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Title: Crystal Structure of the Non-heme Iron Dixoygenase PtlH in Pentalenolactone Biosynthesis

Abstract

The non-heme iron dioxygenase PtlH from the soil organism Streptomyces avermitilis is a member of the iron(II)/{alpha}-ketoglutarate-dependent dioxygenase superfamily and catalyzes an essential reaction in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone. To investigate the structural basis for substrate recognition and catalysis, we have determined the x-ray crystal structure of PtlH in several complexes with the cofactors iron, a-ketoglutarate, and the non-reactive enantiomer of the substrate, ent-1-deoxypentalenic acid, in four different crystal forms to up to 1.31 Angstroms resolution. The overall structure of PtlH forms a double-stranded barrel helix fold, and the cofactor-binding site for iron and a-ketoglutarate is similar to other double-stranded barrel helix fold enzymes. Additional secondary structure elements that contribute to the substrate-binding site in PtlH are not conserved in other double-stranded barrel helix fold enzymes. Binding of the substrate enantiomer induces a reorganization of the monoclinic crystal lattice leading to a disorder-order transition of a C-terminal {alpha}-helix. The newly formed helix blocks the major access to the active site and effectively traps the bound substrate. Kinetic analysis of wild type and site-directed mutant proteins confirms a critical function of two arginine residues in substrate binding, while simulated docking of the enzymatic reaction product reveals the likelymore » orientation of bound substrate.« less

Authors:
; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
959570
Report Number(s):
BNL-82556-2009-JA
Journal ID: ISSN 0021-9258; JBCHA3; TRN: US201016%%714
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Biological Chemistry; Journal Volume: 282; Journal Issue: 50
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; ANTIBIOTICS; ARGININE; BIOSYNTHESIS; CATALYSIS; CRYSTAL LATTICES; CRYSTAL STRUCTURE; ENZYMES; IRON; KINETICS; MUTANTS; ORIENTATION; PROTEINS; RESIDUES; RESOLUTION; SOILS; STREPTOMYCES; SUBSTRATES; national synchrotron light source

Citation Formats

You,Z., Omura, S., Ikeda, H., Cane, D., and Jogl, G.. Crystal Structure of the Non-heme Iron Dixoygenase PtlH in Pentalenolactone Biosynthesis. United States: N. p., 2007. Web. doi:10.1074/jbc.M706358200.
You,Z., Omura, S., Ikeda, H., Cane, D., & Jogl, G.. Crystal Structure of the Non-heme Iron Dixoygenase PtlH in Pentalenolactone Biosynthesis. United States. doi:10.1074/jbc.M706358200.
You,Z., Omura, S., Ikeda, H., Cane, D., and Jogl, G.. Mon . "Crystal Structure of the Non-heme Iron Dixoygenase PtlH in Pentalenolactone Biosynthesis". United States. doi:10.1074/jbc.M706358200.
@article{osti_959570,
title = {Crystal Structure of the Non-heme Iron Dixoygenase PtlH in Pentalenolactone Biosynthesis},
author = {You,Z. and Omura, S. and Ikeda, H. and Cane, D. and Jogl, G.},
abstractNote = {The non-heme iron dioxygenase PtlH from the soil organism Streptomyces avermitilis is a member of the iron(II)/{alpha}-ketoglutarate-dependent dioxygenase superfamily and catalyzes an essential reaction in the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone. To investigate the structural basis for substrate recognition and catalysis, we have determined the x-ray crystal structure of PtlH in several complexes with the cofactors iron, a-ketoglutarate, and the non-reactive enantiomer of the substrate, ent-1-deoxypentalenic acid, in four different crystal forms to up to 1.31 Angstroms resolution. The overall structure of PtlH forms a double-stranded barrel helix fold, and the cofactor-binding site for iron and a-ketoglutarate is similar to other double-stranded barrel helix fold enzymes. Additional secondary structure elements that contribute to the substrate-binding site in PtlH are not conserved in other double-stranded barrel helix fold enzymes. Binding of the substrate enantiomer induces a reorganization of the monoclinic crystal lattice leading to a disorder-order transition of a C-terminal {alpha}-helix. The newly formed helix blocks the major access to the active site and effectively traps the bound substrate. Kinetic analysis of wild type and site-directed mutant proteins confirms a critical function of two arginine residues in substrate binding, while simulated docking of the enzymatic reaction product reveals the likely orientation of bound substrate.},
doi = {10.1074/jbc.M706358200},
journal = {Journal of Biological Chemistry},
number = 50,
volume = 282,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}