טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentKaustov Lilia
SubjectSolid State MMR Structural and Mechanistic Investigation
of KDO8P Synthase
DepartmentDepartment of Chemistry
Supervisor Professor Asher Schmidt


Abstract

The enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS), being essential for the biosynthesis of the lipopolysaccharides (LPS) in Gram-negative bacteria, is an attractive target for the design of novel antibacterial drugs. However, the mechanistic details of the catalytic reaction are yet not fully resolved. In this work solid state NMR complemented by 31P solution NMR were employed to characterize the active site of KDO8PS, and to elucidate its mechanism.

Identification and characterization of active site residues of KDO8PS was accomplished by applying rotational-echo double-resonance (REDOR) solid state NMR experiments to lyophilized binary complexes of uniformly and selectively 15N-labeled KDO8PS with each of its two natural substrates, and with a potent bisubstrate inhibitor (Inh). 15N{31P} REDOR data showed two sets of enzyme residues in two (adjacent) subsites capable of independent and sufficiently strong binding of the substrates. In particular, recognition and binding of phosphoenolpyruvate (PEP) is facilitated by structurally rigid and chemically distinct set of Lys and Arg residues which are so preserved in the apoenzyme. Finding that arabinose-5-phosphate (A5P) binds in its cyclic form, enabled to propose a new analog that may become a potent mechanistic probe as well as a new antibacterial drug.

REDOR NMR along with 31P solution NMR data of the enzyme-Inh and enzyme-Inh-PEP complexes identified the inhibitor as an A5P-based inhibitor. The high potency of the inhibitor is attributed to its A5P moiety, while the PEP mimicking moiety binds weaker, and may interfere with PEP binding only indirectly. Substantiation of this determination was possible via the development and application of a DANTE-based, frequency-selective REDOR experiment.