טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentYaniv Orit
SubjectCloning, Expression, and Biochemical Characterization of
the Enzyme KDO8P Synthase from the
Hyperthermophilic Bacterium
Aquifex Pyrophilus
DepartmentDepartment of Biotechnology
Supervisors Professor Timor Baasov
Professor Yuval Shoham
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

3-Deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase catalyzes an aldol-type condensation between phsosphoenolpyruvate (PEP) and D-arabinose-5-phosphate (A5P) to produce KDO8P and inorganic phosphate. KDO8P is a precursor of the unusual eight carbon atom sugar, KDO, which is an essential constituent of the lipopolysaccharide of all Gram-negative bacteria. KDO plays a crucial role in the assembly process of lipopolysaccharide, and therefore is an attractive target for the development of novel antibacterial drugs. The KDO8PS gene from the heperthermophilic bacterium Aquifex pyrophilus was cloned from a λDASHII genomic library by using the kdsA gene from Escherichia coli as a DNA probe.  One of the positive clones was isolated and contained a 15.5 kb chromosomal segment.  Sequence analysis of the segment revealed 15 open reading frames, among them was the kdsA gene. The kdsA gene from A. pyrophilus encodes for a 267-amino-acid protein with a calculated molecular mass of 29,696 Da, that exhibits 87% identity (262 amino acids overlap) to KDO8PS from Aquifex aeolicus and 45% identity (262 amino acids overlap) to the KDO8PS from Escherichia coli.  The kdsA gene was expressed in E. coli using the T7 polymerase expression system. The purification procedure included two steps, heat treatment and anion exchange chromatography, yielding 14 mg protein per 1.8 liter culture with an overall yield of 40%. The purified KDO8PS was used for crystal structure determination and biochemical characterization. The enzyme was most active at 80°C with a pH optimum between 5.5-6.0. Its thermostability was characterized with half-lives of 0.5, 2.25, and 8.0 h at 90°C, 80°C, and 70°C, respectively. Kinetic constants at 60°C were KmA5P=70 µ­M, KmPEP =290 µ­M, and kcat= 4 s-1. Treatment with EDTA eliminates enzyme activity that can be restored by addition of several divalent metal ions such as Cd+2 and Mn+2, suggesting that the KDO8PS from A. pyrophilus  is a metalloenzyme.