PMID- 10406467
OWN - NLM
STAT- MEDLINE
DCOM- 19990921
LR  - 20161124
IS  - 0888-8809 (Print)
IS  - 0888-8809 (Linking)
VI  - 13
IP  - 7
DP  - 1999 Jul
TI  - Molecular modeling of human P450c17 (17alpha-hydroxylase/17,20-lyase): insights
      into reaction mechanisms and effects of mutations.
PG  - 1169-82
AB  - P450c17 (17alpha-hydroxylase/17,20-lyase) catalyzes steroid 17alpha-hydroxylase
      and 17,20-lyase activities in the biosynthesis of androgens and estrogens. These 
      two activities are differentially regulated in a tissue-specific and
      developmentally programmed manner. To visualize the active site topology of human
      P450c17 and to study the structural basis of its substrate specificity and
      catalytic selectivity, we constructed a second-generation computer-graphic model 
      of human P450c17. The energetics of the model are comparable to those of the
      principal template of the model, P450BMP, as determined from its crystallographic
      coordinates. The protein structure analysis programs PROCHECK, WHATIF, and SurVol
      indicate that the predicted P450c17 structure is reasonable. The hydrophobic
      active site accommodates both delta4 and delta5 steroid substrates in a
      catalytically favorable orientation. The predicted contributions of positively
      charged residues to the redox-partner binding site were confirmed by
      site-directed mutagenesis. Molecular dynamic simulations with pregnenolone,
      17-OH-pregnenolone, progesterone, and 17-OH-progesterone docked into the
      substrate-binding pocket demonstrated that regioselectivity of the hydroxylation 
      reactions is determined both by proximity of hydrogens to the iron-oxo complex
      and by the stability of the carbon radicals generated after hydrogen abstraction.
      The model explains the activities of all known naturally occurring and synthetic 
      human P450c17 mutants. The model predicted that mutation of lysine 89 would
      disrupt 17,20-lyase activity to a greater extent than 17alpha-hydroxylase
      activity; expression of a test mutant, K89N, in yeast confirmed this prediction. 
      Hydrogen peroxide did not support catalysis of the 17,20-lyase reaction, as would
      be predicted by mechanisms involving a ferryl peroxide. Our present model and
      biochemical data suggest that both the hydroxylase and lyase activities proceed
      from a common steroid-binding geometry by an iron oxene mechanism. This model
      will facilitate studies of sex steroid synthesis and its disorders and the design
      of specific inhibitors useful in chemotherapy of sex steroid-dependent cancers.
FAU - Auchus, R J
AU  - Auchus RJ
AD  - Department of Pediatrics, University of California, San Francisco 94143-0978,
      USA.
FAU - Miller, W L
AU  - Miller WL
LA  - eng
GR  - DK-37922/DK/NIDDK NIH HHS/United States
GR  - DK-42154/DK/NIDDK NIH HHS/United States
GR  - U54-HD-34449/HD/NICHD NIH HHS/United States
GR  - etc.
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Mol Endocrinol
JT  - Molecular endocrinology (Baltimore, Md.)
JID - 8801431
RN  - 0 (Bacterial Proteins)
RN  - 42VZT0U6YR (Heme)
RN  - 73R90F7MQ8 (Pregnenolone)
RN  - 9035-51-2 (Cytochrome P-450 Enzyme System)
RN  - EC 1.- (Mixed Function Oxygenases)
RN  - EC 1.14.14.19 (Steroid 17-alpha-Hydroxylase)
RN  - EC 1.6.2.4 (NADPH-Ferrihemoprotein Reductase)
RN  - EC 1.6.2.4 (flavocytochrome P450 BM3 monoxygenases)
SB  - IM
MH  - Amino Acid Sequence
MH  - *Bacterial Proteins
MH  - Binding Sites
MH  - Cytochrome P-450 Enzyme System/chemistry
MH  - Heme/metabolism
MH  - Humans
MH  - Mixed Function Oxygenases/chemistry
MH  - Models, Molecular
MH  - Molecular Sequence Data
MH  - Mutagenesis, Site-Directed
MH  - *Mutation
MH  - NADPH-Ferrihemoprotein Reductase
MH  - Pregnenolone/metabolism
MH  - Protein Conformation
MH  - Sequence Homology, Amino Acid
MH  - Steroid 17-alpha-Hydroxylase/*chemistry/genetics/*metabolism
EDAT- 1999/07/16 00:00
MHDA- 1999/07/16 00:01
CRDT- 1999/07/16 00:00
PHST- 1999/07/16 00:00 [pubmed]
PHST- 1999/07/16 00:01 [medline]
PHST- 1999/07/16 00:00 [entrez]
AID - 10.1210/mend.13.7.0326 [doi]
PST - ppublish
SO  - Mol Endocrinol. 1999 Jul;13(7):1169-82. doi: 10.1210/mend.13.7.0326.