GAS2 encodes a 2-oxoglutarate dependent dioxygenase involved in ABA catabolism

Liu et al.1 recently reported the characterization of Arabidopsis thaliana GAS2 (Gain of Function in ABA-modulated Seed Germination 2), which was described as an enzyme that catalyzes the stereospecific hydration of GA12 to produce GA12 16, 17-dihydro-16-ol (DHGA12). However, as previously reported2, we did not find conversion of [17-14C]-labeled or [1-,7-,12-,18-14C4]-labeled GA12 by GAS2. Furthermore, the authors1 state the isolation of endogenous DHGA12 from dry Arabidopsis seeds, which we cannot confirm by our attempts to isolate this compound from 0.5 g dry Arabidopsis Col-0 seeds (data not shown). Instead, we present here data showing that the recombinant GAS2 enzyme is able to catabolize abscisic acid (ABA) to phaseic acid (PA) and further to a second product, putative 8-carboxy-ABA (Fig. 1a). O_FIG O_LINKSMALLFIG WIDTH=141 HEIGHT=200 SRC="FIGDIR/small/516706v1_fig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@18fe917org.highwire.dtl.DTLVardef@11006a1org.highwire.dtl.DTLVardef@1235c94org.highwire.dtl.DTLVardef@930b98_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 1.C_FLOATNO GAS2 is an ABA catabolizing oxidase. (a) The proposed ABA catabolic pathway catalyzed by GAS2. (b) Metabolism of d6-ABA incubated with 70 l cell-lysate of recombinant ATGA2ox1 and cofactors (negative control, upper lane). Metabolism of d6-ABA incubated with different volumes of cell-lysates containing recombinant GAS2 and cofactors as described in Methods. Chromatograms of characteristic single ions are shown in the first row for ABA (194 Da), in the middle row for PA (125 DA), and in the right row for compound A (194 Da). Products that were identified on the basis of their mass spectra of the methyl ester derivatives are labelled in red. (c) Top lane: Representative mass spectra of the d6-ABA substrate (KRI = 2087), and of its incubation products, PA (KRI = 2141) and compound A (KRI = 2197), by recombinant GAS2 enzyme. Bottom lane: Mass spectra of unlabeled PA substrate (KRI = 2146), and of the incubation product, compound A (KRI = 2201), by recombinant GAS2 enzyme. C_FIG