Substrate channeling of retinoic acid between cellular retinoic acid binding proteins (CRABPs) and CYP26 enzymes

Abstract

CYP26s metabolize all‐trans‐retinoic acid (atRA) efficiently. In cells atRA is bound to two binding proteins, CRABP‐I and CRABP‐II (Kd<1nM). CRABP‐II channels atRA to nuclear retinoic acid receptors but the effect of CRABPs on CYP26 mediated metabolism of atRA has not been characterized. The aim of this study was to determine whether CRABP‐I and/or ‐II channel atRA directly to CYP26s. Using holoCRABP‐II as a substrate and with 2‐fold excess of apoCRABP, atRA hydroxylation by CYP26B1 was observed at similar rate but metabolism by CYP3A4 and CYP2C8 was completely eliminated when compared to free atRA. With holoCRABP‐I atRA metabolism by CYP26B1 was decreased by 50% (in presence of apoCRABP) and abolished by CYP3A4 and CYP2C8. Using isotope dilution with atRA‐D5 or atRA bound to CRABP‐I and CRABP‐II an equal ratio of labeled and unlabeled products was obtained demonstrating that CYP26B1 can obtain its substrate directly from CRABPs. holo‐CRABP‐I and holoCRABP‐II inhibited CYP26B1 mediated 9cis‐RA hydroxylation with a 100nM IC50 suggesting that CRABPs interact with CYP26B1 directly. Finally, excess of apoCRABP‐I decreased the Vmax of atRA hydroxylation but had no effect on Km suggesting that CRABP‐I interacts directly with CYP26B1. These results show that CRABP‐I and CRABP‐II channel atRA to CYP26B1 and demonstrate that ligand delivery to P450 enzymes by small binding proteins is possible.