Comprehensive cross‐genome survey and phylogeny of glycoside hydrolase family 16 members reveals the evolutionary origin of EG16 and XTH proteins in plant lineages

Summary Carbohydrate‐active enzymes (CAZymes) are central to the biosynthesis and modification of the plant cell wall. An ancient clade of bifunctional plant endo‐glucanases (EG16 members) was recently revealed and proposed to represent a transitional group uniting plant xyloglucan endo‐transglycosylase/hydrolase (XTH) gene products and bacterial mixed‐linkage endo‐glucanases in the phylogeny of glycoside hydrolase family 16 (GH16). To gain broader insights into the distribution and frequency of EG16 and other GH16 members in plants, the PHYTOZOME, PLAZA, NCBI and 1000 PLANTS databases were mined to build a comprehensive census among 1289 species, spanning the broad phylogenetic diversity of multiple algae through recent plant lineages. EG16, newly identified EG16‐2 and XTH members appeared first in the green algae. Extant EG16 members represent the early adoption of the β‐jellyroll protein scaffold from a bacterial or early‐lineage eukaryotic GH16 gene, which is characterized by loop deletion and extension of the N terminus (in EG16‐2 members) or C terminus (in XTH members). Maximum‐likelihood phylogenetic analysis of EG16 and EG16‐2 sequences are directly concordant with contemporary estimates of plant evolution. The lack of expansion of EG16 members into multi‐gene families across green plants may point to a core metabolic role under tight control, in contrast to XTH genes that have undergone the extensive duplications typical of cell‐wall CAZymes. The present census will underpin future studies to elucidate the physiological role of EG16 members across plant species, and serve as roadmap for delineating the closely related EG16 and XTH gene products in bioinformatic analyses of emerging genomes and transcriptomes. Significance Statement This comprehensive census of recently expanded ‘omic resources for nearly 1300 algal and plant species reveals the very early origins of plant endo‐glucanase 16 (EG16) and xyloglucan endo‐transglycosylase/hydrolase (XTH) gene products within the phylogeny of Glycoside Hydrolase Family 16 (GH16), serves as bioinformatic roadmap for delineating these closely related proteins from emerging genomes and transcriptomes, and will underpin future studies to elucidate the physiological role of EG16 members in plant species.