Abstract
AMPylation is a novel post-translational modification (PTM) involving covalent attachment of an AMP moiety to threonine/tyrosine side chains of a protein. AMPylating enzymes belonging to three different families, namely Fic/Doc, GS-ATase and DrrA have been experimentally characterized. Involvement of these novel enzymes in a myriad of biological processes makes them interesting candidates for genome-wide search. We have used SVM and HMM to develop a computational protocol for identification of AMPylation domains and their classification into various functional subfamilies catalyzing AMPylation, deAMPylation, phosphorylation and phosphocholine transfer. Our analysis has not only identified novel PTM catalyzing enzymes among unannotated proteins, but has also revealed how this novel enzyme family has evolved to generate functional diversity by subtle changes in sequence/structures of the proteins. Phylogenetic analysis of Fic/Doc has revealed three new isofunctional subfamilies, thus adding to their functional divergence. Also, frequent occurrence of Fic/Doc proteins on highly mobile and unstable genomic islands indicated their evolution via extensive horizontal gene transfers. On the other hand phylogenetic analyses indicate lateral evolution of GS-ATase family and an early duplication event responsible for AMPylation and deAMPylation activity of GS-ATase. Our analysis also reveals molecular basis of substrate specificity of DrrA proteins.
Highlights
AMPylation or adenylylation is a post-translational modification (PTM) involving the covalent attachment of AMP moiety onto the side chains of threonine/tyrosine residues of proteins[1]
Even though iterative profile based searches increases the repertoire of Fic/Doc proteins compared to pair-wise BLAST search, it is often found that such methods cannot distinguish between the subfamilies
We wanted to investigate if machine learning based SVM classifiers and sensitive profile based Hidden Markov Models (HMM) can be used for identification and classification of AMPylation domains
Summary
AMPylation or adenylylation is a post-translational modification (PTM) involving the covalent attachment of AMP moiety onto the side chains of threonine/tyrosine residues of proteins[1]. It has been shown that AMPylation is catalyzed by three different families of enzymes, namely, Fic (Filamentation induced by cAMP), DrrA and GS-ATase (Glutamine Synthetase Adenylyltransferase)[2,5,6]. Recent studies have shown that Doc domains catalyze phosphorylation[15,16] reaction similar to kinases Another type III effector protein, avirulence protein B (AvrB) from Pseudomonas syringae shares structural similarity with Fic/Doc family of proteins. Analysis of phylogeny and synteny of these AMPylation domains suggested evolution of AMPylating enzymes, Fic domains by extensive horizontal gene transfer This was confirmed by the presence of many Fic proteins on highly unstable Genomic Islands. We have attempted to decipher sequence determinants of the substrate specificity of DrrA type AMPylation domains towards a variety of Rab proteins
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