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Determining the Structure and Kinetics of Desferroxiamine D Variants: E445A and H443A
Author(s) -
Rosales Vincent,
Gahir Paven,
Hoffmann Katherine
Publication year - 2020
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.2020.34.s1.06102
Subject(s) - siderophore , streptomyces coelicolor , cofactor , mutagenesis , enzyme , chemistry , stereochemistry , substrate (aquarium) , biochemistry , combinatorial chemistry , binding site , bacteria , biology , mutant , genetics , gene , ecology
Virulent bacteria such as MRSA and anthrax produce siderophores which are successful in competing for and chelating iron from their host environment. These are synthesized by a family of enzymes called NIS synthetases, of which DesD is a model enzyme out of Streptomyces coelicolor . DesD contributes to the catalytic formation of siderophore Desferrioxamine E, a siderophore formed by iterative combination of three molecules of hydroxysuccinylcadaverine along with stoichiometric amounts of cofactor ATP. We have previously characterized the kinetic constants for ATP and the largest substrate, called dfoG, as well as binding studies for the cofactors and analogs and structural models of the apo and analog‐bound enzyme. The purpose of the current study is to determine the sidechains involved in catalysis by using site directed mutagenesis, kinetic studies, binding studies and crystallography to analyse the impact of the mutation. This work will focus on two variants: E445A and H445A which we hypothesize will be critical for cofactor binding (E445A) and catalysis (H445A) based on alignments and structural analysis. The current work will present overexpression and purification of these two variants, along with the biochemical characterization of their functions. Support or Funding Information This research was supported in part by the National Science Foundation (NSF‐RUI grant 1716986 to K.M.H.).