Enzymatic Aromatization of Steroids. I. Effects of Oxygen and Carbon Monoxide on the Intermediate Steps of Estrogen Biosynthesis

Meigs, Robert A.; Ryan, Kenneth J.

doi:10.1097/00006254-197107000-00016

Cited by 9 publications

(11 citation statements)

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“…A second possibility is that it may be difficult for CO to coordinate with the heme iron center at the active site of these mutants. A phenomenon similar to that proposed in this hypothesis was observed during the analysis of the spectrum of the catalytically active human CYP19 (8,16,20). The introduced branched-chain amino acids might hamper the formation of a CO-enzyme complex.…”

Section: Discussionsupporting

confidence: 72%

Biotechnological Production of Caffeic Acid by Bacterial Cytochrome P450 CYP199A2

Furuya

Arai

Kino

2012

Appl Environ Microbiol

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ABSTRACTCaffeic acid is a biologically active molecule that has various beneficial properties, including antioxidant, anticancer, and anti-inflammatory activities. In this study, we explored the catalytic potential of a bacterial cytochrome P450, CYP199A2, for the biotechnological production of caffeic acid. When the CYP199A2 enzyme was reacted withp-coumaric acid, it stoichiometrically produced caffeic acid. The crystal structure of CYP199A2 shows that Phe at position 185 is situated directly above, and only 6.35 Å from, the heme iron. This F185 residue was replaced with hydrophobic or hydroxylated amino acids using site-directed mutagenesis to create mutants with novel and improved catalytic properties. In whole-cell assays with the known substrate of CYP199A2, 2-naphthoic acid, only the wild-type enzyme hydroxylated 2-naphthoic acid at the C-7 and C-8 positions, whereas all of the active F185 mutants exhibited a preference for C-5 hydroxylation. Interestingly, several F185 mutants (F185V, F185L, F185I, F185G, and F185A mutants) also acquired the ability to hydroxylate cinnamic acid, which was not hydroxylated by the wild-type enzyme. These results demonstrate that F185 is an important residue that controls the regioselectivity and the substrate specificity of CYP199A2. Furthermore,Escherichia colicells expressing the F185L mutant exhibited 5.5 times higher hydroxylation activity forp-coumaric acid than those expressing the wild-type enzyme. By using the F185L whole-cell catalyst, the production of caffeic acid reached 15 mM (2.8 g/liter), which is the highest level so far attained in biotechnological production of this compound.

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Section: Discussionsupporting

confidence: 72%

Biotechnological Production of Caffeic Acid by Bacterial Cytochrome P450 CYP199A2

Furuya

Arai

Kino

2012

Appl Environ Microbiol

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“…was coined for each of these numbered P450 isoforms [83] CO binding involves the donation of electrons from the carbon to the iron through a σ-bond as well as back-donation of electrons from the occupied ferrous iron d-orbitals to the empty antibonding π-orbitals of the ligand [84] Early studies with model ferroporphyrins indicated that only those with a thiolate ligand trans to the CO yielded the 450-nm absorption, thereby providing key evidence for the presence of a thiolate fifth ligand in P450 [85] The 450 nm absorption maximum of the ferrous P450-CO complex is proposed by some to reflect the red-shifted hyperporphyrin split Soret peak of the P450 heme iron-CO complex [76] CO inhibition is a diagnostic test of P450-catalyzed processes, although the sensitivities of different P450 isoforms to CO differ [86] and a few P450-catalyzed reactions are resistant to its inhibition [87][88][89] Moreover, the CO sensitivity to inhibition of P450 enzymes such as aromatase (CYP19) [90,91] and P450 scc (CYP11A1) [92] with multistep catalytic cascades, is drastically reduced as they traverse the conformational and ligand states inherent in each of those catalytic processes The susceptibility of different families to CO inhibition also varies, appearing to decrease in the order CYP2D>CYP2C>CYP3A among the major drug-metabolizing subfamilies of human liver P450 isoforms [86] …”

Section: Coordination To P450 Ferrous Hemementioning

confidence: 99%

Inhibition of Cytochrome P450 Enzymes

Correia

Hollenberg

2015

Cytochrome P450

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“…Our current results on ferric P450arom indicate that the effects of substrate binding on the structural environment of the heme in P450arom are similar among the substrates and analogue. Each substrate-bound P450arom, however, exhibits different reactivity to the molecular oxygen and different sensitivity to the inhibition by CO (40), allowing us to speculate that the heme environment of P450arom would depend on the substrates. To confirm this, we measured the absorption spectra for the ferrous-CO form of P450arom and tried to detect the CO-associated vibrations that are sensitive to the distal environment and the electronic property of the proximal ligand (29)(30)(31).…”

Section: Uv/vis Absorption Spectra Of Ferric P450arommentioning

confidence: 99%

Raman Evidence for Specific Substrate-Induced Structural Changes in the Heme Pocket of Human Cytochrome P450 Aromatase during the Three Consecutive Oxygen Activation Steps

et al. 2006

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Specific substrate-induced structural changes in the heme pocket are proposed for human cytochrome P450 aromatase (P450arom) which undergoes three consecutive oxygen activation steps. We have experimentally investigated this heme environment by resonance Raman spectra of both substratefree and substrate-bound forms of the purified enzyme. The Fe-CO stretching mode (ν Fe-CO ) of the CO complex and Fe 3+ -S stretching mode (ν Fe-S ) of the oxidized form were monitored as a structural marker of the distal and proximal sides of the heme, respectively. The ν Fe-CO mode was upshifted from 477 to 485 and to 490 cm -1 by the binding of androstenedione and 19-aldehyde-androstenedione, substrates for the first and third steps, respectively, whereas ν Fe-CO was not observed for P450arom with 19-hydroxyandrostenedione, a substrate for the second step, indicating that the heme distal site is very flexible and changes its structure depending on the substrate. The 19-aldehyde-androstenedione binding could reduce the electron donation from the axial thiolate, which was evident from the low-frequency shift of ν Fe-S by 5 cm -1 compared to that of androstenedione-bound P450arom. Changes in the environment in the heme distal site and the reduced electron donation from the axial thiolate upon 19-aldehydeandrostenedione binding might stabilize the ferric peroxo species, an active intermediate for the third step, with the suppression of the formation of compound I (Fe 4+ dO porphyrin +• ) that is the active species for the first and second steps. We, therefore, propose that the substrates can regulate the formation of alternative reaction intermediates by modulating the structure on both the heme distal and proximal sites in P450arom.

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Enzymatic Aromatization of Steroids. I. Effects of Oxygen and Carbon Monoxide on the Intermediate Steps of Estrogen Biosynthesis

Cited by 9 publications

References 0 publications

Biotechnological Production of Caffeic Acid by Bacterial Cytochrome P450 CYP199A2

Biotechnological Production of Caffeic Acid by Bacterial Cytochrome P450 CYP199A2

Inhibition of Cytochrome P450 Enzymes

Raman Evidence for Specific Substrate-Induced Structural Changes in the Heme Pocket of Human Cytochrome P450 Aromatase during the Three Consecutive Oxygen Activation Steps

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