Evidence of Allosteric Coupling between Substrate Binding and Adx Recognition in the Vitamin D Carbon-24 Hydroxylase CYP24A1

Kumar, Amit; Wilderman, P. Ross; Tu, Chengjian; Shen, Shichen; Qu, Jun; Estrada, D. Fernando

doi:10.1021/acs.biochem.0c00107

Cited by 12 publications

(20 citation statements)

References 40 publications

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“…More recently, Ivanov and associates used surface plasmon resonance (SPR) to estimate K d values for POR and Adx interactions with 12 different P450s [12]. The effect of substrate binding on the P450-Adx complex has been examined recently both by SPR [13] and solution NMR [14,15]. Solution NMR indicated that substrate binding altered the interaction of microsomal P450 17A1 with another auxiliary protein, cytochrome b 5 [16], which is consistent with more recent SPR studies [13].…”

Section: Introductionsupporting

confidence: 55%

Functional interactions of adrenodoxin with several human mitochondrial cytochrome P450 enzymes

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et al. 2020

Archives of Biochemistry and Biophysics

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Seven of the 57 human cytochrome P450 (P450) enzymes are mitochondrial and carry out important reactions with steroids and vitamins A and D. These seven P450s utilize an electron transport chain that includes NADPH, NADPH-adrenodoxin reductase (AdR), and adrenodoxin (Adx) instead of the diflavin NADPH-P450 reductase (POR) used by the other P450s in the endoplasmic reticulum. Although numerous studies have been published involving mitochondrial P450 systems, the experimental conditions vary considerably. We compared human Adx and bovine Adx, a commonly used component, and found very similar catalytic activities in reactions catalyzed by human P450s 11B2, 27A1, and 27C1. Binding constants of 6-200 nM were estimated for Adx binding to these P450s using microscale thermophoresis. All P450 catalytic reactions were saturated at 10 μM Adx, and higher concentrations were not inhibitory up to at least 50 μM. Collectively these studies demonstrate the tight binding of Adx (both human and bovine) to AdR and to several mitochondrial P450s and provide guidance for optimization of Adxdependent P450 reactions.

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Section: Introductionsupporting

confidence: 55%

Functional interactions of adrenodoxin with several human mitochondrial cytochrome P450 enzymes

Child

Reddish

Glass

et al. 2020

Archives of Biochemistry and Biophysics

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“…We have previously reported on the presence of long-range communication between the active site and the proximal Adx-binding surface of the corresponding interaction in rCYP24A1. 13,14 An interesting feature of hCYP24A1 is that, in contrast to other mitochondrial CYPs, 24,25 titration with unfused hAdx alone does not introduce a detectible high-spin character of the heme (Figure S2). This is also true for the spectra of the uncleaved hCYP24A1-hAdx protein.…”

Section: ■ Resultsmentioning

confidence: 99%

Characterization of a Cleavable Fusion of Human CYP24A1 with Adrenodoxin Reveals the Variable Role of Hydrophobics in Redox Partner Binding

2022

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The improper maintenance of the bioactivated form of vitamin-D (1α,25-(OH) 2 D) may result in vitamin-D insufficiency and therefore compromise the absorption of dietary calcium. A significant regulator of vitamin-D metabolism is the inactivating function of the mitochondrial enzyme cytochrome P450 24A1 (CYP24A1). In humans, CYP24A1 carries out hydroxylation of carbon-23 (C23) or carbon-24 (C24) of the 1α,25(OH) 2 D side chain, eventually resulting in production of either an antagonist of the vitamin-D receptor (C23 pathway) or calcitroic acid (C24 pathway). Despite its importance to human health, the human isoform (hCYP24A1) remains largely uncharacterized due in part to the difficulty in producing the enzyme using recombinant means. In this study, we utilize a cleavable fusion with the cognate redox partner, human Adx (hAdx), to stabilize hCYP24A1 during production. The subsequent cleavage and isolation of active hCYP24A1 allowed for an investigation of substrate and analog binding, enzymatic activity, and redox partner recognition. We demonstrate involvement of a nonpolar contact involving Leu-80 of hAdx and a nonconserved proximal surface of hCYP24A1. Interestingly, shortening the length of this residue (L80V) results in enhanced binding between the CYP-Adx complex and 1α,25(OH) 2 D yet unexpectedly results in decreased catalysis. The same mutation has a negligible effect on rat CYP24A1 (a C24-hydroxylase), indicating the presence of a species-specific requirement that may correlate with differences in regioselectivity of the reaction. Taken together, this work presents an example of production of a challenging human CYP as well as providing details regarding hydrophobic modulation of a CYP-Adx complex that is critical to human vitamin-D metabolism.

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“…Mutation of adrenodoxin Leu80 to lysine nearly abolished 11β-hydroxylase activity in both CYP11B enzymes ( 14 ). Conversely, for vitamin D 3 -metabolizing rat CYP24A1, the mutation L80K led to a tighter binding of adrenodoxin and did not abolish enzyme activity ( 33 ). In CYP11B1, mutation of the conserved Val441 to glycine is reported to abolish cortisol production in patients ( 34 ) and this could also be due to decreased interactions.…”

Section: Discussionmentioning

confidence: 99%

Structural and functional insights into aldosterone synthase interaction with its redox partner protein adrenodoxin

Brixius‐Anderko

Scott

2021

Journal of Biological Chemistry

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Evidence of Allosteric Coupling between Substrate Binding and Adx Recognition in the Vitamin D Carbon-24 Hydroxylase CYP24A1

Cited by 12 publications

References 40 publications

Functional interactions of adrenodoxin with several human mitochondrial cytochrome P450 enzymes

Functional interactions of adrenodoxin with several human mitochondrial cytochrome P450 enzymes

Characterization of a Cleavable Fusion of Human CYP24A1 with Adrenodoxin Reveals the Variable Role of Hydrophobics in Redox Partner Binding

Structural and functional insights into aldosterone synthase interaction with its redox partner protein adrenodoxin

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