Abstract
BackgroundEnzymes in the radical SAM (rSAM) domain family serve in a wide variety of biological processes, including RNA modification, enzyme activation, bacteriocin core peptide maturation, and cofactor biosynthesis. Evolutionary pressures and relationships to other cellular constituents impose recognizable grammars on each class of rSAM-containing system, shaping patterns in results obtained through various comparative genomics analyses.ResultsAn uncharacterized gene cluster found in many Actinobacteria and sporadically in Firmicutes, Chloroflexi, Deltaproteobacteria, and one Archaeal plasmid contains a PqqE-like rSAM protein family that includes Rv0693 from Mycobacterium tuberculosis. Members occur clustered with a strikingly well-conserved small polypeptide we designate "mycofactocin," similar in size to bacteriocins and PqqA, precursor of pyrroloquinoline quinone (PQQ). Partial Phylogenetic Profiling (PPP) based on the distribution of these markers identifies the mycofactocin cluster, but also a second tier of high-scoring proteins. This tier, strikingly, is filled with up to thirty-one members per genome from three variant subfamilies that occur, one each, in three unrelated classes of nicotinoproteins. The pattern suggests these variant enzymes require not only NAD(P), but also the novel gene cluster. Further study was conducted using SIMBAL, a PPP-like tool, to search these nicotinoproteins for subsequences best correlated across multiple genomes to the presence of mycofactocin. For both the short chain dehydrogenase/reductase (SDR) and iron-containing dehydrogenase families, aligning SIMBAL's top-scoring sequences to homologous solved crystal structures shows signals centered over NAD(P)-binding sites rather than over substrate-binding or active site residues. Previous studies on some of these proteins have revealed a non-exchangeable NAD cofactor, such that enzymatic activity in vitro requires an artificial electron acceptor such as N,N-dimethyl-4-nitrosoaniline (NDMA) for the enzyme to cycle.ConclusionsTaken together, these findings suggest that the mycofactocin precursor is modified by the Rv0693 family rSAM protein and other enzymes in its cluster. It becomes an electron carrier molecule that serves in vivo as NDMA and other artificial electron acceptors do in vitro. Subclasses from three different nicotinoprotein families show "only-if" relationships to mycofactocin because they require its presence. This framework suggests a segregated redox pool in which mycofactocin mediates communication among enzymes with non-exchangeable cofactors.
Highlights
Enzymes in the radical SAM domain family serve in a wide variety of biological processes, including RNA modification, enzyme activation, bacteriocin core peptide maturation, and cofactor biosynthesis
Partial Phylogenetic Profiling (PPP), a data mining tool that searches for phylogenetically correlated proteins by tuning sets of working definitions of families centered on those proteins for optimal match to the query profile [8], revealed strong matches to several neighboring proteins, including a glycosyltransferase (Rv0696) and a protein of unknown function (Rv0692) with no homologs outside of these neighborhoods
Among the 1450 genomes included in the PPP data set, we determined that the three always occur together, or not at all, both within and outside the Actinobacteria
Summary
Enzymes in the radical SAM (rSAM) domain family serve in a wide variety of biological processes, including RNA modification, enzyme activation, bacteriocin core peptide maturation, and cofactor biosynthesis. The system will tend not to be essential, so that one strain may have the system while others lack it, yet distantly related species inhabiting similar environments may share it. These and other commonalities reflect underlying constraints on the relationships among the different parts of a cell, a biological grammar that is shared among analogous systems even if they show no common ancestry and no sequence similarities. For sequences outside of the Actinobacteria, these peaks dramatically outscore the apex of the triangular heat map, the single point that represents the full length of the protein. The SIMBAL result, in which a local hotspot outscores the full-length sequence, suggests that PPP may be understating the strength of the linkage between the N-terminal region and the mycofactocin system
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