Cloned (Comment) | Organism |
---|---|
gene kdnA, encoded in the Kdo8N biosynthetic cluster, DNA and amino acid sequence determination and analysis, subcloning and expression in Escherichia coli strains EC100D and WBB06 at 37°C, recombinant expression of the gene cluster for 8-amino-3,8-dideoxy-D-manno-octulosonic acid biosynthesis from Shewanella oneidensis in Escherichia coli results in lipid A containing 8-amino-3,8-dideoxy-D-manno-octulosonic acid, and in vitro assays confirm the enzymatic functionality converting 3-deoxy-D-manno-octulosonic acid to 8-amino-3,8-dideoxy-D-manno-octulosonic acid, with incorporation into the Kdo8N-lipid A domain of lipopolysaccharide by a metal-dependent oxidase followed by a glutamate-dependent aminotransferase, recombinant expression of His-tagged enzyme in Escherichia coi strain C41 | Shewanella oneidensis |
Protein Variants | Comment | Organism |
---|---|---|
additional information | enzyme knockout by in-frame deletion, chromosomal deletion of kdnA/kdnB, overview. The knock-out strain shows increased sensitivity to polymyxin B (3fold) and bile salts (2fold) | Shewanella oneidensis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
8-dehydro-3-deoxy-D-manno-octulosonic acid + L-glutamate | Shewanella oneidensis | i.e. Kdo | 8-amino-3,8-dideoxy-D-manno-octulosonic acid + 2-oxoglutarate | i.e. Kdo8N | ? | |
8-dehydro-3-deoxy-D-manno-octulosonic acid + L-glutamate | Shewanella oneidensis MR-1 / ATCC 700550 | i.e. Kdo | 8-amino-3,8-dideoxy-D-manno-octulosonic acid + 2-oxoglutarate | i.e. Kdo8N | ? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Shewanella oneidensis | Q8EEB1 | gene kdnA or SO_2476 | - |
Shewanella oneidensis MR-1 / ATCC 700550 | Q8EEB1 | gene kdnA or SO_2476 | - |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged enzyme from Escherichia coi strain C41 by nickel affinity chromatography and dialysis, to homogeneity | Shewanella oneidensis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
8-dehydro-3-deoxy-D-manno-octulosonic acid + L-glutamate | i.e. Kdo | Shewanella oneidensis | 8-amino-3,8-dideoxy-D-manno-octulosonic acid + 2-oxoglutarate | i.e. Kdo8N | ? | |
8-dehydro-3-deoxy-D-manno-octulosonic acid + L-glutamate | i.e. Kdo, direct conversion of Kdo to Kdo8N prior to its incorporation into the Kdo8N-lipid A domain of lipopolysaccharide by a metal-dependent oxidase KdnB, EC 1.1.3.48, followed by a glutamate-dependent aminotransferase KdnA, EC 2.3.1.09, the electron acceptor is molecular oxygen | Shewanella oneidensis | 8-amino-3,8-dideoxy-D-manno-octulosonic acid + 2-oxoglutarate | i.e. Kdo8N | ? | |
8-dehydro-3-deoxy-D-manno-octulosonic acid + L-glutamate | i.e. Kdo | Shewanella oneidensis MR-1 / ATCC 700550 | 8-amino-3,8-dideoxy-D-manno-octulosonic acid + 2-oxoglutarate | i.e. Kdo8N | ? | |
8-dehydro-3-deoxy-D-manno-octulosonic acid + L-glutamate | i.e. Kdo, direct conversion of Kdo to Kdo8N prior to its incorporation into the Kdo8N-lipid A domain of lipopolysaccharide by a metal-dependent oxidase KdnB, EC 1.1.3.48, followed by a glutamate-dependent aminotransferase KdnA, EC 2.3.1.09, the electron acceptor is molecular oxygen | Shewanella oneidensis MR-1 / ATCC 700550 | 8-amino-3,8-dideoxy-D-manno-octulosonic acid + 2-oxoglutarate | i.e. Kdo8N | ? | |
additional information | the reaction catalyzed by KdnB, EC 1.1.3.48, is thermodynamically unfavorable and requires the second reaction catalyzed by KdnA to drive product formation, both enzymes are required for product formation. Production of H2O2 when Mn-KdnB and PLP-KdnA are incubated with Kdo and L-Glu | Shewanella oneidensis | ? | - |
? | |
additional information | the reaction catalyzed by KdnB, EC 1.1.3.48, is thermodynamically unfavorable and requires the second reaction catalyzed by KdnA to drive product formation, both enzymes are required for product formation. Production of H2O2 when Mn-KdnB and PLP-KdnA are incubated with Kdo and L-Glu | Shewanella oneidensis MR-1 / ATCC 700550 | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
kdnA | - |
Shewanella oneidensis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Shewanella oneidensis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Shewanella oneidensis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
additional information | NAD+ and NADP+ are not required for activity | Shewanella oneidensis | |
pyridoxal 5'-phosphate | required for activity | Shewanella oneidensis |
General Information | Comment | Organism |
---|---|---|
evolution | 8-amino-3,8-dideoxy-D-manno-octulosonic acid (Kdo8N) is found exclusively in marine bacteria of the genus Shewanella | Shewanella oneidensis |
malfunction | creation of an Shewanella oneidensis kdnA/kdnB in-frame deletion strain shows increased sensitivity to the cationic antimicrobial peptide polymyxin as well as bile salts | Shewanella oneidensis |
metabolism | 8-amino-3,8-dideoxy-D-manno-octulosonic acid (Kdo8N) biosynthesis pathway, 8-dehydro-3-deoxy-D-manno-octulosonic acid is directly converted to Kdo8N followed by incorporation into lipid A, overview. The entire gene cluster is required for 8-amino-3,8-dideoxy-D-manno-octulosonic acid (Kdo8N) biosynthesis | Shewanella oneidensis |
physiological function | the first sugar bridging the hydrophobic lipid A and the polysaccharide domain is 3-deoxy-D-manno-octulosonic acid (Kdo), and thus it is critically important for lipopolysaccharide biosynthesis. KdnA is a pyridoxal 5'-phosphate-dependent aminotransferase that utilizes L-glutamate and may play a role in outer membrane integrity | Shewanella oneidensis |