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

ABSTRACT: Nonribosomal peptide synthetases (NRPSs) and NRPS-like enzymes have diverse functions in primary and secondary metabolisms. By using a structure-guided approach, we uncovered the function of a NRPS-like enzyme with unusual domain architecture, catalyzing two sequential two-electron reductions of glycine betaine to choline. Structural analysis based on the homology model suggests cation-? 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 kingdom fungi. 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 insight in understanding fungal choline metabolism. The use of an NRPS-like enzyme for reductive choline formation is energetically efficient compared with known pathways. Our discovery also underscores the capabilities of the structure-guided approach in assigning functions of uncharacterized multidomain proteins, which can potentially aid functional discovery of new enzymes by genome mining.

SUBMITTER: Hai Y

PROVIDER: S-EPMC6535007 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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

Hai Yang Y Huang Arthur M AM Tang Yi Y

Proceedings of the National Academy of Sciences of the United States of America 20190506 21

Nonribosomal peptide synthetases (NRPSs) and NRPS-like enzymes have diverse functions in primary and secondary metabolisms. By using a structure-guided approach, we uncovered the function of a NRPS-like enzyme with unusual domain architecture, catalyzing two sequential two-electron reductions of glycine betaine to choline. Structural analysis based on the homology model suggests cation-π interactions as the major substrate specificity determinant, which was verified using substrate analogs and i ...[more]

PMID: 31061132

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Project description:Fluctuations in osmolarity are one of the most prevalent stresses to which bacteria must adapt, both hypo- and hyperosmotic conditions. Most bacteria cope with high osmolarity by accumulating compatible solutes (osmolytes) in the cytoplasm to maintain the turgor pressure of the cell. Vibrio parahaemolyticus, a halophile, utilizes at least six compatible solute transporters for the uptake of osmolytes: two ABC family ProU transporters and four betaine-carnitine-choline transporter (BCCT) family transporters. The full range of compatible solutes transported by this species has yet to be determined. Using an osmolyte phenotypic microarray plate for growth analyses, we expanded the known osmolytes used by V. parahaemolyticus to include N,N-dimethylglycine (DMG), among others. Growth pattern analysis of four triple-bccT mutants, possessing only one functional BCCT, indicated that BccT1 (VP1456), BccT2 (VP1723), and BccT3 (VP1905) transported DMG. BccT1 was unusual in that it could take up both compounds with methylated head groups (glycine betaine [GB], choline, and DMG) and cyclic compounds (ectoine and proline). Bioinformatics analysis identified the four coordinating amino acid residues for GB in the BccT1 protein. In silico modeling analysis demonstrated that GB, DMG, and ectoine docked in the same binding pocket in BccT1. Using site-directed mutagenesis, we showed that a strain with all four residues mutated resulted in the loss of uptake of GB, DMG, and ectoine. We showed that three of the four residues were essential for ectoine uptake, whereas only one of the residues was important for GB uptake. Overall, we have demonstrated that DMG is a highly effective compatible solute for Vibrio species and have elucidated the amino acid residues in BccT1 that are important for the coordination of GB, DMG, and ectoine transport.IMPORTANCEVibrio parahaemolyticus possesses at least six osmolyte transporters, which allow the bacterium to adapt to high-salinity conditions. In this study, we identified several additional osmolytes that were utilized by V. parahaemolyticus We demonstrated that the compound DMG, which is present in the marine environment, was a highly effective osmolyte for Vibrio species. We determined that DMG is transported via BCCT family carriers, which have not been shown previously to take up this compound. BccT1 was a carrier for GB, DMG, and ectoine, and we identified the amino acid residues essential for the coordination of these compounds. The data suggest that for BccT1, GB is more easily accommodated than ectoine in the transporter binding pocket.

Dataset Information

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

Publications

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

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