Structural basis for the role in protein folding of conserved proline-rich regions in cytochromes P450

Kemper, Byron

doi:10.1016/j.taap.2003.11.030

Cited by 42 publications

(25 citation statements)

References 21 publications

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“…SW disease is also a feature of the P30E mutation. In this case, the close contact (2.4 Å) between the carbon of P30 in the N-terminal loop and the side chain of Y376 within the β5-β6 hairpin loop is critical for protein folding (18). P30E disrupts this interaction, leading to structural instability.…”

Section: Resultsmentioning

confidence: 99%

Structure–phenotype correlations of human CYP21A2 mutations in congenital adrenal hyperplasia

Haider

Islam

D’Atri

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

114

130

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Mutations in the cytochrome p450 (CYP)21A2 gene, which encodes the enzyme steroid 21-hydroxylase, cause the majority of cases in congenital adrenal hyperplasia, an autosomal recessive disorder. To date, more than 100 CYP21A2 mutations have been reported. These mutations can be associated either with severe salt-wasting or simple virilizing phenotypes or with milder nonclassical phenotypes. Not all CYP21A2 mutations have, however, been characterized biochemically, and the clinical consequences of these mutations remain unknown. Using the crystal structure of its bovine homolog as a template, we have constructed a humanized model of CYP21A2 to provide comprehensive structural explanations for the clinical manifestations caused by each of the known disease-causing missense mutations in CYP21A2. Mutations that affect membrane anchoring, disrupt heme and/or substrate binding, or impair stability of CYP21A2 cause complete loss of function and salt-wasting disease. In contrast, mutations altering the transmembrane region or conserved hydrophobic patches cause up to a 98% reduction in enzyme activity and simple virilizing disease. Mild nonclassical disease can result from interference in oxidoreductase interactions, saltbridge and hydrogen-bonding networks, and nonconserved hydrophobic clusters. A simple in silico evaluation of previously uncharacterized gene mutations could, thus, potentially help predict the often diverse phenotypes of a monogenic disorder.structure-phenotype correlation | molecular modeling | CYP21A2 structural analysis

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Section: Resultsmentioning

confidence: 99%

Structure–phenotype correlations of human CYP21A2 mutations in congenital adrenal hyperplasia

Haider

Islam

D’Atri

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

114

130

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“…This amino acid is the first proline residue in the N-terminal prolinerich motif (PPGPXPXPXXGN), which has been suggested to play an important role in the proper folding of the P450 enzyme (Kemper, 2004). Substitutions of the first proline residue in CYP2C2 resulted in reduced (substitutions with Ala, Ile, and Leu) or null (with Asp, Glu, Gly, and Val) enzymatic activities.…”

Section: Discussionmentioning

confidence: 99%

“…When the CYP2C2 protein was expressed in insect cells, the Pro to Ala substitution at this site was reported to result in the reduced expression of functional cytochrome P450 hemoprotein and the increased expression of inactive cytochrome P420 (Chen et al, 1998). The amino acid alteration at this position was suggested to hamper the proper folding of CYP2C2 protein possibly by preventing interactions with other polypeptide regions (Kemper, 2004). A polymorphism of the first Pro site has also been reported in CYP2D6 as the *10 allele (Kagimoto et al, 1990).…”

Section: Discussionmentioning

confidence: 99%

Functional analysis of three CYP1A2 variants found in a Japanese population

Saito

Hanioka

Maekawa

et al. 2005

Drug Metabolism and Disposition

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ABSTRACT:Human cytochrome P450 1A2 (CYP1A2) catalyzes the metabolism of many important drugs and environmental chemicals. We previously reported three naturally occurring genetic polymorphisms (125C>G, Pro42Arg, CYP1A2*15; 1130G>A, Arg377Gln, *16; and 1367G>A, Arg456His, *8) found in a Japanese population. In this study, these variant enzymes were expressed in Chinese hamster V79 cells, and their mRNA and protein expression levels as well as catalytic activities were determined. All three variant enzymes showed reduced protein expression levels (66% for Pro42Arg and approximately 30% for Arg377Gln and Arg456His) compared with that of the wild type (WT) without any change in mRNA expression levels. Kinetic analysis for 7-ethoxyresorufin O-deethylation revealed that V max and V max /K m of all three variants were less than 3 and 1% of the WT, respectively, although the K m value was significantly increased only in the Arg377Gln variant (approximately a 9-fold increase). Markedly reduced activities of the three variants were also observed for phenacetin O-deethylation. In the reduced CO difference spectral analysis using recombinant proteins produced in the Sf21/baculovirus system, the peak at 450 nm seen in the WT protein was hardly observed in the three variants, suggesting marked reductions in their hemoprotein formation. These results suggest that Pro42, Arg377, and Arg456 are critical amino acids for the production of catalytically active CYP1A2 holoenzyme.

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“…To date, several functionally significant regions and residues in the P450s have been identified based on homology modeling and site-directed mutagenesis studies. For instance, the prolinerich motif (PPGPXPXPXXGN motif) in the N-terminal region of P450s plays a significant role in the proper assembly of the native enzyme (Kemper, 2004). In addition, Kerdpin et al (2004) have reported that SRS1 and SRS5 of CYP2C8 are important for paclitaxel binding, and that S114 in SRS1 is especially critical for the interaction with the substrate.…”

Section: Functional Characterization Of Cyp2c8 Variantsmentioning

confidence: 99%

Functional Characterization of Five Novel Cyp2c8 Variants, G171s, R186x, R186g, K247r, and K383n, Found in a Japanese Population

Hichiya¹,

Tanaka-Kagawa²,

Soyama³

et al. 2005

Drug Metab Dispos

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ABSTRACT:Cytochrome P450 2C8 is one of the primary enzymes responsible for the metabolism of a wide range of drugs such as paclitaxel, cerivastatin, and amiodarone. We have sequenced the CYP2C8 gene from 201 Japanese subjects and found five novel nonsynonymous single nucleotide polymorphisms (SNPs): 511G>A (G171S), 556C>T (R186X; X represents the translational stop codon), 556C>G (R186G), 740A>G (K247R), and 1149G>T (K383N), with the allele frequency of 0.0025. The CYP2C8 variants were heterologously expressed in COS-1 cells and functionally characterized in terms of expression level, paclitaxel 6␣-hydroxylase activity, and intracellular localization. The prematurely terminated R186X variant was undetectable by Western blotting and inactive toward paclitaxel 6␣-hydroxylation. The G171S, K247R, and K383N variants exhibited properties similar to those of the wild-type CYP2C8. Paclitaxel 6␣-hydroxylase activity of the R186G transfectant was only 10 to 20% that of wild-type CYP2C8. Furthermore, the R186G variant displayed a lower level of protein expression in comparison to the wild type, which was restored by the addition of a proteasome inhibitor (MG-132; Z-Leu-Leu-Leu-aldehyde). The reduced CO-difference spectral analysis using recombinant proteins from an insect cell/baculovirus system revealed that the R186G variant has a minor peak at 420 nm in addition to the characteristic Soret peak at 450 nm, suggesting the existence of improperly folded protein. These results indicate that the novel CYP2C8 SNPs, 556C>T (R186X) and 556C>G (R186G), could influence the metabolism of CYP2C8 substrates such as paclitaxel and cerivastatin.

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Structural basis for the role in protein folding of conserved proline-rich regions in cytochromes P450

Cited by 42 publications

References 21 publications

Structure–phenotype correlations of human CYP21A2 mutations in congenital adrenal hyperplasia

Structure–phenotype correlations of human CYP21A2 mutations in congenital adrenal hyperplasia

Functional analysis of three CYP1A2 variants found in a Japanese population

Functional Characterization of Five Novel Cyp2c8 Variants, G171s, R186x, R186g, K247r, and K383n, Found in a Japanese Population

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