Adam Zalewski1, Nina S Ma1, Balazs Legeza1, Nora Renthal1, Christa E Flück1, Amit V Pandey1. 1. Division of Pediatric Endocrinology, Diabetology, and Metabolism (A.Z., B.L., C.E.F., A.V.P.), Department of Pediatrics, University Children's Hospital, Inselspital, Bern, and Department of Clinical Research, University of Bern, CH-3010 Bern, Switzerland; and Division of Endocrinology (N.S.M., N.R.), Boston Children's Hospital, and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02114.
Abstract
CONTEXT: CYP27B1 converts 25-hydroxyvitamin D3 to active 1,25-dihydroxyvitamin D3, playing a vital role in calcium homeostasis and bone growth. Vitamin D-dependent rickets type 1 (VDDR-1) is a rare autosomal recessive disorder caused by mutations in CYP27B1. OBJECTIVE: The objective of the study was an enzymatic and structural analysis of mutations in a patient with calcipenic rickets. Design, Setting, Patient, and Intervention: Two siblings presented with calcipenic rickets and normal 1,25-dihydroxyvitamin D3 levels. CYP27B1 gene analysis showed compound heterozygous mutations confirming VDDR-1. We studied wild-type CYP27B1 and mutations H441Y and R459L by computational homology modeling, molecular dynamics simulations, and functional studies using a luciferase assay. The patients were successfully treated with calcitriol. MAIN OUTCOME: The main outcomes of the study were novel mutations leading to a severe loss of CYP27B1 activities for metabolism of 25-hydroxyvitamin D3. RESULTS: Mitochondrial cytochrome P450s require adrenodoxin (FDX1) and adrenodoxin reductase. We created models of CYP27B1-FDX1 complex, which revealed negative effects of mutations H441Y and R459L. Upon structural analysis, near-identical folds, protein contact areas, and orientations of heme/iron-sulfur cluster suggested that both mutations may destabilize the CYP27B1-FDX1 complex by negating directional interactions with adrenodoxin. This system is highly sensitive to small local changes modulating the binding/dissociation of adrenodoxin, and electron-transporting efficiency might change with mutations at the surface. Functional assays confirmed this hypothesis and showed severe loss of activity of CYP27B1 by both mutations. CONCLUSIONS: This is the first report of mutations in CYP27B1 causing VDDR-1 by affecting protein-protein interactions with FDX1 that results in reduced CYP27B1 activities. Detailed characterization of mutations in CYP27B1 is required for understanding the novel molecular mechanisms causing VDDR-1.
CONTEXT: CYP27B1 converts 25-hydroxyvitamin D3 to active 1,25-dihydroxyvitamin D3, playing a vital role in calcium homeostasis and bone growth. Vitamin D-dependent rickets type 1 (VDDR-1) is a rare autosomal recessive disorder caused by mutations in CYP27B1. OBJECTIVE: The objective of the study was an enzymatic and structural analysis of mutations in a patient with calcipenic rickets. Design, Setting, Patient, and Intervention: Two siblings presented with calcipenic rickets and normal 1,25-dihydroxyvitamin D3 levels. CYP27B1 gene analysis showed compound heterozygous mutations confirming VDDR-1. We studied wild-type CYP27B1 and mutations H441Y and R459L by computational homology modeling, molecular dynamics simulations, and functional studies using a luciferase assay. The patients were successfully treated with calcitriol. MAIN OUTCOME: The main outcomes of the study were novel mutations leading to a severe loss of CYP27B1 activities for metabolism of 25-hydroxyvitamin D3. RESULTS: Mitochondrial cytochrome P450s require adrenodoxin (FDX1) and adrenodoxin reductase. We created models of CYP27B1-FDX1 complex, which revealed negative effects of mutations H441Y and R459L. Upon structural analysis, near-identical folds, protein contact areas, and orientations of heme/iron-sulfur cluster suggested that both mutations may destabilize the CYP27B1-FDX1 complex by negating directional interactions with adrenodoxin. This system is highly sensitive to small local changes modulating the binding/dissociation of adrenodoxin, and electron-transporting efficiency might change with mutations at the surface. Functional assays confirmed this hypothesis and showed severe loss of activity of CYP27B1 by both mutations. CONCLUSIONS: This is the first report of mutations in CYP27B1 causing VDDR-1 by affecting protein-protein interactions with FDX1 that results in reduced CYP27B1 activities. Detailed characterization of mutations in CYP27B1 is required for understanding the novel molecular mechanisms causing VDDR-1.
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