TY - JOUR
T1 - Hybrid homology modeling and mutational analysis of cytochrome P450C24A1 (CYP24A1) of the Vitamin D pathway
T2 - Insights into substrate specificity and membrane bound structure-function
AU - Annalora, Andrew J.
AU - Bobrovnikov-Marjon, Ekaterina
AU - Serda, Rita
AU - Pastuszyn, Andrzej
AU - Graham, Sandra E.
AU - Marcus, Craig B.
AU - Omdahl, John L.
N1 - Copyright:
Copyright 2009 Elsevier B.V., All rights reserved.
PY - 2007/4/15
Y1 - 2007/4/15
N2 - Cytochrome P450C24A1 (CYP24A1), a peripheral inner mitochondrial membrane hemoprotein and candidate oncogene, regulates the side-chain metabolism and biological function of vitamin D and many of its related analog drugs. Rational mutational analysis of rat CYP24A1 based on hybrid (2C5/BM-3) homology modeling and affinity labeling studies clarified the role of key domains (N-terminus, A′, A, and F-helices, β3a strand, and β5 hairpin) in substrate binding and catalysis. The scope of our study was limited by an inability to purify stable mutant enzyme targeting soluble domains (B′, G, and I-helices) and suggested greater conformational flexibility among CYP24A1's membrane-associated domains. The most notable mutants developed by modeling were V391T and I500A, which displayed defective-binding function and profound metabolic defects for 25-hydroxylated vitamin D3 substrates similar to a non-functional F-helix mutant (F249T) that we previously reported. Val-391 (β3a strand) and Ile-500 (β5 hairpin) are modeled to interact with Phe-249 (F-helix) in a hydrophobic cluster that directs substrate-binding events through interactions with the vitamin D cis-triene moiety. Prior affinity labeling studies identified an amino-terminal residue (Ser-57) as a putative active-site residue that interacts with the 3β-OH group of the vitamin D A-ring. Studies with 3-epi and 3-deoxy-1,25(OH)2D3 analogs confirmed interactions between the 3β-OH group and Ser-57 effect substrate recognition and trafficking while establishing that the trans conformation of A-ring hydroxyl groups (1α and 3β) is obligate for high-affinity binding to rat CYP24A1. Our work suggests that CYP24A1's amphipathic nature allows for monotopic membrane insertion, whereby a pw2d-like substrate access channel is formed to shuttle secosteroid substrate from the membrane to the active-site. We hypothesize that CYP24A1 has evolved a unique amino-terminal membrane-binding motif that contributes to substrate specificity and docking through coordinated interactions with the vitamin D A-ring.
AB - Cytochrome P450C24A1 (CYP24A1), a peripheral inner mitochondrial membrane hemoprotein and candidate oncogene, regulates the side-chain metabolism and biological function of vitamin D and many of its related analog drugs. Rational mutational analysis of rat CYP24A1 based on hybrid (2C5/BM-3) homology modeling and affinity labeling studies clarified the role of key domains (N-terminus, A′, A, and F-helices, β3a strand, and β5 hairpin) in substrate binding and catalysis. The scope of our study was limited by an inability to purify stable mutant enzyme targeting soluble domains (B′, G, and I-helices) and suggested greater conformational flexibility among CYP24A1's membrane-associated domains. The most notable mutants developed by modeling were V391T and I500A, which displayed defective-binding function and profound metabolic defects for 25-hydroxylated vitamin D3 substrates similar to a non-functional F-helix mutant (F249T) that we previously reported. Val-391 (β3a strand) and Ile-500 (β5 hairpin) are modeled to interact with Phe-249 (F-helix) in a hydrophobic cluster that directs substrate-binding events through interactions with the vitamin D cis-triene moiety. Prior affinity labeling studies identified an amino-terminal residue (Ser-57) as a putative active-site residue that interacts with the 3β-OH group of the vitamin D A-ring. Studies with 3-epi and 3-deoxy-1,25(OH)2D3 analogs confirmed interactions between the 3β-OH group and Ser-57 effect substrate recognition and trafficking while establishing that the trans conformation of A-ring hydroxyl groups (1α and 3β) is obligate for high-affinity binding to rat CYP24A1. Our work suggests that CYP24A1's amphipathic nature allows for monotopic membrane insertion, whereby a pw2d-like substrate access channel is formed to shuttle secosteroid substrate from the membrane to the active-site. We hypothesize that CYP24A1 has evolved a unique amino-terminal membrane-binding motif that contributes to substrate specificity and docking through coordinated interactions with the vitamin D A-ring.
KW - 1,25-dihydroxyvitamin D3
KW - 25-hydroxyvitamin D3 24R-hydroxylase
KW - Affinity labeling
KW - Calcitriol
KW - CYP24A1
KW - CYPs
KW - Cytochrome P450
KW - Homology modeling
KW - Monotopic membrane protein
KW - P450C24A1
KW - Site-directed mutagenesis
KW - Vitamin D
KW - Vitamin D inactivation
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UR - http://www.scopus.com/inward/citedby.url?scp=34247130103&partnerID=8YFLogxK
U2 - 10.1016/j.abb.2006.11.018
DO - 10.1016/j.abb.2006.11.018
M3 - Article
C2 - 17207766
AN - SCOPUS:34247130103
VL - 460
SP - 262
EP - 273
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
SN - 0003-9861
IS - 2
ER -