Role of THR-252 in Cytochrome P450CAM: A Study with Unnatural Amino Acid Mutagenesis

Kimata, Yoko; Shimada, Hideo; Hirose, Tatsuro; Ishimura, Yuzuru

doi:10.1006/bbrc.1995.1310

Cited by 122 publications

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“…On the basis of the Thr switch model and our current results, we propose the heme active site structure of the oxygen complex of P450arom as shown in Figure 8. Because extensive mechanistic studies on P450cam demonstrated that compound I formation requires protonation of the distal oxygen atom in the oxygen complex by the water molecule hydrogenbonded to the conserved Thr (56,60,61), the O 2 ligand would be hydrogen-bonded to the water molecule in the first and second oxidative steps in P450arom (Figure 8a). On the other hand, the plausible hydrogen-bonding interaction between the aldehyde group in 19-aldo-AD and Thr-310 could induce the displacement of Thr-310 and the water molecule, which could cause the disruption of the hydrogenbonding interaction between ligated O 2 and the water molecule (Figure 8b).…”

Section: Structural Effects Of Substrate Binding On the Heme Environmmentioning

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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Section: Structural Effects Of Substrate Binding On the Heme Environmmentioning

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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Section: Discussionmentioning

confidence: 99%

“…However, the two catalytic water molecules are retained in the crystal structure of the T252A mutant, which suggests that the role of Thr252 is not to hold the active site water molecules in place, and that uncoupling does not result from disordering of the two active site water molecules [21]. The early finding that the Thr252 side chain hydroxyl group can be replaced by a methoxy group without a significant change in the coupling efficiency indicates that Thr252 does not serve as a hydrogen bond or proton donor in the dioxygen activation process [7]. It is therefore likely that Thr252 serves as a hydrogen bond acceptor from the ferric hydroperoxy intermediate.…”

Section: Discussionmentioning

confidence: 99%

“…The crystal structures of ferrous camphor-and CO-bound P450 cam suggest that Thr252 is a key catalytic residue. This inference is substantiated by the fact that mutation of Thr252 to an Ala, Val, or Leu produces enzymes in which substrate hydroxylation is highly uncoupled from electron transfer [4][5][6][7], whereas replacement by a Ser yields a catalytically active enzyme [6]. In one instance is the conserved Thr replaced by a non-hydrogen bonding moiety.…”

mentioning

confidence: 99%

“…As noted in the introduction, several roles can be envisioned for the conserved distal Thr [11]. The finding that replacement of the hydroxyl of Thr252 with a methoxy group in P450 cam yields an active, coupled enzyme indicates that the role of the hydroxyl is likely to be as a proton acceptor rather than proton donor [7].To investigate the molecular basis for the retention of coupling efficiency in the T252N mutant, we have carried out a modeling study using the crystal structures of the ferrous dioxygen complexes of wild-type P450 cam and its T252A mutant [2,21]. Proton transfer is required for scission of the iron-bound dioxygen bond to give the Fe(IV)=O hydroxylating species and it was proposed that the required protons are delivered by a proton shuttle involving a hydrogenbonding network that includes two water molecules [3].…”

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Efficient catalytic turnover of cytochrome P450cam is supported by a T252N mutation

Kim

Heo

Montellano

2008

Archives of Biochemistry and Biophysics

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A Thr (or Ser) residue on the I-helix is a highly conserved structural feature of cytochrome P450 enzymes. It is believed to be indispensable as a proton delivery shuttle in the oxygen activation process. Previous work showed that P450 cin (CYP176A1), which contains an Asn instead of the conserved Thr, is fully functional in the catalytic oxidation of cineole [Hawkes, D.B. et al. (2002) J. Biol. Chem. 277, 27725-27732]. To determine whether the substitution of Asn for Thr is specific or general, the conserved Thr252 in P450 cam (CYP101) was mutated to generate the T252N, T252N/ V253T, and T252A mutants. Steady-state kinetic analysis of the oxidation of camphor by these mutants indicated that the T252N and T252N/V253T mutants have comparable turnover numbers but higher K m values relative to the wild-type enzyme. Spectroscopic binding assays indicate that the higher K m values reflect a decrease in the camphor binding affinity. Non-productive H 2 O 2 generation was negligible with the T252N and T252N/V253T mutants, but, as previously observed, was dominant in the T252A mutant. Our results, and a structure model based on the crystal structures of the ferrous dioxygen complexes of P450 cam and its T252A mutant, suggest that Asn252 can stabilize the ferric hydroperoxy intermediate, preventing premature release of H 2 O 2 and enabling addition of the second proton to the distal oxygen to generate the catalytic ferryl species.The cytochrome P450 (CYP or P450) family of enzymes is involved in oxidative metabolism of a wide range of endo-and exogenous chemicals [1]. P450 cam (CYP101) from Pseudomonas putida, the structurally and biochemically best characterized P450 enzyme [2], catalyzes the regio-and stereospecific hydroxylation of camphor to 5-exo-hydroxycamphor. As first established for P450 cam , the general P450 catalytic cycle involves (a) substrate binding, (b) reduction of the iron from the ferric to the ferrous state, (c) Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Abbreviations used: CYP (P450), cytochrome P450; PCR, polymerase chain reaction; ORF, open reading frames; Pd, putidaredoxin; PdR, putidaredoxin reductase; TB, Terrific Broth; GC-MS, gas chromatography-mass spectrometry. K d indicates a dissociation constant estimated by measurement of the UV-visible changes in the P450 heme spectrum. An acid-alcohol side-chain pair, typically an Asp and Thr, is thought in most P450 enzymes to be a requirement for productive dioxygen activation in P450 catalysis [3]. The crystal structures of ferrous camphor-and CO-bound P450 cam suggest that Thr252 is a key catal...

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Inhibitors of cytochrome P450 catalyzed insecticide metabolism: A rational approach

Keserü

Kolossváry²,

Szekely³

1999

Int. J. Quant. Chem.

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ABSTRACT:The inhibition of cytochrome P450 mediated oxidative metabolism affected by methylenedioxyphenyl synergists was studied by theoretical methods. Binding w Ž . conformations of safrole-based inhibitors piperonyl butoxide PBO , safrole, and x izosafrole were obtained by the recently developed low-mode conformational search within the active site of cytochrome P450 . Increased activity of PBO was rationalized cam by the steric block created by its long side chain in the substrate access channel of the Ž . enzyme. Stability of the Fe II ᎐carbene complex was confirmed by quantum chemical calculations. In addition to the effect of back-donation, the role of the carbene orientation Ž . was explored. Molecular dynamics MD simulations revealed that hydrogen bonding Ž . between the carbene oxygen and Thr252-OH might stabilize the Fe II complex. Changes in the coordination of axial thiolate during carbene complexation were found to be crucial for the inhibitory action. Decreasing redox potential of the enzyme is caused by the increasing thiol character of the proposed drifting of the axial ligand stabilized by NH...S hydrogen bonds with Gly359 and Leu358.

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Role of THR-252 in Cytochrome P450CAM: A Study with Unnatural Amino Acid Mutagenesis

Cited by 122 publications

References 0 publications

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

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

Efficient catalytic turnover of cytochrome P450cam is supported by a T252N mutation

Inhibitors of cytochrome P450 catalyzed insecticide metabolism: A rational approach

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