Biological Systems Discovery In Silico: Radical S -Adenosylmethionine Protein Families and Their Target Peptides for Posttranslational Modification

Abstract

Data mining methods in bioinformatics and comparative genomics commonly rely on working definitions of protein families from prior computation. Partial phylogenetic profiling (PPP), by contrast, optimizes family sizes during its searches for the cooccurring protein families that serve different roles in the same biological system. In a large-scale investigation of the incredibly diverse radical S-adenosylmethionine (SAM) enzyme superfamily, PPP aided in building a collection of 68 TIGRFAMs hidden Markov models (HMMs) that define nonoverlapping and functionally distinct subfamilies. Many identify radical SAM enzymes as molecular markers for multicomponent biological systems; HMMs defining their partner proteins also were constructed. Newly found systems include five groupings of protein families in which at least one marker is a radical SAM enzyme while another, encoded by an adjacent gene, is a short peptide predicted to be its substrate for posttranslational modification. The most prevalent, in over 125 genomes, featuring a peptide that we designate SCIFF (six cysteines in forty-five residues), is conserved throughout the class Clostridia, a distribution inconsistent with putative bacteriocin activity. A second novel system features a tandem pair of putative peptidemodifying radical SAM enzymes associated with a highly divergent family of peptides in which the only clearly conserved feature is a run of His-Xaa-Ser repeats. A third system pairs a radical SAM domain peptide maturase with selenocysteine-containing targets, suggesting a new biological role for selenium. These and several additional novel maturases that cooccur with predicted target peptides share a C-terminal additional 4Fe4S-binding domain with PqqE, the subtilosin A maturase AlbA, and the predicted mycofactocin and Nif11-class peptide maturases as well as with activators of anaerobic sulfatases and quinohemoprotein amine dehydrogenases. Radical SAM enzymes with this additional domain, as detected by TIGR04085, significantly outnumber lantibiotic synthases and cyclodehydratases combined in reference genomes while being highly enriched for members whose apparent targets are small peptides. Interpretation of comparative genomics evidence suggests unexpected (nonbacteriocin) roles for natural products from several of these systems.

X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification

“…According to the designation by TIGR04085, the SPASM domain initiates at C261, not C255 (the first Fe ligating cysteine in anSMEcpe). However, both cysteines are conserved in anSMEs and it is common for SPASM domain-containing proteins to have a proximal upstream cysteine (22). In the anSMEcpe structures, C255 and C261 ligate the first auxiliary cluster, Auxiliary Cluster I (Aux I), before the backbone folds into a beta hairpin (V266-Y274).…”

“…In the case of BtrN, one auxiliary cluster has been identified (21), while anSMEs have two (10,17). For anSME, the sequence surrounding these two clusters, including a previously identified 7-cysteine motif (CX 9-15 GX 4 C-gap-CX 2 CX 5 CX 3 C-gap-C) (17), places it in a ∼1,400-membered AdoMet radical subfamily that was recently described by Haft and Basu through bioinformatic analysis and thought to function in the modification of ribosomally translated peptides (22). This subfamily has been designated TIGR04085 and named SPASM for its biochemically characterized founding members AlbA, PqqE, anSMEs, and MtfC, which are involved in subtilosin A, pyrroloquinoline quinone, anaerobic sulfatase, and mycofactocin maturation, respectively (22,23).…”

“…For anSME, the sequence surrounding these two clusters, including a previously identified 7-cysteine motif (CX 9-15 GX 4 C-gap-CX 2 CX 5 CX 3 C-gap-C) (17), places it in a ∼1,400-membered AdoMet radical subfamily that was recently described by Haft and Basu through bioinformatic analysis and thought to function in the modification of ribosomally translated peptides (22). This subfamily has been designated TIGR04085 and named SPASM for its biochemically characterized founding members AlbA, PqqE, anSMEs, and MtfC, which are involved in subtilosin A, pyrroloquinoline quinone, anaerobic sulfatase, and mycofactocin maturation, respectively (22,23). While the function of these auxiliary clusters is unknown, the 7-cysteine motif prompted speculation that members of the SPASM subfamily, including anSMEs, use an available ligation site on one of the [4Fe-4S] clusters for substrate binding (10).…”

X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry

“…SPASM motifs are found in more than 280 AdoMet radical enzymes, all of which are putatively involved in the maturation of ribosomally translated peptides (21). The name SPASM derives from the biochemically characterized members of this subfamily, AlbA (22), PqqE (23), anSMEs (9,19), and MtfC (24), involved in subtilosin A, pyrroloquinoline quinone, anaerobic sulfatase, and mycofactocin maturation, respectively.…”

“…These two Aux clusters in anSMEs are surrounded by a C-terminal sequence motif, "CX 9-15 GX 4 C-gap-CX 2 CX 5 CX 3 Cgap-C" (21), that is referred to as a SPASM motif or domain. SPASM motifs are found in more than 280 AdoMet radical enzymes, all of which are putatively involved in the maturation of ribosomally translated peptides (21).…”