Cloning, Yeast Expression, and Characterization of the Coupling of Two Distantly Related Arabidopsis thalianaNADPH-Cytochrome P450 Reductases with P450 CYP73A5

Abstract

Two NADPH-cytochrome P450 reductase-encoding cDNAs were isolated from an <i>Arabidopsis</i> cDNA library by metabolic interference in a <i>Saccharomyces cerevisiae</i> mutant disrupted for its endogenous <i>cpr1</i>gene. <i>ATR1</i> encodes a protein of 692 amino acids, while<i>ATR2</i> encodes either a 712-residue protein (ATR2-1), or a 702-residue protein (ATR2-2) depending on the choice of the initiation codon. Comparative analysis of ATR1 and ATR2-1 indicates 64% amino acid sequence identity and the absence of conservation in the third base of conserved amino acid codons. The two <i>Arabidopsis</i>reductases are encoded by distinct genes whose divergence is expected an early event in angiosperms evolution. A poly(Ser/Thr) stretch reminiscent of a plant chloroplastic targeting signal is present at the ATR2-1 N-terminal end but absent in ATR1. The cDNA open reading frames were expressed in yeast. The recombinant polypeptides were found present in the yeast endoplasmic reticulum membrane and exhibited a high specific NADPH-cytochrome <i>c</i> reductase activity. To gain more insight into the respective functions of the two reductases, the <i>Arabidopsis</i> cDNA encoding cinnamate 4-hydroxylase (CYP73A5) was cloned and co-expressed with ATR1 or ATR2 in yeast. Biochemical characterization of the <i>Arabidopsis</i>ATR1/CYP73A5 and ATR2-1/CYP73A5 systems demonstrates that the two distantly related <i>Arabidopsis</i> reductases similarly support the first oxidative step of the phenylpropanoid general pathway.