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Title: Functional Insights into Human HMG-CoA Lyase from Structures of Acyl-CoA-containing Ternary Complexes

Abstract

HMG-CoA lyase (HMGCL) is crucial to ketogenesis, and inherited human mutations are potentially lethal. Detailed understanding of the HMGCL reaction mechanism and the molecular basis for correlating human mutations with enzyme deficiency have been limited by the lack of structural information for enzyme liganded to an acyl-CoA substrate or inhibitor. Crystal structures of ternary complexes of WT HMGCL with the competitive inhibitor 3-hydroxyglutaryl-CoA and of the catalytically deficient HMGCL R41M mutant with substrate HMG-CoA have been determined to 2.4 and 2.2 {angstrom}, respectively. Comparison of these {beta}/{alpha}-barrel structures with those of unliganded HMGCL and R41M reveals substantial differences for Mg{sup 2+} coordination and positioning of the flexible loop containing the conserved HMGCL 'signature' sequence. In the R41M-Mg{sup 2+}-substrate ternary complex, loop residue Cys{sup 266} (implicated in active-site function by mechanistic and mutagenesis observations) is more closely juxtaposed to the catalytic site than in the case of unliganded enzyme or the WT enzyme-Mg{sup 2+}-3-hydroxyglutaryl-CoA inhibitor complex. In both ternary complexes, the S-stereoisomer of substrate or inhibitor is specifically bound, in accord with the observed Mg{sup 2+} liganding of both C3 hydroxyl and C5 carboxyl oxygens. In addition to His{sup 233} and His{sup 235} imidazoles, other Mg{sup 2+} ligands are the Asp{supmore » 42} carboxyl oxygen and an ordered water molecule. This water, positioned between Asp{sup 42} and the C3 hydroxyl of bound substrate/inhibitor, may function as a proton shuttle. The observed interaction of Arg{sup 41} with the acyl-CoA C1 carbonyl oxygen explains the effects of Arg{sup 41} mutation on reaction product enolization and explains why human Arg{sup 41} mutations cause drastic enzyme deficiency.« less

Authors:
; ; ; ;  [1]
  1. MCW
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE
OSTI Identifier:
1002603
Resource Type:
Journal Article
Journal Name:
J. Biol. Chem.
Additional Journal Information:
Journal Volume: 285; Journal Issue: 2010; Journal ID: ISSN 0021-9258
Country of Publication:
United States
Language:
ENGLISH
Subject:
36 MATERIALS SCIENCE; CARBONYLS; CRYSTAL STRUCTURE; ENZYMES; FUNCTIONALS; IMIDAZOLES; LYASES; MUTAGENESIS; MUTANTS; MUTATIONS; OXYGEN; POSITIONING; PROTONS; REACTION KINETICS; RESIDUES; SUBSTRATES; WATER

Citation Formats

Fu, Zhuji, Runquist, Jennifer A, Montgomery, Christa, Miziorko, Henry M, Kim, Jung-Ja P, and UMKC). Functional Insights into Human HMG-CoA Lyase from Structures of Acyl-CoA-containing Ternary Complexes. United States: N. p., 2010. Web. doi:10.1074/jbc.M110.139931.
Fu, Zhuji, Runquist, Jennifer A, Montgomery, Christa, Miziorko, Henry M, Kim, Jung-Ja P, & UMKC). Functional Insights into Human HMG-CoA Lyase from Structures of Acyl-CoA-containing Ternary Complexes. United States. https://doi.org/10.1074/jbc.M110.139931
Fu, Zhuji, Runquist, Jennifer A, Montgomery, Christa, Miziorko, Henry M, Kim, Jung-Ja P, and UMKC). 2010. "Functional Insights into Human HMG-CoA Lyase from Structures of Acyl-CoA-containing Ternary Complexes". United States. https://doi.org/10.1074/jbc.M110.139931.
@article{osti_1002603,
title = {Functional Insights into Human HMG-CoA Lyase from Structures of Acyl-CoA-containing Ternary Complexes},
author = {Fu, Zhuji and Runquist, Jennifer A and Montgomery, Christa and Miziorko, Henry M and Kim, Jung-Ja P and UMKC)},
abstractNote = {HMG-CoA lyase (HMGCL) is crucial to ketogenesis, and inherited human mutations are potentially lethal. Detailed understanding of the HMGCL reaction mechanism and the molecular basis for correlating human mutations with enzyme deficiency have been limited by the lack of structural information for enzyme liganded to an acyl-CoA substrate or inhibitor. Crystal structures of ternary complexes of WT HMGCL with the competitive inhibitor 3-hydroxyglutaryl-CoA and of the catalytically deficient HMGCL R41M mutant with substrate HMG-CoA have been determined to 2.4 and 2.2 {angstrom}, respectively. Comparison of these {beta}/{alpha}-barrel structures with those of unliganded HMGCL and R41M reveals substantial differences for Mg{sup 2+} coordination and positioning of the flexible loop containing the conserved HMGCL 'signature' sequence. In the R41M-Mg{sup 2+}-substrate ternary complex, loop residue Cys{sup 266} (implicated in active-site function by mechanistic and mutagenesis observations) is more closely juxtaposed to the catalytic site than in the case of unliganded enzyme or the WT enzyme-Mg{sup 2+}-3-hydroxyglutaryl-CoA inhibitor complex. In both ternary complexes, the S-stereoisomer of substrate or inhibitor is specifically bound, in accord with the observed Mg{sup 2+} liganding of both C3 hydroxyl and C5 carboxyl oxygens. In addition to His{sup 233} and His{sup 235} imidazoles, other Mg{sup 2+} ligands are the Asp{sup 42} carboxyl oxygen and an ordered water molecule. This water, positioned between Asp{sup 42} and the C3 hydroxyl of bound substrate/inhibitor, may function as a proton shuttle. The observed interaction of Arg{sup 41} with the acyl-CoA C1 carbonyl oxygen explains the effects of Arg{sup 41} mutation on reaction product enolization and explains why human Arg{sup 41} mutations cause drastic enzyme deficiency.},
doi = {10.1074/jbc.M110.139931},
url = {https://www.osti.gov/biblio/1002603}, journal = {J. Biol. Chem.},
issn = {0021-9258},
number = 2010,
volume = 285,
place = {United States},
year = {Mon Aug 16 00:00:00 EDT 2010},
month = {Mon Aug 16 00:00:00 EDT 2010}
}