Implications of divergence of methionine adenosyltransferase in archaea

Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenosylmethionine from L-methionine and adenosine triphosphate. MAT enzymes are ancient, believed to share a common ancestor, and are highly conserved in all three domains of life. However, the sequences of archaeal MATs show considerable divergence compared to their bacterial and eukaryotic counterparts. Furthermore, the structural and functional significance of this sequence divergence are not well understood. In the present study, we employed structural analysis and ancestral sequence reconstruction (ASR) to investigate archaeal MAT divergence. We observed that the dimer interface containing the active site (which is usually well-conserved) diverged considerably between the bacterial/eukaryotic MATs and archaeal MAT. A detailed investigation of the available structures supports the sequence analysis outcome: the protein domains and subdomains of bacterial and eukaryotic MAT are more similar than those of archaea. Finally, we resurrected archaeal MAT ancestors. Interestingly, archaeal MAT ancestors show substrate specificity, which is lost during evolution. This observation supports the hypothesis of a common MAT ancestor for the three domains of life. In conclusion, we have demonstrated that archaeal MAT is an ideal system for studying an enzyme family that evolved differently in one domain compared to others while maintaining the same catalytic activity

On the emergence of the hemD-like fold and its use for fold-chimeragenesis

Whilst the structural diversity of proteins may appear endless, even large protein complexes can be decomposed into their independently folding units, the domains. Little is known about domain emergence. Structural and sequence evidence suggests they evolved through combination of subdomain-sized fragments. To investigate this hypothesis, we searched for homologous regions among domains with a broadly different topology (fold), employing the α/β-class as defined in the Structural Classification Of Proteins (SCOP), which is believed to contain the oldest domains. We compared their sequence profiles all-against-all and found that in spite of their globally different architectures a large number of them share local homologous regions ranging from a dozen to >200 amino acids. An interesting hit constitutes the hemD-like fold, whose profile alignments provide strong evidence for its emergence via flavodoxin-like gene duplication, insertion and segment-swapping. To test this hypothesis experimentally, we reverted these evolutionary events, finding that the obtained protein in fact adopts the canonical flavodoxin-like fold. These results illustrate a way how Nature recycles a limited repertoire of building blocks, which provides a successful strategy to reach diversity at a lower molecular cost than creating every unit de novo. Such building units may have overcome a selective pressure through the course of evolution due to their function and/or intrinsic stability that allowed them to be modified and extended. Inspired by this naturally occurring strategy, I designed a cobalamin-binding chimera, extracting a portion of the binding pocket of a cobalamin-binding domain and exchanged it against its homologous region in the hemD-like fold. The resulting chimera expresses solubly, is well folded and binds cobalamin, illustrating that mimicking Nature’s combinatorial approach is a good source of soluble and well-folded proteins and may be employed as an alternative strategy to design novel functionalities

Reconstructing the Remote Origins of a Fold Singleton from a Flavodoxin-Like Ancestor

Evolutionary processes that led to the emergence of structured protein domains left footprints in the sequences of modern proteins. We searched for such hints employing state-of-the-art sequence analysis and found evidence that the HemD-like fold emerged from the flavodoxin-like fold through segment swap and gene duplication. To verify this hypothesis, we reverted these evolutionary steps experimentally, constructing a HemD-half that resulted in a protein with the canonical flavodoxin-like architecture. These results of fold reconstruction from the sequence of a different fold strongly support our hypothesis of common ancestry. It further illustrates the plasticity of modern proteins to form new folded proteins