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Structural and Functional Substrate Binding in an Iterative Non‐ribosomal Peptide Synthetase Independent Siderophore (NIS) Enzyme from Streptomyces coelicolor
Author(s) -
Hoffmann Katherine M
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.836.5
Subject(s) - streptomyces coelicolor , isothermal titration calorimetry , chemistry , enzyme , peptide , stereochemistry , biochemistry , ribosome , active site , ribosomal rna , peptide bond , siderophore , binding site , rna , mutant , gene
Prokaryotes scavenge iron using small molecule chelators called siderophores, made through a novel chemistry of peptide bond formation outside of a ribosome. One type of siderophore synthesis uses N on‐ribosomal peptide siderophore I ndependent S ynthesis enzymes (NIS synthetases) to form amide bonds (peptide bonds) between metabolic intermediates in NIS siderophore biosynthesis pathways. Recently, primary structure analysis, correlated with known activity, has identified four subtypes (A, A′, B and C), only two of which have been structurally described. While subtype is based on the primary substrate specificity, an additional remarkable kinetic behavior exists in a sub‐category of enzymes whereby multiple iterative bonds may be catalyzed on the same substrate. Prior work in the field has identified a novel protein fold (including a novel ATP binding motif,) but several sub‐types and iterative proteins are yet to be structurally or biochemically described. The iterative activity is correlated with broad substrate specificity, but the binding affinity has never been quantified. In fact, iterative enzyme kinetic parameters have been elusive, as the first published biochemical characterization found the reporters limited the speed of the assays. We are structurally and functionally characterizing a representative iterative NIS enzyme, DesD, from Streptomyces coelicolor , using the methodologies of X‐ray crystallography and isothermal titration calorimetry (ITC). Our results suggest a firm order of binding and significantly more substrate specificity than previously thought. Additionally, the innovative use of calorimetry to measure kinetic parameters has allowed a reporter‐free assay that may be broadly applicable to other processive enzymes. Finally, we are structurally characterizing the apo, partial complex, and full complex of DesD bound to it's high‐specificity substrates, to fully map binding interactions. We hope future work will include the design and synthesis of inhibitors, as well as site‐directed mutagenesis, to probe mechanism of the novel chemistry.