3VRM

Structure of cytochrome P450 Vdh mutant T107A with bound vitamin D3

Summary for 3VRM

• Entry DOI: 10.2210/pdb3vrm/pdb
• Related: 3A4G 3A4H 3A4Z 3A50 3A51
• Descriptor: Vitamin D(3) 25-hydroxylase, PROTOPORPHYRIN IX CONTAINING FE, (1S,3Z)-3-[(2E)-2-[(1R,3AR,7AS)-7A-METHYL-1-[(2R)-6-METHYLHEPTAN-2-YL]-2,3,3A,5,6,7-HEXAHYDRO-1H-INDEN-4-YLIDENE]ETHYLI DENE]-4-METHYLIDENE-CYCLOHEXAN-1-OL, ... (4 entities in total)
• Functional Keywords: cytochrome p450, vitamin d3, monooxygenase, oxidoreductase, p450 fold, hemoprotein
• Biological source: Pseudonocardia autotrophica (Amycolata autotrophica)
• Cellular location: Cytoplasm (By similarity): C4B644
• Total number of polymer chains: 1
• Total formula weight: 46458.70

Authors

Nishioka, T.,Yasutake, Y.,Tamura, T. (deposition date: 2012-04-12, release date: 2013-04-17, Last modification date: 2023-11-08)

Primary citation

Yasutake, Y.,Nishioka, T.,Imoto, N.,Tamura, T.
A single mutation at the ferredoxin binding site of p450 vdh enables efficient biocatalytic production of 25-hydroxyvitamin d3.
Chembiochem, 14:2284-2291, 2013

PubMed Abstract: Vitamin D3 hydroxylase (Vdh) from Pseudonocardia autotrophica is a cytochrome P450 monooxygenase that catalyzes the two-step hydroxylation of vitamin D3 (VD3 ) to produce 25-hydroxyvitamin D3 (25(OH)VD3 ) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2 VD3 ). These hydroxylated forms of VD3 are useful as pharmaceuticals for the treatment of conditions associated with VD3 deficiency and VD3 metabolic disorder. Herein, we describe the creation of a highly active T107A mutant of Vdh by engineering the putative ferredoxin-binding site. Crystallographic and kinetic analyses indicate that the T107A mutation results in conformational change from an open to a closed state, thereby increasing the binding affinity with ferredoxin. We also report the efficient biocatalytic synthesis of 25(OH)VD3 , a promising intermediate for the synthesis of various hydroxylated VD3 derivatives, by using nisin-treated Rhodococcus erythropolis cells containing VdhT107A . The gene-expression cassette encoding Bacillus megaterium glucose dehydrogenase-IV was inserted into the R. erythropolis chromosome and expressed to avoid exhaustion of NADH in a cytoplasm during bioconversion. As a result, approximately 573 μg mL(-1) 25(OH)VD3 was successfully produced by a 2 h bioconversion.

PubMed: 24115473
DOI: 10.1002/cbic.201300386

Experimental method

X-RAY DIFFRACTION (2.57 Å)