Cloned (Comment) | Organism |
---|---|
expression in Escherichia coli | Shewanella oneidensis |
gene kdnB, 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 25°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 LPS 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 |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
EDTA | - |
Shewanella oneidensis | |
additional information | no inhibition by NADH | Shewanella oneidensis | |
NAD+ | slight inhibition | Shewanella oneidensis |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Fe2+ | or Mn2+, required | Shewanella oneidensis | |
Fe2+ | activates, KdnB contains 0.51 mol of iron/mol of enzyme | Shewanella oneidensis | |
Mn2+ | activates, required for activity | Shewanella oneidensis | |
Mn2+ | or Fe2+, required | Shewanella oneidensis | |
additional information | KdnB is a metal-dependent enzyme | Shewanella oneidensis | |
Zn2+ | KdnB contains 0.24 mol of iron/mol of enzyme | Shewanella oneidensis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
3-deoxy-alpha-D-manno-octulopyranosonate + O2 | Shewanella oneidensis | - |
3,8-dideoxy-8-oxo-alpha-D-manno-octulosonate + H2O2 | enzyme oxidizes an alcohol using a metal and molecular oxygen | ? | |
3-deoxy-alpha-D-manno-octulopyranosonate + O2 | Shewanella oneidensis MR-1 / ATCC 700550 | - |
3,8-dideoxy-8-oxo-alpha-D-manno-octulosonate + H2O2 | enzyme oxidizes an alcohol using a metal and molecular oxygen | ? | |
3-deoxy-alpha-D-manno-octulosonic acid + O2 | Shewanella oneidensis | i.e. Kdo | 3,8-dideoxy-8-oxo-alpha-D-manno-octulosonic acid + H2O2 | - |
? | |
3-deoxy-alpha-D-manno-octulosonic acid + O2 | Shewanella oneidensis MR-1 / ATCC 700550 | i.e. Kdo | 3,8-dideoxy-8-oxo-alpha-D-manno-octulosonic acid + H2O2 | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Shewanella oneidensis | - |
- |
- |
Shewanella oneidensis | Q8EEB0 | gene kdnB or SO_2477 | - |
Shewanella oneidensis MR-1 / ATCC 700550 | - |
- |
- |
Shewanella oneidensis MR-1 / ATCC 700550 | Q8EEB0 | gene kdnB or SO_2477 | - |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged enzyme from Escherichia coi strain C41 by nickel affinity chromatography, dialysis, and gel filtration, to homogeneity | Shewanella oneidensis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
3-deoxy-alpha-D-manno-octulopyranosonate + O2 | - |
Shewanella oneidensis | 3,8-dideoxy-8-oxo-alpha-D-manno-octulosonate + H2O2 | enzyme oxidizes an alcohol using a metal and molecular oxygen | ? | |
3-deoxy-alpha-D-manno-octulopyranosonate + O2 | - |
Shewanella oneidensis MR-1 / ATCC 700550 | 3,8-dideoxy-8-oxo-alpha-D-manno-octulosonate + H2O2 | enzyme oxidizes an alcohol using a metal and molecular oxygen | ? | |
3-deoxy-alpha-D-manno-octulopyranosonate + O2 + L-Glu | - |
Shewanella oneidensis | 3,8-dideoxy-8-oxo-alpha-D-manno-octulosonate + H2O2 | enzyme oxidizes an alcohol using a metal and molecular oxygen | ? | |
3-deoxy-alpha-D-manno-octulopyranosonate + O2 + L-Glu | - |
Shewanella oneidensis MR-1 / ATCC 700550 | 3,8-dideoxy-8-oxo-alpha-D-manno-octulosonate + H2O2 | enzyme oxidizes an alcohol using a metal and molecular oxygen | ? | |
3-deoxy-alpha-D-manno-octulosonic acid + O2 | i.e. Kdo | Shewanella oneidensis | 3,8-dideoxy-8-oxo-alpha-D-manno-octulosonic acid + H2O2 | - |
? | |
3-deoxy-alpha-D-manno-octulosonic acid + O2 | i.e. Kdo, direct conversion of Kdo to 8-amino-3,8-dideoxy-D-manno-octulosonic acid (Kdo8N) prior to its incorporation into the Kdo8N-lipid A domain of lipopolysaccharide by a metal-dependent oxidase KdnB followed by a glutamate-dependent aminotransferase KdnA, EC 2.3.1.09, the electron acceptor is molecular oxygen | Shewanella oneidensis | 3,8-dideoxy-8-oxo-alpha-D-manno-octulosonic acid + H2O2 | - |
? | |
3-deoxy-alpha-D-manno-octulosonic acid + O2 | i.e. Kdo | Shewanella oneidensis MR-1 / ATCC 700550 | 3,8-dideoxy-8-oxo-alpha-D-manno-octulosonic acid + H2O2 | - |
? | |
3-deoxy-alpha-D-manno-octulosonic acid + O2 | i.e. Kdo, direct conversion of Kdo to 8-amino-3,8-dideoxy-D-manno-octulosonic acid (Kdo8N) prior to its incorporation into the Kdo8N-lipid A domain of lipopolysaccharide by a metal-dependent oxidase KdnB followed by a glutamate-dependent aminotransferase KdnA, EC 2.3.1.09, the electron acceptor is molecular oxygen | Shewanella oneidensis MR-1 / ATCC 700550 | 3,8-dideoxy-8-oxo-alpha-D-manno-octulosonic acid + H2O2 | - |
? | |
additional information | the reaction catalyzed by KdnB is thermodynamically unfavorable and requires the second reaction catalyzed by KdnA to drive product formation, both enzymes are required for product formation. Enzyme KdnB appears to be an alcohol oxidase as opposed to an alcohol dehydrogenase, production of H2O2 when Mn-KdnB and PLP-KdnA, EC 2.6.1.109, are incubated with Kdo and L-Glu | Shewanella oneidensis | ? | - |
? | |
additional information | the reaction catalyzed by KdnB is thermodynamically unfavorable and requires the second reaction catalyzed by KdnA to drive product formation, both enzymes are required for product formation. Enzyme KdnB appears to be an alcohol oxidase as opposed to an alcohol dehydrogenase, production of H2O2 when Mn-KdnB and PLP-KdnA, EC 2.6.1.109, are incubated with Kdo and L-Glu | Shewanella oneidensis MR-1 / ATCC 700550 | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
kdnB | - |
Shewanella oneidensis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Shewanella oneidensis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Shewanella oneidensis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
additional information | NAD(P) is not required, and no product inhibition is observed for NADH | Shewanella oneidensis | |
additional information | NAD+ and NADP+ are not required for activity | Shewanella oneidensis |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme belongs to a putative distinct class of metal-dependent alcohol oxidases | 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 by 3fold and 2fold, respectively | 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 | a KdnB knock-out strain shows 3fold increased sensitivity to polymyxin B and 2fold increased sensitivity to bile salts | Shewanella oneidensis |
physiological function | endotoxin lipopolysaccharide is composed of a hydrophobic anchor, known as lipid A, an inner core oligosaccharide, and a repeating O-antigen polysaccharide. The first sugar bridging the hydrophobic lipid A and the polysaccharide domain is 3-deoxy-D-manno-octulosonic acid. Derivative 8-amino-3,8-dideoxy-Dmanno-octulosonic acid is found exclusively in marine bacteria of the genus Shewanella. Data are consistent with direct conversion of 3-deoxy-D-manno-octulosonic acid to 8-amino-3,8-dideoxy-D-manno-octulosonic acid prior to its incorporation into the 8-amino-3,8-dideoxy-D-manno-octulosonic acid-lipid A domain of lipopolysaccharide by a metal-dependent oxidase followed by a glutamate-dependent aminotransferase | 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 | Shewanella oneidensis |