Structure-guided function discovery of an NRPS-like glycine betaine reductase for choline biosynthesis in fungi

Abstract

Nonribosomal peptide synthetases (NRPS) and NRPS-like enzymes have diverse functions in primary and secondary metabolism. By using a structure-guided approach, we uncovered the function of an NRPS-like enzyme with unusual domain architecture, catalyzing two sequential two-electron reductions of glycine betaine to choline. Structural analysis based on homology model suggests cation-p interactions as the major substrate specificity determinant, which was verified using substrate analogs and inhibitors. Bioinformatic analysis indicates this NRPS-like glycine betaine reductase is highly conserved and widespread in fungi kingdom. Genetic knockout experiments confirmed its role in choline biosynthesis and maintaining glycine betaine homeostasis in fungi. Our findings demonstrate that the oxidative choline-glycine betaine degradation pathway can operate in a fully reversible fashion and provide new insights in understanding fungal choline metabolism. The use of an NRPS-like enzyme for reductive choline formation is energetically efficient compared to known pathways. Our discovery also underscores the capabilities of structure-guided approach in assigning function of uncharacterized multidomain proteins, which can potentially aid functional discovery of new enzymes by genome mining.

Glycine betaine uptake and metabolism in marine microbial communities

“…Glycerophosphocholine is made from choline‐phospholipids, a lipid class predominantly produced by eukaryotic organisms in marine plankton communities (Van Mooy and Fredricks, 2010; Popendorf et al ., 2011). A non‐ribosomal peptide synthetase‐like enzyme (ATRR) that catalyses the reduction of GBT to choline was recently characterized in fungi (Hai et al ., 2019). In the metatranscriptome data, transcription of atrr gene homologues was dominated by members of Dinophyceae, Stramenopiles, Haptophyta and Amoebozoa (Fig.…”

“…1). A non‐ribosomal peptide synthetase‐like glycine betaine reductase (ATRR) responsible for this transformation was identified in fungi (Hai et al ., 2019), though the presence of this pathway in marine microbes is unexplored. The third characterized pathway of GBT metabolism is GBT reduction to trimethylamine (TMA) via betaine reductase (GrdHI) (3 in Fig.…”