FAD-dependent oxidoreductase CpaO catalyzes an unusual ring formation in cyclopiazonic acid biosynthesis

Ting Cheng (鄭婷) 1, Chin-Yuan Chang1

1 Department of Biological Science and Technology, National Yang Ming Chiao Tung University 

Cyclopiazonic acid (CPA), a nanomolar inhibitor, inhibits sarcoplasmic or endoplasmic reticulum calcium-dependent ATPase (SERCA) to affect the muscle contraction-relaxation cycle, resulting in an increase muscle contraction. CPA is a pentacyclic indole tetramic acid neurotoxin, which is produced by the Aspergillus and Penicillum species. Previous studies have reported that CPA is derived from a precursor l-tryptophan (l-Trp), which is modified with the cyclo-acetoacetyl and prenyl groups by a hybrid PKS/NRPS (polyketide synthase/nonribosomal peptide synthetase) CpaS and a prenyl transferase CpaD, respectively, to form β-CPA. CpaO is a FAD-dependent oxidoreductase, which was proposed to catalyzes dehydrogenation of β-CPA resulting in a pentacyclic ring system; however, the enzyme catalytic mechanism about the unusual intramolecular ring closure is still a mystery. In this study, we used a combination of biochemical characterization, mutagenesis analysis, and structural biology approaches to unravel the catalytic mechanism of CpaO for the ring formation. According to the crystal structures of CapO in complex with the substrate β-CPA and the product α-CPA, respectively, we propose a new CpaO catalytic mechanism. This study revises the catalytic mechanism of CpaO involved in CPA biosynthesis and provides new insights into the ring formation of the l-Trp-based natural products.