chy1, an Arabidopsis mutant with impaired beta-oxidation, is defective in a peroxisomal beta-hydroxyisobutyryl-CoA hydrolase

The Arabidopsis chy1 mutant is resistant to indole-3-butyric acid, a natura lly occurring form of the plant hormone auxin. Because the mutant also has defects in peroxisomal beta -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% ide ntical to a mammalian valine catabolic enzyme that hydrolyzes beta -hydroxy isobutyryl-CoA. We demonstrated that a human beta -hydroxyisobutyryl-CoA hy drolase 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 beta -hy droxyisobutyryl-CoA. Mutagenesis studies showed that a glutamate that is ca talytically essential in homologous enoyl-CoA hydratases was also essential in CHY1. Mutating a residue that is differentially conserved between hydro lases and hydratases established that this position is relevant to the cata lytic distinction between the enzyme classes. It is likely, that CHY1 acts in peroxisomal valine catabolism and that accumulation of a toxic intermedi ate, methacrylyl-CoA, causes the altered beta -oxidation phenotypes of the chy1 mutant. Our results support the hypothesis that the energy-intensive s equence unique to valine catabolism, where an intermediate CoA ester is hyd rolyzed and a new CoA ester is formed two steps later, avoids methacrylyl-C oA accumulation.