Application | Comment | Organism |
---|---|---|
synthesis | L-galactonate shows potential applications as an additive to nutrients and cosmetics. AnGar1 variants are developed that represent the a GalA reductases with a higher preference for NADH compared to NADPH. The altered cofactor-specificity enables the coupling of GalA reduction to glycolysis, resulting in higher yields of GalOA when glucose is used as a redox donor. The engineered AnGar1 should prove valuable for D-galacturonic acid utilization in pectin-rich hydrolysates, which contain neutral sugars such as glucose, galactose, or arabinose, all of which are funneled into glycolysis. The NADH-dependent GalA reductases can facilitate the coupling of L-galactonate production to the oxidation of glycerol, an abundant waste product that can be supplemented to pectin-rich hydrolysates | Aspergillus niger |
Cloned (Comment) | Organism |
---|---|
expression in Saccharomyces cerevisie | Aspergillus niger |
gene gaaA, recombinant expression of wild-type enzyme in Saccharomyces cerevisiae strain SiHY001 | Aspergillus niger |
gene gar1, recombinant expression of wild-type and mutant enzymes in Saccharomyces cerevisiae strain SiHY001, real-time PCR expression analysis | Aspergillus niger |
Protein Variants | Comment | Organism |
---|---|---|
K261M | site-directed mutagenesis, the recombinant yeast strain expressing the enzyme mutant shows increased activity with NADH in L-galacturonate reduction compared to wild-type | Aspergillus niger |
K261M | the mutation confers AnGar1 the ability to accept NADH in addition to NADPH. Mutation partly abolishes the AnGar1 activity with NADPH as the cofactor | Aspergillus niger |
K261M/R267L | mutation confers NADH specificity to the enzyme. Mutation almost fully abolishes the AnGar1 activity with NADPH as the cofactor | Aspergillus niger |
K261M/R267L | site-directed mutagenesis, the recombinant yeast strain expressing the enzyme mutant shows increased activity with NADH in L-galacturonate reduction compared to wild-type | Aspergillus niger |
additional information | establishment of the production of L-galactonate (GalOA) or the full GalA catabolic pathway in Saccharomyces cerevisiae using the enzyme mutant K261M/R267L with increased NADH activity, and coupling the reduction of GalA to the oxidation of the sugar alcohol sorbitol that has a higher reduction state compared to glucose for yielding the necessary redox cofactors. By choosing a suitable sorbitol dehydrogenase, yeast strains are designed in which the sorbitol metabolism yields a surplus of either NADPH or NADH | Aspergillus niger |
R267L | site-directed mutagenesis, the recombinant yeast strain expressing the enzyme mutant shows increased activity with NADH in L-galacturonate reduction compared to wild-type | Aspergillus niger |
R267L | the mutation confers AnGar1 the ability to accept NADH in addition to NADPH. Mutation partly abolishes the AnGar1 activity with NADPH as the cofactor | Aspergillus niger |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
NADP+ | mutant enzyme R267L shows a considerable sensitivity at the lower (0.16 mM) but not at the higher (0.8 mM) NADH concentration | Aspergillus niger |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-galacturonate + NADPH + H+ | Aspergillus niger | - |
L-galactonate + NADP+ | - |
? | |
D-galacturonate + NADPH + H+ | Aspergillus niger CBS 513.88 | - |
L-galactonate + NADP+ | - |
? | |
L-galactonate + NAD+ | Aspergillus niger | - |
D-galacturonate + NADH + H+ | - |
r | |
L-galactonate + NADP+ | Aspergillus niger | - |
D-galacturonate + NADPH + H+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Aspergillus niger | - |
- |
- |
Aspergillus niger | A2R7U3 | - |
- |
Aspergillus niger | A8DRH9 | - |
- |
Aspergillus niger CBS 513.88 | A2R7U3 | - |
- |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-galacturonate + NADPH + H+ | - |
Aspergillus niger | L-galactonate + NADP+ | - |
? | |
D-galacturonate + NADPH + H+ | - |
Aspergillus niger CBS 513.88 | L-galactonate + NADP+ | - |
? | |
L-galactonate + NAD+ | - |
Aspergillus niger | D-galacturonate + NADH + H+ | - |
r | |
L-galactonate + NADP+ | - |
Aspergillus niger | D-galacturonate + NADPH + H+ | - |
r |
Synonyms | Comment | Organism |
---|---|---|
AnGaaA | - |
Aspergillus niger |
AnGar1 | - |
Aspergillus niger |
D-galacturonic acid reductase | - |
Aspergillus niger |
D-galacturonic acid reductases | - |
Aspergillus niger |
gaaA | - |
Aspergillus niger |
GalA reductase | - |
Aspergillus niger |
gar1 | - |
Aspergillus niger |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
additional information | the enzyme accepts NADPH as well as NADH as cofactor. But its catalytic efficiency for GalA reduction is approximately 50fold higher with NADPH than with NADH | Aspergillus niger | |
additional information | the wild-type enzyme Gar1 accepts NADPH but not NADH as cofactor | Aspergillus niger | |
NAD+ | - |
Aspergillus niger | |
NADH | - |
Aspergillus niger | |
NADP+ | - |
Aspergillus niger | |
NADPH | - |
Aspergillus niger |
General Information | Comment | Organism |
---|---|---|
evolution | AnGaaA is identified as the bona fide GalA reductase in Aspergillus niger. AnGaaA is not related to Gar1 proteins | Aspergillus niger |