Recently, while investigating the methionine salvage pathway of the bacterium Klebsiella pneumoniae , we discovered a novel dioxygenase metalloenzyme which catalyzes the oxidative decomposition of the keto-acid precursor of methionine, 2-keto-4-methylthiobutyric acid. This new enzyme, known as E2, does not show significant sequence homology to any protein with a known structure, but appears to be eminently suitable for NMR structural work. The 20 kDa enzyme is monomeric, consisting of a single 181-residue polypeptide. ESR and NMR studies of the enzyme clearly indicate the presence of a paramagnetic metal ion. However, different metals can be bound to the enzyme, including Mg'1' Mn*,2 , Ni+2 and Fe +2 , and the products of the decomposition reaction catalyzed by E2 differ depending upon which metal ion is bound. Other subtle structural differences are evident as a function of metal ion binding; clearly, the metal site responds to the metal ion bound there, resulting in structural perturbations which are then reflected in the reactivity of the enzyme. We have acquired preliminary 750 MHz 2D (HSQC, NOESY, TOCSY, DQF-COSY) and 3D spectra (NOESYHSQC, TOCSY-HSQC) of unlabeled and 15N-labeled enzyme which clearly indicates the suitability of NMR methods for structural characterization and solution structure-function experiments.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR000995-23
Application #
6279665
Study Section
Project Start
1998-05-01
Project End
1999-04-30
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
23
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Type
DUNS #
City
Cambridge
State
MA
Country
United States
Zip Code
02139
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