Studies of 3-Deoxy-D-manno-octulosonate 8-Phosphate Phosphatase: MechanisticInsights and a Gene Fusion Example.

[摘要] 3-Deoxy-D-manno-octulosonate (KDO) is a carbohydrate molecule required for integrity of Gram-negative bacterial outer membrane, therefore its biosynthesis is a potential antibiotic target. In addition to serving as a linker molecule in the lipopolysaccharide layer on the outer membrane, KDO is also found as a building block of the capsular polysaccharide. Four enzymes encoded by the kds genes are required for the biosynthesis of the activated form of KDO. The third enzyme, 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) phosphatase (KdsC), catalyzes the hydrolysis of KDO8P to form KDO and inorganic phosphate. Subsequently, KDO is activated to cytidine monophosphate-KDO by the cytidylyltransferase (KdsB).To gain mechanistic insights in order to design inhibitors, substrate or products-bound KdsC structures are necessary. However, none of these structures have been reported for KdsC. After observing a dramatic conformational change in the C-terminal tail region of E. coli KdsC from our initial crystallization studies, a tail deletion mutant (KdsCΔ8) was constructed. A products-bound structure was obtained for the first time by co-crystallizing KdsCΔ8 and KDO8P. Detailed analysis on this structure suggests an important role of tetramerization in defining the substrate specificity of KdsC and assigns a potential regulatory role of the C-terminal tail region in the catalytic cycle. Through structural analysis as well as biochemical characterization of tailed and tail-less KdsCs, a catalytic cycle model of KdsC is proposed which involves the active participation of the tail, through conformational changes, in the product releasing step.Through a genomic database search, the first gene fusion example in the KDO pathway was revealed from Pseudomonas putida F1 as a fusion of kdsC and kdsB (kdsBC). This fusion gene not only uniquely clusters with other kds genes, but also resides in a capsular expression-related kps gene cluster. A combined colorimetric method was established to monitor phosphate released by KdsC and pyrophosphate released by KdsB, independently and simultaneously. The kdsBC gene was proved to encode a bifunctional enzyme displaying both KdsC and KdsB activities. The full-length bifunctional enzyme and each of its active domains were fully characterized.

[发布机构] University of Michigan

[效力级别] Lipopolysaccharide

[关键词] KDO Biosynthetic Pathway;Lipopolysaccharide;Haloacid Dehalogenase Superfamily;3-deoxy-D-manno-Octulosonate 8-phosphate Phosphatase "KDO8P"Fusion Gene;Biological Chemistry;Science;Chemistry