Abstract
BackgroundPlant ALDH10 enzymes are aminoaldehyde dehydrogenases (AMADHs) that oxidize different ω-amino or trimethylammonium aldehydes, but only some of them have betaine aldehyde dehydrogenase (BADH) activity and produce the osmoprotectant glycine betaine (GB). The latter enzymes possess alanine or cysteine at position 441 (numbering of the spinach enzyme, SoBADH), while those ALDH10s that cannot oxidize betaine aldehyde (BAL) have isoleucine at this position. Only the plants that contain A441- or C441-type ALDH10 isoenzymes accumulate GB in response to osmotic stress. In this work we explored the evolutionary history of the acquisition of BAL specificity by plant ALDH10s.ResultsWe performed extensive phylogenetic analyses and constructed and characterized, kinetically and structurally, four SoBADH variants that simulate the parsimonious intermediates in the evolutionary pathway from I441-type to A441- or C441-type enzymes. All mutants had a correct folding, average thermal stabilities and similar activity with aminopropionaldehyde, but whereas A441S and A441T exhibited significant activity with BAL, A441V and A441F did not. The kinetics of the mutants were consistent with their predicted structural features obtained by modeling, and confirmed the importance of position 441 for BAL specificity. The acquisition of BADH activity could have happened through any of these intermediates without detriment of the original function or protein stability. Phylogenetic studies showed that this event occurred independently several times during angiosperms evolution when an ALDH10 gene duplicate changed the critical Ile residue for Ala or Cys in two consecutive single mutations. ALDH10 isoenzymes frequently group in two clades within a plant family: one includes peroxisomal I441-type, the other peroxisomal and non-peroxisomal I441-, A441- or C441-type. Interestingly, high GB-accumulators plants have non-peroxisomal A441- or C441-type isoenzymes, while low-GB accumulators have the peroxisomal C441-type, suggesting some limitations in the peroxisomal GB synthesis.ConclusionOur findings shed light on the evolution of the synthesis of GB in plants, a metabolic trait of most ecological and physiological relevance for their tolerance to drought, hypersaline soils and cold. Together, our results are consistent with smooth evolutionary pathways for the acquisition of the BADH function from ancestral I441-type AMADHs, thus explaining the relatively high occurrence of this event.
Highlights
Plant ALDH10 enzymes are aminoaldehyde dehydrogenases (AMADHs) that oxidize different ω-amino or trimethylammonium aldehydes, but only some of them have betaine aldehyde dehydrogenase (BADH) activity and produce the osmoprotectant glycine betaine (GB)
It can be observed that primitive plants with a known genome like Ostrococcus tauri, O. lucimarinus, Micromonas pusilla, Chlamydomonas reinhardtii, Volvox carteri (Chlorophyta), Physcomitrella patents (Briophyta) and Selaginella moellendorffii (Lycopodiophyta) contain only one ALDH10 enzyme. All these enzymes possess Ile at position equivalent to 441 (SoBADH numbering), which is the residue most frequently found in ALDH10 enzymes of the other plant families (Figures 1B and 1C)
Structural characterization of the Spinach BADH (SoBADH) A441 mutants Since the two main aspects that determine the evolution of a protein are function and protein stability, we investigated whether the changes made at position 441 affect the structure and/or thermal stability of the mutant enzymes
Summary
Plant ALDH10 enzymes are aminoaldehyde dehydrogenases (AMADHs) that oxidize different ω-amino or trimethylammonium aldehydes, but only some of them have betaine aldehyde dehydrogenase (BADH) activity and produce the osmoprotectant glycine betaine (GB). By means of X-ray crystallography, in silico model building, site-directed mutagenesis, and kinetic studies of the ALDH10 enzyme from spinach (SoBADH), we found that only an amino acid residue at position 441 is critical for an ALDH10 enzyme being able to accept or reject BAL as a substrate [19] This residue, located in the second sphere of interaction with the substrate behind the indole group of the tryptophan equivalent to W456 in SoBADH, determines the size of the pocket formed by the Trp and Tyr residues equivalent to Y160 and W456 (SoBADH numbering) where the bulky trimethylammonium group of BAL binds. This conclusion was drawn by Díaz-Sánchez et al [19] by comparing the crystal structures of the SoBADH (PDB code 4A0M) with those of the two pea AMADH enzymes, which do not have
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