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Altering the substrate specificity of the Escherichia coli E1 Component of the 2‐Oxoglutarate Dehydrogenase Multienzyme Complex
Author(s) -
Shim Da Jeong,
Nemeria Natalia S.,
Balakrishnan Anand,
Jordan Frank,
Farinas Edgardo T.
Publication year - 2010
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.24.1_supplement.645.6
Subject(s) - decarboxylation , substrate (aquarium) , chemistry , dehydrogenase , biochemistry , stereochemistry , mutagenesis , thiamine , saturated mutagenesis , oxoglutarate dehydrogenase complex , enzyme , transferase , escherichia coli , biology , branched chain alpha keto acid dehydrogenase complex , catalysis , mutation , ecology , gene , mutant
The substrate specificity of the 2‐oxoglutarate dehydrogenase multienzyme complex (OGDH) was engineered to accept unnatural substrates. OGDH catalyzes the rate limiting step in the citric acid cycle. It contains three components: a thiamine diphosphate dependent oxoglutarate decarboxylase (E1), a dihydrolipoylsuccinyl transferase (E2), and a dihydrolipoyl dehydrogenase (E3). The crystal structure, kinetic studies, and site directed mutagenesis analysis of the E1 component indicate that His260 and His298 are important for recognizing the distal carboxylate of 2‐oxoglutarate. Saturation mutagenesis libraries of His260, His298, and His260/His298 were constructed and screened for activity towards 2‐oxovalerate. Several E1 variants were isolated that are active towards 2‐oxovalerate, and the kinetic parameters were determined. In addition, circular dichroism investigations were performed that identified the formation of the pre‐decarboxylation thiamin‐bound intermediate via formation of the 1’, 4’‐imino tautomer of ThDP. The activity of the overall complex on reconstitution with the E2–E3 sub‐complex shows that substrate specificity is controlled at both the E1 and E2 levels.