Site-directed mutageneses of rat liver cytochrome P-450d: axial ligand and heme incorporation

Shimizu, Tôru; Hirano, Kenji; Takahashi, Masae; Hatano, Masahiro; Fujii‐Kuriyama, Yoshiaki

doi:10.1021/bi00411a035

Cited by 88 publications

(60 citation statements)

References 14 publications

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“…2A,B, lane 3). These results suggest that the Cys 441 of human PGI2 synthase is a critical residue for the catalytic activity of the enzyme as shown by P450d [20] and thromboxane synthase [21].…”

Section: Resultsmentioning

confidence: 74%

“…2C, lanes 2,4). Similarly, it was reported that the substitution of histidine for this cysteine in rat P450d caused a loss of catalytic activity of the enzyme [20]. In contrast, Tomura et al [22] reported that the substitution of histidine for this cysteine in Fusariurn oxysporum P450nor retained a weak but significant level of the enzymatic activity.…”

Section: Resultsmentioning

confidence: 97%

“…As shown in Fig. 1, comparison of the amino acid sequence of human PGIe synthase with P450s shows that the PGI2 synthase has a significant sequence similarity to other P450s in the C-terminal region including the Cys-pocket near the helix L. The cysteine residue in the pocket has been shown to constitute the fifth ligand of the heme iron [17][18][19][20][21]. Thus, it is very possible that this invariant cysteine residue with a polar side chain at position 441 of the PGI2 synthase plays an important role in the catalytic activity of the enzyme.…”

Section: Resultsmentioning

confidence: 99%

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Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys⁴⁴¹ of the Cys‐pocket, and Glu³⁴⁷ and Arg³⁵⁰ of the EXXR motif

et al. 1996

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The possible active site Cys 441 in the Cys-pocket and Glu 347 and Arg ~s° of the EXXR motif of the human prostacyclin synthase, which catalyzes the conversion of prostaglandin H2 to prostacyclin, were subjected to site-directed mutagenesis in order to understand the role of these residues in expressing the enzymatic activity. Five expression vectors encoding the mutant enzymes with a single replacement, Cys441Ala, Cys441Ser, Cys441His, Glu347Ala and Arg3S°Ala, as well as the wild-type enzyme were expressed in 293 cells. The microsomal fraction of the cells expressing the wild-type enzyme showed a specific activity of 96 nmol 6-keto-PGF1Jmin per mg protein. All of the mutant enzymes examined showed no detectable enzyme activity, although immunoblot analysis demonstrated that levels of all the expressed mutant enzymes were similar to that of the wild-type enzyme. These results indicated that the Cys 441 in the Cyspocket, and Glu 347 and Arg 35° of the EXXR motif of human prostacyclin synthase are important for expressing the enzymatic activity.

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Section: Resultsmentioning

confidence: 74%

Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys⁴⁴¹ of the Cys‐pocket, and Glu³⁴⁷ and Arg³⁵⁰ of the EXXR motif

et al. 1996

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“…Several groups have tried to replace the axial cysteine in P450, but their attempts were unsuccessful because of the low expression level of the mutants [24,25]. Accordingly, we developed a new expression system that produces apo-P450 cam as the inclusion bodies in E. coli [18].…”

mentioning

confidence: 99%

Proximal cysteine residue is essential for the enzymatic activities of cytochrome P450cam

Shiro¹,

Takahashi²,

Hori³

et al. 2001

Eur J Biochem

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To investigate the functional and structural roles of the proximal thiolate ligand in cytochrome P450 cam , we prepared the C357H mutant of the enzyme in which the axial cysteine residue (Cys357) was replaced with a histidine residue. We obtained the unstable C357H mutant by developing a new preparation procedure involving in vitro folding of P450 cam from the inclusion bodies. The C357H mutant in the ferrous-CO form exhibited the Soret peak at 420 nm and the Fe-CO stretching line at 498 cm 21 , indicating a neutral histidine residue as the axial ligand. However, another internal ligand is coordinated to the heme iron as the sixth ligand in the ferric and ferrous forms of the C357H mutant, suggesting the collapse of the substratebinding site. The C357H mutant showed no catalytic activity for camphor hydroxylation and the reduced heterolytic/ homolytic ratio of the O±O bond scission in the reaction with cumene hydroperoxide. The present observations indicate that the thiolate coordination in P450 cam is important for the construction of the heme pocket and the heterolysis of the O±O bond.Keywords: P450; CPO; P420; oxygen activation; axial push effect.Heme-thiolate enzymes such as cytochrome P450 (P450) and chloroperoxidase from Caldariomyces fumago (CPO) activate heme-bound dioxygen and hydrogen peroxide and insert oxygen atoms into various hydrocarbons [1±4]. The enzymes have attracted much interest because the heterolytic O±O bond scission of the heme-bound dioxygen and the C±H bond activation of inactivated hydrocarbons are difficult in mild conditions [1,5]. As the enzymes have an anionic thiolate as the axial heme ligand [6,7], the strong electron donation of the thiolate is supposed to facilitate the heterolysis of the dioxygen that coordinates trans to the thiolate ligand [8,9]. The importance of the thiolate coordination for the activities of these enzymes is exemplified by their inactivated forms (P420 and C420) which lack the thiolate coordination [10,11]. However, the roles of Fe±S bond that are important to understand the molecular mechanism of the enzyme activities are still controversial [12,13].To clarify the roles of the thiolate coordination in the heme-thiolate enzymes, we replaced the axial histidine of myoglobin (Mb) with cysteine (H93C), and investigated its catalytic reactivities [14,15]. We observed that the thiolate ligand of Mb H93C accelerated the heterolysis of the O±O bond of cumene hydroperoxide and enhanced the hydrogen peroxide supported epoxidation of styrene [15]. These observations allowed us to propose that the thiolate ligation in heme proteins encourages the heterolytic cleavage of the O±O bond [15]. Our proposal is further confirmed by Higuchi et al. who demonstrated that the ligation of alkyl thiolate to iron porphyrin facilitates the O±O bond heterolysis of alkyl peracid in hydrophobic solvent [16,17]. These model works indicate the importance of the thiolate coordination in the heterolytic O±O bond scission.We have also shown experimentally that the thiolate ligand control...

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“…5) (16). In all the known CYP51 genes isolated to date the same triplet encodes arginine, but other members of the superfamily have lysine at the same position where it was found in the mutant protein (16). This may explain the retention of catalytic activity by the mutant protein altered in this important region of all cytochrome P450 molecules, i.e., this alteration is compatible with a functional hemebinding environment.…”

Section: Discussionmentioning

confidence: 93%

The R467K Amino Acid Substitution in Candida albicans Sterol 14α-Demethylase Causes Drug Resistance through Reduced Affinity

Lamb

Kelly

White

et al. 2000

Antimicrob Agents Chemother

117

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The cytochrome P450 sterol 14␣-demethylase (CYP51) of Candida albicans is involved in an essential step of ergosterol biosynthesis and is the target for azole antifungal compounds. We have undertaken site-directed mutation of C. albicans CYP51 to produce a recombinant mutant protein with the amino acid substitution R467K corresponding to a mutation observed clinically. This alteration perturbed the heme environment causing an altered reduced-carbon monoxide difference spectrum with a maximum at 452 nm and reduced the affinity of the enzyme for fluconazole, as shown by ligand binding studies. The specific activity of CYP51(R467K) for the release of formic acid from 3␤-[32-3 H]hydroxylanost-7-en-32-ol was 70 pmol/nmol of P450/min for microsomal protein compared to 240 pmol/nmol of P450/min for microsomal fractions expressing wild-type CYP51. Furthermore, inhibition of activity by fluconazole revealed a 7.5-fold-greater azole resistance of the recombinant protein than that of the wild type. This study demonstrates that resistance observed clinically can result from the altered azole affinity of the fungal CYP51 enzyme.

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Site-directed mutageneses of rat liver cytochrome P-450d: axial ligand and heme incorporation

Cited by 88 publications

References 14 publications

Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys⁴⁴¹ of the Cys‐pocket, and Glu³⁴⁷ and Arg³⁵⁰ of the EXXR motif

Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys⁴⁴¹ of the Cys‐pocket, and Glu³⁴⁷ and Arg³⁵⁰ of the EXXR motif

Proximal cysteine residue is essential for the enzymatic activities of cytochrome P450cam

The R467K Amino Acid Substitution in Candida albicans Sterol 14α-Demethylase Causes Drug Resistance through Reduced Affinity

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Site-directed mutageneses of rat liver cytochrome P-450d: axial ligand and heme incorporation

Cited by 88 publications

References 14 publications

Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys441 of the Cys‐pocket, and Glu347 and Arg350 of the EXXR motif

Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys441 of the Cys‐pocket, and Glu347 and Arg350 of the EXXR motif

Proximal cysteine residue is essential for the enzymatic activities of cytochrome P450cam

The R467K Amino Acid Substitution in Candida albicans Sterol 14α-Demethylase Causes Drug Resistance through Reduced Affinity

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Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys⁴⁴¹ of the Cys‐pocket, and Glu³⁴⁷ and Arg³⁵⁰ of the EXXR motif

Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys⁴⁴¹ of the Cys‐pocket, and Glu³⁴⁷ and Arg³⁵⁰ of the EXXR motif