Evidence for distinct rate-limiting steps in the cleavage of alkenes by carotenoid cleavage dioxygenases

Abstract

Edited by F. Peter Guengerich Carotenoid cleavage dioxygenases (CCDs) use a nonheme Fe(II) cofactor to split alkene bonds of carotenoid and stilbenoid substrates. The iron centers of CCDs are typically five-coordinate in their resting states, with solvent occupying an exchangeable site. The involvement of this iron-bound solvent in CCD catalysis has not been experimentally addressed, but computational studies suggest two possible roles. 1) Solvent dissociation provides a coordination site for O 2 , or 2) solvent remains bound to iron but changes its equilibrium position to allow O 2 binding and potentially acts as a proton source. To test these predictions, we investigated isotope effects (H 2 O versus D 2 O) on two stilbenoidcleaving CCDs, Novosphingobium aromaticivorans oxygenase 2 (NOV2) and Neurospora crassa carotenoid oxygenase 1 (CAO1), using piceatannol as a substrate. NOV2 exhibited an inverse isotope effect (k H /k D ϳ 0.6) in an air-saturated buffer, suggesting that solvent dissociates from iron during the catalytic cycle. By contrast, CAO1 displayed a normal isotope effect (k H /k D ϳ 1.7), suggesting proton transfer in the rate-limiting step. X-ray absorption spectroscopy on NOV2 and CAO1 indicated that the protonation states of the iron ligands are unchanged within pH 6.5-8.5 and that the Fe(II)-aquo bond is minimally altered by substrate binding. We pinpointed the origin of the differential kinetic behaviors of NOV2 and CAO1 to a single amino acid difference near the solvent-binding site of iron, and X-ray crystallography revealed that the substitution alters binding of diffusible ligands to the iron center. We conclude that solvent-iron dissociation and proton transfer are both associated with the CCD catalytic mechanism. Carotenoid cleavage dioxygenases (CCDs) 2 constitute a family of nonheme iron enzymes that catalyze the cleavage of alk-This work was supported by National Institutes of Health Grants R01EY009339 (to P. D. K.) and R01EY020551 (to J. v. L.), Department of Veterans Affairs Grant IK2BX002683 (to P. D. K.), and Burroughs Wellcome Fund Award 1015187 (to P. D. K.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Department of Veterans Affairs. This article contains Figs. S1-S8 and Tables S1 and S2.

A modular pathway engineering strategy for the high-level production of β-ionone in Yarrowia lipolytica

“…In the subsequent 3-L bioreactor fermentation, it was found that the DO was a very important factor for the biosynthesis of β-ionone. This is consistent with a recent report [47] and is related to the last metabolic step of β-ionone biosynthesis, which is catalyzed by carotenoid cleavage dioxygenase and requires two oxygen molecules to produce two β-ionone molecules. Thus, with the optimization of DO level at 15% the yield of β-ionone increased twofold compared to the bioreactor fermentation performed with DO at 0-5%.…”

Structural and mechanistic aspects of carotenoid cleavage dioxygenases (CCDs)

“…The normal sKIE observed for CAO1 was suggestive of proton transfer during the rate-limiting step of the reaction, although the exact proton source could not be pinpointed. Interestingly, inverse sKIE behavior could be conferred to CAO1 by substitution of a single amino acid residue (Leu509) located near the iron-solvent compex to Val as is found in NOV2 [76].…”

“…This issue was later revisited using purified protein and optimized reaction conditions with results clearly supporting dioxygenase chemistry [43]. More recently, solvent kinetic isotope effect (sKIE) studies have been carried out on NOV2 and CAO1 to determine the impact of deuterium substitution on steady-state kinetics [76]. These studies revealed a striking difference in behavior with NOV2 and CAO1 exhibiting inverse and normal sKIEs on k cat , respectively.…”

Efficient production of the β-ionone aroma compound from organic waste hydrolysates using an engineered Yarrowia lipolytica strain

“…The cleavage of a β-carotene by CCD1 requires two oxygen molecules to produce two β-ionone molecules. The presence of considerable amounts of β-carotene seen in our experiments suggests that despite oxygen supply in flasks during fermentation may be insufficient, limiting the activity of PhCCD1 ( Khadka et al, 2019 ; Liu et al, 2021 ). This explanation raises the possibility that both the YLBI3120 and YLBI3121 strains have the potential to produce even higher concentrations of β-ionone under optimal fermentation conditions.…”