Crystal structure of the non-heme iron dioxygenase 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, alpha-ketoglutarate, and the non-reactive enantiomer of the substrate, ent-1-deoxypentalenic acid, in four different crystal forms to up to 1.31 A resolution. The overall structure of PtlH forms a double-stranded barrel helix fold, and the cofactor-binding site for iron and alpha-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.

Figure 1

The PtlH reaction (A) The sesquiterpenoid antibiotic pentalenolactone produced by S. avermitilis. (B) The enzymatic reaction catalyzed by PtlH. (C) The unreactive substrate homolog ent-1-deoxypentalenic acid.

Figure 2

Mechanism of oxidation by Fe(II)/a-ketoglutarate-dependent dioxygenases. Intermediate G is the highly reactive ferryl-oxo complex.

Figure 3

Structure of PtlH (A) Stereo diagram showing a schematic representation of the structure of PtlH. The Fe(II) ion is shown as a light-green sphere, α-ketoglutarate is shown as sticks with carbon atoms in gray, and ent-1PL is shown as sticks with carbon atoms in blue. (B) Topology diagram of PtlH. The position of the β-strand not present in the PtlH DSBH fold is indicated with a gray arrow. (C) Final 2Fo-Fc electron density map (contoured at 1σ) of the active site region. Electron density for Fe(II), α-ketoglutarate, ent-1PL and the Fe(II)-coordinated solvent water molecule is shown in green, gray, blue and red, respectively.

Figure 4

The PtlH Active Site (A) Stereo diagram of Fe(II)/α-ketoglutarate binding site. The Fe(II) ion is shown as a green sphere, α-ketoglutarate is shown as sticks with carbon atoms in gray and the iron-coordinated solvent water molecule is shown as a small red sphere. (B) Coordination of ent-1PL in the PtlH active site. ent-1PL is shown in sticks with blue carbon atoms and solvent water molecules are shown as small salmon-colored spheres. (C, D) Surface illustration of PtlH colored by electrostatic potential in the absence of helix α10 (c, crystal form II) and with helix α10 shown as cartoon representation to indicate the closure of the active site in the presence of this helix. The bound ent-1PL molecule is shown in blue sticks; bound α-ketoglutarate is shown in gray sticks.

Figure 5

Structural Model of Product Binding (A) Orientation of ent-1PL in the PtlH active site (B) Model of the orientation of the reaction product, 11β-hydroxy-1-deoxypentalenic acid in the PtlH active site. The iron-coordinated water molecule (salmon) is not part of the model but is shown for reference.

Figure 6

Comparison of PtlH with Phytanoyl-CoA 2-Hydroxylase (A) Cartoon representation of a least-squares alignment of PtlH with phytanoyl-CoA 2-hydroxylase (Pdb Code: 2A1X). PtlH is colored in orange with the iron ion shown in green, α-ketoglutarate in gray sticks and ent-1PL in cyan sticks. Phytanoyl-CoA 2-hydroxylase and bound cofactors are shown in blue. (B) Comparison of the active site region after local least-squares alignment of Fe(II) and Fe(II)-coordinating histidine and aspartate residues. Color scheme as in (A).