Theoretical and Experimental Studies of the Conversion of Chromopyrrolic Acid to an Antitumor Derivative by Cytochrome P450 StaP: The Catalytic Role of Water Molecules

Abstract

Chromopyrrolic acid (CPA) oxidation by cytochrome P450 StaP is a key process in the biosynthesis of antitumor drugs (Onaka, H.; Taniguchi, S.; Igarashi, Y.; Furumai, T. Biosci. Biotechnol. Biochem. 2003, 67, 127-138), which proceeds by an unusual C-C bond coupling. Additionally, because CPA is immobilized by a hydrogen-bonding array, it is prohibited from undergoing direct reaction with Compound I, the active species of P450. As such, the mechanism of P450 StaP poses a puzzle. In the present Article, we resolve this puzzle by combination of theory, using QM/MM calculations, and experiment, using crystallography and reactivity studies. Theory shows that the hydrogen-bonding machinery of the pocket deprotonates the carboxylic acid groups of CPA, while the nearby His(250) residue and the crystal waters, Wat(644) and Wat(789), assist the doubly deprotonated CPA to transfer electron density to Compound I; hence, CPA is activated toward proton-coupled electron transfer that sets the entire mechanism in motion. The ensuing mechanism involves a step of C-C bond formation coupled to a second electron transfer, four proton-transfer and tautomerization steps, and four steps where Wat(644) and Wat(789) move about and mediate these events. Experiments with the dichlorinated substrate, CCA, which expels Wat(644), show that the enzyme loses its activity. H250A and H250F mutations of P450 StaP show that His(250) is important, but in its absence Wat(644) and Wat(789) form a hydrogen-bonding diad that mediates the transformation. Thus, the water diad emerges as the minimal requisite element that endows StaP with function. This highlights the role of water molecules as biological catalysts that transform a P450 to a peroxidase-type (Derat, E.; Shaik, S. J. Am. Chem. Soc. 2006, 128, 13940-13949).

Proton-Coupled Electron Transfer in Organic Synthesis: Fundamentals, Applications, and Opportunities

“…Wenger's model polypyridyl system with spacers, Ru-O distances, and corresponding rates of concerted PCET [141]. Various Ruthenium complexes used to induce oxidative PCET, as well as a Rhenium complex featuring an unconjugated phenol ligand which readily forms the phenoxyl in the presence of base [148] Miller et al Oxidation of CPA (41) by StaP P450 Cpd1 into a precursor (42) to staurosporine with a schematic for the PCET activation step proposed by Shaik [16]. A highly conserved hydrogen bonding triad Trp48, Asp237, and His118 [166] in Ribonucleotide Reductase (RNR).…”

Recent density functional theory model calculations of drug metabolism by cytochrome P450

“…ab initio, DFT, or semiempirical method) treatment of atoms involved in the active site with a MM description of the native protein environment [135][136][137]. QM/MM approach is becoming increasingly popular for the study of biochemical reactions [138][139][140].…”

Understanding the determinants of selectivity in drug metabolism through modeling of dextromethorphan oxidation by cytochrome P450

“…However, because the size of models that can feasibly be treated is limited, they may not account for important steric factors and binding preferences. QM/MM calculations offer the potential to do this, because larger and more extensive models can be treated, and a number of studies of issues relating to mechanisms and selectivity have started to appear (16,(21)(22)(23). However such studies remain very challenging.…”