Human P450 CYP17A1: Control of Substrate Preference by Asparagine 202. Biochemistry 2018 Feb 06;57(5):764-771
Date
12/29/2017Pubmed ID
29283561Pubmed Central ID
PMC5801141DOI
10.1021/acs.biochem.7b01067Scopus ID
2-s2.0-85041471825 (requires institutional sign-in at Scopus site) 11 CitationsAbstract
CYP17A1 is a key steroidogenic enzyme known to conduct several distinct chemical transformations on multiple substrates. In its hydroxylase activity, this enzyme adds a hydroxyl group at the 17α position of both pregnenolone and progesterone at approximately equal rates. However, the subsequent 17,20 carbon-carbon scission reaction displays variable substrate specificity in the numerous CYP17A1 isozymes operating in vertebrates, manifesting as different Kd and kcat values when presented with 17α-hydroxypregnenlone (OHPREG) versus 17α-hydroxyprogesterone (OHPROG). Here we show that the identity of the residue at position 202 in human CYP17A1, thought to form a hydrogen bond with the A-ring alcohol substituent on the pregnene- nucleus, is a key driver of this enzyme's native preference for OHPREG. Replacement of asparagine 202 with serine completely reverses the preference of CYP17A1, more than doubling the rate of turnover of the OHPROG to androstenedione reaction and substantially decreasing the rate of formation of dehydroepiandrosterone from OHPREG. In a series of resonance Raman experiments, it was observed that, in contrast with the case for the wild-type protein, in the mutant the 17α alcohol of OHPROG tends to form a H-bond with the proximal rather than terminal oxygen of the oxy-ferrous complex. When OHPREG was a substrate, the mutant enzyme was found to have a H-bonding interaction with the proximal oxygen that is substantially weaker than that of the wild type. These results demonstrate that a single-point mutation in the active site pocket of CYP17A1, even when far from the heme, has profound effects on steroidogenic selectivity in androgen biosynthesis.
Author List
Gregory MC, Mak PJ, Khatri Y, Kincaid JR, Sligar SGAuthor
James Kincaid PhD Department Chair and Professor, Biophysical Chemistry in the Chemistry department at Marquette UniversityMESH terms used to index this publication - Major topics in bold
17-alpha-Hydroxypregnenolone17-alpha-Hydroxyprogesterone
Amino Acid Sequence
Amino Acid Substitution
Androstenedione
Animals
Catalysis
Catalytic Domain
Conserved Sequence
Dehydroepiandrosterone
Genes, Synthetic
Humans
Hydrogen Bonding
Mammals
Models, Molecular
Mutation, Missense
Point Mutation
Protein Binding
Protein Conformation
Recombinant Proteins
Sequence Alignment
Sequence Homology, Amino Acid
Steroid 17-alpha-Hydroxylase
Substrate Specificity
