UC Berkeley

The GH3 family of adenylating enzymes conjugate acyl substrates such as the growth hormone indole-3-acetic acid (IAA) to amino acids via a two-step reaction of acyl substrate adenylation followed by amino acid conjugation. Arabidopsis thaliana GH3.5 was previously shown to be unusual in that it could adenylate both IAA and the defense hormone salicylic acid (SA, 2-hydroxybenzoate). Our detailed studies of the kinetics of GH3.5 on a variety of auxin and benzoate substrates provides insight into the acyl preference and reaction mechanism of GH3.5. For example, we found GH3.5 activity on substituted benzoates is not defined by the substitution position as it is for GH3.12/PBS3. Most importantly, we show that GH3.5 strongly prefers Asp as the amino acid conjugate and that the concentration of Asp dictates the functional activity of GH3.5 on IAA vs. SA. Not only is Asp used in amino acid biosynthesis, but it also plays an important role in nitrogen mobilization and in the production of downstream metabolites, including pipecolic acid which propagates defense systemically. During active growth, [IAA] and [Asp] are high and the catalytic efficiency (k_cat/K_m) of GH3.5 for IAA is 360-fold higher than with SA. GH3.5 is expressed under these conditions and conversion of IAA to inactive IAA-Asp would provide fine spatial and temporal control over local auxin developmental responses. By contrast, [SA] is dramatically elevated in response to (hemi)-biotrophic pathogens which also induce GH3.5 expression. Under these conditions, [Asp] is low and GH3.5 has equal affinity (K_m) for SA and IAA with similar catalytic efficiencies. However, the concentration of IAA tends to be very low, well below the K_m for IAA. Therefore, GH3.5 catalyzed formation of SA-Asp would occur, fine-tuning localized defensive responses through conversion of active free SA to SA-Asp. Taken together, we show how GH3.5, with dual activity on IAA and SA, can integrate cellular metabolic status via Asp to provide fine control of growth vs. defense outcomes and hormone homeostasis.