Substrate Specificity and Metal Requirements of 3-Deoxy-D-manno-octulosonate8-Phosphate Synthase (KDOPS).

[摘要] 3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDOPS) catalyzes the aldol-type condensation of D-arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) and inorganic phosphate.This reaction is a key step in the biosynthetic pathway of 3-deoxy-D-manno-octulosonate (KDO), a site-specific constituent of the lipopolysaccharide layer (LPS) in the Gram-negative (G-) bacteria cell envelope.The inhibition of KDO biosynthesis has been shown to lead to incomplete LPS formation, and an arrest in cell growth.Therefore, KDOPS represents an ideal target for the development of novel G- antibiotics.A functionally related enyme to KDOPS, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) catalyzes a similar condensation reaction of PEP with D-erythrose 4-phosphate (E4P).E4P is one -CHOH- unit shorter than A5P, the substrate of KDOPS.KDOPS and DAHPS share many similarities in structure and mechanism.Based on phylogenetic analysis, KDOPSs have been separated into two classes which differ in their metal requirements: Class I are non-metallo enzymes while Class II are metalloenzymes.All known DAHPSs are metallo enzymes.The focus of this dissertation is to study the substrate specificity and metal requirements of KDOPS in order to gain more mechanistic insight into KDOPS.Multiple approaches were undertaken to study and characterize KDOPS utilizing a combination of techniques in molecular biology, enzymology, biochemistry, and analytical chemistry.Three methodologies including structure-based engineering, half-barrel swapping and directed evolution were used to attempt to alter the substrate specificity of KDOPS from A5P to E4P.The important residues/loop involved in carbohydrate substrate binding in KDOPS were identified in this approach.In order to select an alternate substrate for KDOPS, several A5P analogues were tested.Arabinose 5-difluoromethylenephosphonate and 2-deoxy-ribose 5-phosphate were identified as alternate substrates for KDOPS, but had a much slower rates than the natural substrate A5P.Finally, mutagenesis experiments were perfomed to change the metal requirements of KDOPS.The A. aeolicus KDOPS C11N mutant successfully converted a metallo KDOPS to a non-metallo enzyme by single amino acid substitution.X-ray crystallography studies on this mutant suggest that the metal plays an important structural role in KDOPS.

[发布机构] University of Michigan

[效力级别] Substrate Specificity

[关键词] KDOPS;Substrate Specificity;Metal Requirements;Chemistry;Science;Chemistry