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Literature summary extracted from

  • Harth, S.; Wagner, J.; Sens, T.; Choe, J.Y.; Benz, J.P.; Weuster-Botz, D.; Oreb, M.
    Engineering cofactor supply and NADH-dependent D-galacturonic acid reductases for redox-balanced production of L-galactonate in Saccharomyces cerevisiae (2020), Sci. Rep., 10, 19021 .
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
1.1.1.365 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(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.365 expression in Saccharomyces cerevisie Aspergillus niger
1.1.1.365 gene gaaA, recombinant expression of wild-type enzyme in Saccharomyces cerevisiae strain SiHY001 Aspergillus niger
1.1.1.365 gene gar1, recombinant expression of wild-type and mutant enzymes in Saccharomyces cerevisiae strain SiHY001, real-time PCR expression analysis Aspergillus niger

Protein Variants

EC Number Protein Variants Comment Organism
1.1.1.365 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
1.1.1.365 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
1.1.1.365 K261M/R267L mutation confers NADH specificity to the enzyme. Mutation almost fully abolishes the AnGar1 activity with NADPH as the cofactor Aspergillus niger
1.1.1.365 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
1.1.1.365 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
1.1.1.365 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
1.1.1.365 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

EC Number Inhibitors Comment Organism Structure
1.1.1.365 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/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.1.365 D-galacturonate + NADPH + H+ Aspergillus niger
-
 L-galactonate + NADP+
-
 ?
1.1.1.365 D-galacturonate + NADPH + H+ Aspergillus niger CBS 513.88
-
 L-galactonate + NADP+
-
 ?
1.1.1.365 L-galactonate + NAD+ Aspergillus niger
-
 D-galacturonate + NADH + H+
-
 r
1.1.1.365 L-galactonate + NADP+ Aspergillus niger
-
 D-galacturonate + NADPH + H+
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.365 Aspergillus niger
-
-
-
1.1.1.365 Aspergillus niger A2R7U3
-
-
1.1.1.365 Aspergillus niger A8DRH9
-
-
1.1.1.365 Aspergillus niger CBS 513.88 A2R7U3
-
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.365 D-galacturonate + NADPH + H+
-
 Aspergillus niger L-galactonate + NADP+
-
 ?
1.1.1.365 D-galacturonate + NADPH + H+
-
 Aspergillus niger CBS 513.88 L-galactonate + NADP+
-
 ?
1.1.1.365 L-galactonate + NAD+
-
 Aspergillus niger D-galacturonate + NADH + H+
-
 r
1.1.1.365 L-galactonate + NADP+
-
 Aspergillus niger D-galacturonate + NADPH + H+
-
 r

Synonyms

EC Number Synonyms Comment Organism
1.1.1.365 AnGaaA
-
 Aspergillus niger
1.1.1.365 AnGar1
-
 Aspergillus niger
1.1.1.365 D-galacturonic acid reductase
-
 Aspergillus niger
1.1.1.365 D-galacturonic acid reductases
-
 Aspergillus niger
1.1.1.365 gaaA
-
 Aspergillus niger
1.1.1.365 GalA reductase
-
 Aspergillus niger
1.1.1.365 gar1
-
 Aspergillus niger

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.365 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
1.1.1.365 additional information the wild-type enzyme Gar1 accepts NADPH but not NADH as cofactor Aspergillus niger
1.1.1.365 NAD+
-
 Aspergillus niger
1.1.1.365 NADH
-
 Aspergillus niger
1.1.1.365 NADP+
-
 Aspergillus niger
1.1.1.365 NADPH
-
 Aspergillus niger

General Information

EC Number General Information Comment Organism
1.1.1.365 evolution AnGaaA is identified as the bona fide GalA reductase in Aspergillus niger. AnGaaA is not related to Gar1 proteins Aspergillus niger