This superfamily comprises structural domains with a topological alpha/beta knot, which is rarely found in protein structures as it poses a folding problem. Members of this superfamily includes SPOUT methyltransferases (such as tRNA:Gm18 MTase TrmH) and tRNA (guanine-N1-)-methyltransferase. The name of the SPOUT superfamily originates from its two founding members, SpoU (now known as TrmH) and TrmD. A characrteristic of this superfamily is the N-terminal Rossmanoidal alpha/beta fold fused to a C-terminal topological knot. This knot forms the binding pocket for the substrate SAM (S-adenosylmethionine), which functions as the methyl donor in the reaction. Most SPOUT methyltransferases are dimeric, with the exception of TrmD.

The TrmH structure has a disc-like shape, composed of a major catalytic domain and a helical subdomain. The catalytic domain consists of a parallel six-stranded beta-sheet, which is flanked by two alpha-helices on one side and by four alpha-helices on the other side. The N-terminal half of the catalytic domain resembles a Rossmann fold or consensus MTase fold, while the C-terminal half has a different topology as compared with that of the consensus MTase fold: it includes a deep trefoil knot. The helical subdomain consists of the N-terminal helix 1 and the C-terminal helix 8, which interact with each other.

Pfam [PfamClan:CL0098], INTERPRO:IPR029026, INTERPRO:IPR029028,PMID:24951554,PMID:15062082