chy1, an Arabidopsis Mutant with Impaired β-Oxidation, Is Defective in a Peroxisomal β-Hydroxyisobutyryl-CoA Hydrolase

Abstract

The Arabidopsis chy1 mutant is resistant to indole-3-butyric acid, a naturally occurring form of the plant hormone auxin. Because the mutant also has defects in peroxisomal ␤-oxidation, this resistance presumably results from a reduced conversion of indole-3-butyric acid to indole-3-acetic acid. We have cloned CHY1, which appears to encode a peroxisomal protein 43% identical to a mammalian valine catabolic enzyme that hydrolyzes ␤-hydroxyisobutyryl-CoA. We demonstrated that a human ␤-hydroxyisobutyryl-CoA hydrolase functionally complements chy1 when redirected from the mitochondria to the peroxisomes. We expressed CHY1 as a glutathione S-transferase (GST) fusion protein and demonstrated that purified GST-CHY1 hydrolyzes ␤-hydroxyisobutyryl-CoA. Mutagenesis studies showed that a glutamate that is catalytically essential in homologous enoyl-CoA hydratases was also essential in CHY1. Mutating a residue that is differentially conserved between hydrolases and hydratases established that this position is relevant to the catalytic distinction between the enzyme classes. It is likely that CHY1 acts in peroxisomal valine catabolism and that accumulation of a toxic intermediate, methacrylyl-CoA, causes the altered ␤-oxidation phenotypes of the chy1 mutant. Our results support the hypothesis that the energy-intensive sequence unique to valine catabolism, where an intermediate CoA ester is hydrolyzed and a new CoA ester is formed two steps later, avoids methacrylyl-CoA accumulation.

Identification and Characterization of Arabidopsis Indole-3-Butyric Acid Response Mutants Defective in Novel Peroxisomal Enzymes

“…We examined IBA-response defects under several conditions, comparing ibr1 and ibr10 with the previously described chy1-3 (Zolman et al 2001a) and ibr3-1 (Zolman et al 2007) mutants. First, we examined the effects of increasing IBA concentrations on root elongation.…”

“…ibr3-1 (Zolman et al 2007) is included for comparison with ibr10 and ibr1. chy1-3 (Zolman et al 2001a) is included as an IBA-resistant control. (C) ibr mutant root elongation on synthetic auxins.…”

“…ibr10-1 was isolated by screening T-DNA insertion lines in the Col-0 accession (pool CS75075; Alonso et al 2003), although our analysis indicated that the T-DNA was not linked to the IBA-resistant phenotype (data not shown). chy1-3 (Zolman et al 2001a) and ibr3-1 (Zolman et al 2007) were described previously and are Col-0 alleles with point mutations in CHY1/ At5g65940 and IBR3/At3g06810, respectively.…”

Conversion of Endogenous Indole-3-Butyric Acid to Indole-3-Acetic Acid Drives Cell Expansion in Arabidopsis Seedlings

“…These genes encode proteins required for proper peroxisome function, such as PEX4 , PEX5 (Zolman et al, 2000), PEX6 (Zolman and Bartel, 2004), and PEX7 (Woodward and Bartel, 2005b;Ramó n and Bartel, 2010); the peroxisomal ABC transporter PXA1, which may transport IBA into the peroxisome for b-oxidation (Zolman et al, 2001b); and enzymes that reside in the peroxisome, including ACX enzymes (Adham et al, 2005), CHY1 (Zolman et al, 2001a), IBR1 (Zolman et al, 2008), IBR3 (Zolman et al, 2007), and IBR10 (Zolman et al, 2008). Here, we have provided biochemical evidence that Arabidopsis seedlings can convert IBA into IAA and that multiple mutants identified in genetic screens for IBA resistance are defective in this conversion (Fig.…”

“…Similarly, mutants defective in the peroxisomal ABC transporter PXA1, which may transport substrates into the peroxisome for b-oxidation (Zolman et al, 2001b), and mutants defective in several peroxisome-targeted enzymes, including the acyl-CoA oxidase double mutant acx1-2 acx2-1 (Adham et al, 2005), the branchedchain amino acid catabolism mutant chy1-10 (Zolman et al, 2001a), and the IBA response triple mutant ibr1-2 ibr3-1 ibr10-1 (Zolman et al, 2008), are IBA resistant and IAA sensitive ( Fig. 1A; Zolman et al, 2001aZolman et al, , 2001bZolman et al, , 2008Adham et al, 2005).…”