides are believed to play key roles in the functioning of the ribonucleoprotein particles in vivo, influencing processes such as maturation of various pre-RNAs, stabilizing the assembly and transport of ribosomes and spliceosomes, and modulating splicing and protein synthesis (9) . However, only a limited number of RNA MTases have been identified and characterized to date, and for only a few of them are sequence data available. Recently, iterative database searches coupled to phylogenetic inference have been successfully applied to identify candidates for novel RNA MTases in genomes of organisms from all three domains of life and to predict their function from amino acid sequence (10 11 12) . A similar approach has also been applied to infer the phylogeny of certain DNA MTase families (13 , 14) .

The amino acid sequences of MTases generating N2-methylguanine (m2G) in RNA remained unknown for a long time; only recently have cloning and sequencing of the Escherichia coli gene coding for the 16S rRNA:m2G1207 MTase (RsmC) revealed its identity with the YjjT ORF (putative open reading frame product) and suggested a function of 16S rRNA:m2G966 MTase for the highly homologous YgjO ORF (15) . No candidate for the ‘missing’ 16S rRNA:m2G1516 MTase has been proposed so far.

As a part of a larger project aiming at resolving evolutionary relationships among all nucleic acids-modifying AdoMet-dependent MTases, I used the sequences of RsmC and YgjO to conduct a search of other putative m2G-generating enzymes in finished and unfinished genomic sequences using PSI-BLAST (16) . All retrieved sequences identified as close homologs of RsmC and YgjO were aligned (Fig. 1 ) and their evolutionary history was inferred. Sequence comparison and phylogenetic analysis of 21 putative 16S rRNA:m2G MTases revealed the presence of three paralogous lineages corresponding to close homologs of YjjT (RsmC), YgjO, and YbiN ORFs (21) , exclusively from Proteobacteria of the {gamma}-subdivision (Fig. 2 ), which suggests that the three orthologous subfamilies might correspond to MTases responsible for specific m2G-modification of the three residues in {gamma}-Proteobacterial 16S rRNA (G966, G1207, and G1516 in E. coli). An alternative hypothesis is that YgjO and/or YbiN might correspond to MTases specific for G1835 and/or G2445 in E. coli 23S rRNA (http://medlib.med.utah.edu/RNAmods/rnarrna.htm).



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  		Figure 1. Conserved sequence blocks in the alignment of putative 16S rRNA:m2G MTases. Only representative sequences are shown from the full-length alignment obtained using CLUSTALX (17) and PSI-BLAST (16) . Sequence names are SwissProt-formatted; the ‘unf’ suffix indicates sequences obtained from the unfinished genome data at the NCBI (http://www.ncbi.nlm.nih.gov/). Conserved motifs are labeled according to the nomenclature proposed by Malone et al. (7) . The residues invariant in the m2G MTase family are shown on the black background; residues with invariant physicochemical character (hydrophobic, small, etc.) are shown on the gray background; residues conserved in the majority of sequences are in boldface. The residues functionally conserved between m6A, m4C and m2G MTases and implicated in binding of the cofactor and the target base are indicated by ‘¤’. In the lower panel the sequences of structurally characterized nucleic acid amino-MTases from the {gamma}-family (7) : DNA:m6A MTase M.TaqI (18) , DNA:m4C MTase M.NgoMXV (19) , and RNA:m6A MTase ErmAM (20) are shown for comparison. The consensus secondary structure is shown at the bottom.



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  		Figure 2. Unrooted evolutionary tree of the putative 16S rRNA:m2G MTase family. The phylogram was inferred based on the full-length alignment using the neighbor-joining algorithm (22) . Branch lengths indicate the evolutionary distances among individual proteins estimated using the JTT matrix (23) . The SwissProt accession numbers are shown; the ‘unf’ suffix indicates sequences obtained from the unfinished genome data. The three paralogous families named after the E. coli open reading frames (21) are indicated.

Neither YbiN nor YgjO homologs were detected in genomic sequences of Haemophilus influenzae (24) , Actinobacillus actinomycetemcomitans (sequenced at Oklahoma University http://www.genome.ou.edu/), and Pasteurella multocida (sequenced at University of Minnesota http://www.cbc.umn.edu/). Of these three human pathogens, only H. influenzae genome has been fully sequenced; however, from the present data it seems likely that the genes coding for the two putative rRNA:m2G MTases (and the corresponding enzymatic activities) have been lost from the DNA of the hypothetical ancestor of the Pasteurellaceae family. This hypothesis could be corroborated by the analysis of Proteobacterial rRNA using thin-layer chromatography or other biochemical methods.

Analysis of the alignment revealed the presence of a characteristic hexapeptide (T/S/C)NPPFH in all putative rRNA:m2G MTase sequences (Fig. 1) . This pattern could be useful in discriminating between this family of enzymes and other proteins, whose putative catalytic center exhibits high similarity to the (D/N/S)P(P/I)(Y/W/F/H) pattern defining motif IV of DNA:cytosine-N4 (m4C) and RNA: or DNA:adenine-N6 (m6A) MTases (3 , 7 , 13 , 25) . To put these sequence similarities into the structural context, I performed the threading analysis using FFAS (Fold and Function Assignment System; ref 26 ) for all sequences analyzed herein. They all showed exceptionally high compatibility of structural elements and catalytically important residues with the atomic coordinates of the DNA:m6A MTase TaqI and slightly lower with the RNA:m6A MTase ErmAM (Fig. 1) , suggesting not only a common evolutionary origin, but also a similar mechanism of methyl group transfer onto the exocyclic amino groups of guanine in RNA and adenine in DNA or RNA (27 , 28) . This hypothesis can be confirmed experimentally by site-directed mutagenesis guided by the alignment presented.

All m2G MTases possess a long stretch of nonconserved sequence at the NH2 terminus, which is missing from all amino-MTases structurally characterized to date. Such nonconserved sequence regions are believed to govern target-specificity in both DNA and RNA MTases (3 , 6 , 7 , 11 , 20) . The putative MTases from the HemK family that were hypothesized to methylate exocyclic amino groups of adenine in nucleic acids exhibit a strikingly similar arrangement of conserved and variable regions (25) , which places both MTase families in the {zeta} class of amino-MTases (7) . From the present study and previous analyses (3 , 4 , 7 , 13 , 15 , 18 19 20 , 25 , 27 , 28) , it can be concluded that the (D/N/S)PP(Y/F/H) motif or its relaxed version (see above) is a hallmark of not only DNA:m4C and m6A MTases as previously thought (and how many putative sequence database entries are annotated) but, more generally, of MTases specific for the exocyclic NH2 group in nucleic acids. It remains to be determined whether other amino-methylating enzymes specific for compounds different that nucleic acids (e.g., some yet undiscovered protein or small molecule MTases) also possess some form of that motif.


   ACKNOWLEDGMENTS
 
I would like to thank Drs. Ashok Bhagwat, Robert Blumenthal, and Monika Radlinska for encouragement, helpful discussions stimulating my interest in evolution of nucleic acid methyltransferases, and critical comments. I also thank all the genome sequencing groups that make their preliminary data publicly available, without which this work could not have been done.

Received for publication April 10, 2000. Accepted for publication May 2, 2000.


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