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Structural analysis of an open active site conformation of nonheme iron halogenase CytC3.

ABSTRACT: CytC3, a member of the recently discovered class of nonheme Fe(II) and alpha-ketoglutarate (alphaKG)-dependent halogenases, catalyzes the double chlorination of L-2-aminobutyric acid (Aba) to produce a known Streptomyces antibiotic, gamma,gamma-dichloroaminobutyrate. Unlike the majority of the Fe(II)-alphaKG-dependent enzymes that catalyze hydroxylation reactions, halogenases catalyze a transfer of halides. To examine the important enzymatic features that discriminate between chlorination and hydroxylation, the crystal structures of CytC3 both with and without alphaKG/Fe(II) have been solved to 2.2 A resolution. These structures capture CytC3 in an open active site conformation, in which no chloride is bound to iron. Comparison of the open conformation of CytC3 with the closed conformation of another nonheme iron halogenase, SyrB2, suggests two important criteria for creating an enzyme-bound Fe-Cl catalyst: (1) the presence of a hydrogen-bonding network between the chloride and surrounding residues, and (2) the presence of a hydrophobic pocket in which the chloride resides.

SUBMITTER: Wong C

PROVIDER: S-EPMC2663892 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Structural analysis of an open active site conformation of nonheme iron halogenase CytC3.

Wong Cintyu C Fujimori Danica Galonić DG Walsh Christopher T CT Drennan Catherine L CL

Journal of the American Chemical Society 20090401 13

CytC3, a member of the recently discovered class of nonheme Fe(II) and alpha-ketoglutarate (alphaKG)-dependent halogenases, catalyzes the double chlorination of L-2-aminobutyric acid (Aba) to produce a known Streptomyces antibiotic, gamma,gamma-dichloroaminobutyrate. Unlike the majority of the Fe(II)-alphaKG-dependent enzymes that catalyze hydroxylation reactions, halogenases catalyze a transfer of halides. To examine the important enzymatic features that discriminate between chlorination and hy ...[more]

PMID: 19281171

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Project description:Nonheme iron- and α-ketoglutarate (αKG)-dependent halogenases (NHFeHals), which catalyze the regio- and stereoselective halogenation of the unactivated C(sp3)-H bonds, exhibit tremendous potential in the challenging asymmetric halogenation. AdeV from Actinomadura sp. ATCC 39365 is the first identified carrier protein-free NHFeHal that catalyzes the chlorination of nucleotide 2'-deoxyadenosine-5'-monophosphate (2'-dAMP) to afford 2'-chloro-2'-deoxyadenosine-5'-monophosphate. Here, we determined the complex crystal structures of AdeV/FeII/Cl and AdeV/FeII/Cl/αKG at resolutions of 1.76 and 1.74 Å, respectively. AdeV possesses a typical β-sandwich topology with H194, H252, αKG, chloride, and one water molecule coordinating FeII in the active site. Molecular docking, mutagenesis, and biochemical analyses reveal that the hydrophobic interactions and hydrogen bond network between the substrate-binding pocket and the adenine, deoxyribose, and phosphate moieties of 2'-dAMP are essential for substrate recognition. Residues H111, R177, and H192 might play important roles in the second-sphere interactions that control reaction partitioning. This study provides valuable insights into the catalytic selectivity of AdeV and will facilitate the rational engineering of AdeV and other NHFeHals for synthesis of halogenated nucleotides. IMPORTANCE Halogenated nucleotides are a group of important antibiotics and are clinically used as antiviral and anticancer drugs. AdeV is the first carrier protein-independent nonheme iron- and α-ketoglutarate (αKG)-dependent halogenase (NHFeHal) that can selectively halogenate nucleotides and exhibits restricted substrate specificity toward several 2'-dAMP analogues. Here, we determined the complex crystal structures of AdeV/FeII/Cl and AdeV/FeII/Cl/αKG. Molecular docking, mutagenesis, and biochemical analyses provide important insights into the catalytic selectivity of AdeV. This study will facilitate the rational engineering of AdeV and other carrier protein-independent NHFeHals for synthesis of halogenated nucleotides.

Dataset Information

Structural analysis of an open active site conformation of nonheme iron halogenase CytC3.

Publications

Structural analysis of an open active site conformation of nonheme iron halogenase CytC3.

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