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Imposing function down a (cupin)-barrel: secondary structure and metal stereochemistry in the ?KG-dependent oxygenases.

ABSTRACT: The Fe(ii)/?ketoglutarate (?KG) dependent oxygenases catalyze a diverse range of reactions significant in biological processes such as antibiotic biosynthesis, lipid metabolism, oxygen sensing, and DNA and RNA repair. Although functionally diverse, the eight-stranded ?-barrel (cupin) and HX(D/E)XnH facial triad motifs are conserved in this super-family of enzymes. Crystal structure analysis of 25 ?KG oxygenases reveals two stereoisomers of the Fe cofactor, Anti and Clock, which differ in the relative position of the exchangeable ligand position and the primary substrate. Herein, we discuss the relationship between the chemical mechanism and the secondary coordination sphere of the ?KG oxygenases, within the constraints of the stereochemistry of the Fe cofactor. Sequence analysis of the cupin barrel indicates that a small subset of positions constitute the second coordination sphere, which has significant ramifications for the structure of the ferryl intermediate. The competence of both Anti and Clock stereoisomers of Fe points to a ferryl intermediate that is 5 coordinate. The small number of conserved close contacts within the active sites of ?KG oxygenases can be extended to chemically related enzymes, such as the ?KG-dependent halogenases SyrB2 and CytC3, and the non-?KG dependent dioxygenases isopenicillin N synthase (IPNS) and cysteine dioxygenase (CDO).

SUBMITTER: Hangasky JA

PROVIDER: S-EPMC4109655 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Imposing function down a (cupin)-barrel: secondary structure and metal stereochemistry in the αKG-dependent oxygenases.

Hangasky John A JA Taabazuing Cornelius Y CY Valliere Meaghan A MA Knapp Michael J MJ

Metallomics : integrated biometal science 20130401 4

The Fe(ii)/αketoglutarate (αKG) dependent oxygenases catalyze a diverse range of reactions significant in biological processes such as antibiotic biosynthesis, lipid metabolism, oxygen sensing, and DNA and RNA repair. Although functionally diverse, the eight-stranded β-barrel (cupin) and HX(D/E)XnH facial triad motifs are conserved in this super-family of enzymes. Crystal structure analysis of 25 αKG oxygenases reveals two stereoisomers of the Fe cofactor, Anti and Clock, which differ in the rel ...[more]

PMID: 23446356

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Project description:Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth. Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction. However, the crystal structure of ectoine synthase is not known and a clear understanding of how its fold contributes to enzyme activity is thus lacking. Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form. Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily. EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure. The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer. We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme. We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency. Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants. An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel.

Dataset Information

Imposing function down a (cupin)-barrel: secondary structure and metal stereochemistry in the ?KG-dependent oxygenases.

Publications

Imposing function down a (cupin)-barrel: secondary structure and metal stereochemistry in the αKG-dependent oxygenases.

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