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)/␣-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, ␣-ketoglutarate, and the non-reactive enantiomer of the substrate, ent-1-deoxypentalenic acid, in four different crystal forms to up to 1.31 Å resolution. The overall structure of PtlH forms a double-stranded barrel helix fold, and the cofactor-binding site for iron and ␣-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 ␣-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.Streptomyces avermitilis is a Gram-positive soil-dwelling organism that is used industrially for the production of the widely used anthelminthic avermectins. Recently, an S. avermitilis gene cluster for the biosynthesis of the sesquiterpenoid antibiotic pentalenolactone ( Fig. 1A) has been characterized (1). Pentalenolactone was first identified from Streptomyces roseogriseus (2) and is also produced by several Streptomyces strains (3-5). The antibiotic is active against Gram-positive and Gram-negative bacteria as well as fungi and protozoa (6). In addition, it has been shown to inhibit viral replication of herpes simplex virus 1 and herpes simplex virus 2 (7) and cell proliferation of rat vascular smooth muscle cells (8). Pentalenolactone inhibits glycolysis (9) by selectively inhibiting glyceraldehyde-3-phosphate dehydrogenase (10). The antibiotic was shown to irreversibly inactivate glyceraldehyde-3-phosphate dehydrogenase by alkylation of the active site cysteine residue (11). An inducible, pentalenolactone-insensitive glyceraldehyde-3-phosphate dehydrogenase isozyme that is part of the biosynthetic gene cluster confers self-resistance against the antibiotic in S. avermitilis and other Streptomyces strains (1,(12)(13)(14).We recently reported the molecular cloning and biochemical characterization of PtlH, an essential non-heme iron-dependent dioxygenase in the pentalenolactone biosynthesis pathway (15). The enzyme catalyzes the Fe(II)-and ␣-ketoglutarate-dependent hydroxylation of 1-deoxypentalenic acid to 11␤-hydroxy-1-deoxypentalenic acid (Fig. 1B). Non-heme...

Oxidative cyclizations in orthosomycin biosynthesis expand the known chemistry of an oxygenase superfamily

“…Despite conservation of fold, the oligomerization state varies, with AviO1 and EvdO2 monomers, EvdO1 a dimer, and HygX a tetramer. Structural similarity searches support our sequence analysis suggesting that the orthosomycin-associated oxygenases are related to the PhyH subfamily of AKG/Fe(II)-dependent oxygenases (16)(17)(18)(19). Of note, the halogenases SyrB2 and CytC3 cluster near the orthosomycin-associated oxygenases (13,21).…”

“…S1). This orthosomycin-associated subfamily is most closely related to the phytanoyl-CoA 2-hydroxylase (PhyH) subfamily of AKG/Fe(II) oxygenases (16)(17)(18)(19). Our analysis further separates the orthosomycin-associated oxygenases into subgroups (Fig.…”

The Interactions in the Carboxyl Terminus of Human 4-Hydroxyphenylpyruvate Dioxygenase Are Critical to Mediate the Conformation of the Final Helix and the Tail to Shield the Active Site for Catalysis

“…Covering of the active site by a C-terminal extension is commonly observed in many 2-oxoglutarate-dependent oxygenases and it is assumed that the C-terminus functions as a gate and controls access to the active site and isolates the bound substrate during catalysis [11], [19]–[22]. Superimposing the crystal structures of P. fluorescens 4-HPPD and S. avermitilis 4-HPPD in complex with NTBC reveal significant differences in the position of C-terminal helix [10], [12].…”

Crystal Structure of Prolyl 4-Hydroxylase from Bacillus anthracis

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