Literature summary for 1.14.13.1 extracted from

Lubbers, R.J.M.; Dilokpimol, A.; Visser, J.; Hilden, K.S.; Maekelae, M.R.; de Vries, R.P.
Discovery and functional analysis of a salicylic acid hydroxylase from Aspergillus niger (2021), Appl. Environ. Microbiol., 87, e02701-20 .

Search for more literature for 1.14.13.1

Cloned(Commentary)

Cloned (Comment)	Organism
gene shyA or NRRL3_9723, DNA and amino acid sequence determination and analysis, enzyme expression analysis, gene NRRL3_9723 shows high amino acid similarity to Shy1 from Fusarium graminearum (FgShy1) (65.1%) and to a putative salicylate hydroxylase (AN2114) from Aspergillus nidulans (77.3%), sequence comparisons and phylogenetic analysis, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)	Aspergillus niger

Cloned (Comment)

Organism

gene shyA or NRRL3_9723, DNA and amino acid sequence determination and analysis, enzyme expression analysis, gene NRRL3_9723 shows high amino acid similarity to Shy1 from Fusarium graminearum (FgShy1) (65.1%) and to a putative salicylate hydroxylase (AN2114) from Aspergillus nidulans (77.3%), sequence comparisons and phylogenetic analysis, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)

Aspergillus niger

Protein Variants

Protein Variants	Comment	Organism
additional information	generation of six deletion mutants, the DELTAshyA, DELTAdhbA, DELTAcrcA, DELTA43 (putative salicylate hydroxylase), DELTA2597 (putative salicylate 3-hydroxylase), and DELTA5330 (putative catechol 1,2-dioxygenase) mutants. The growth of the DELTAshyA mutant is reduced on salicylic acid but not on the other aromatic compounds tested, confirming its role as the salicylate hydroxylase of Aspergillus niger. Growth of the DELTAdhbA mutant is reduced on 2,3-dihydroxybenzoic acid but not on salicylic acid or catechol, confirming its metabolic role. Deletion of crcA results in growth similar to that of the no-carbon source control on salicylic acid, 2,3-dihydroxybenzoic acid, and catechol, while deletion of NRRL3_5330 or hqdA does not result in a phenotype, indicating that crcA encodes the catechol 1,2-dioxygenase of Aspergillus niger. Growth of the DELTA43, DELTA5330, and DELTA2597 mutants do not result in any phenotypes on the tested aromatic compounds	Aspergillus niger

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	the kinetic profile of ShyA, obtained while only varying the salicylic acid concentration at a constant 1 mM concentration of the second substrate (NADH), does not follow Michaelis-Menten kinetics. ShyA reveals a sigmoidal curve. This may be caused by inhibition or inactivation due to low salicylate concentrations and relatively high NADH concentrations	Aspergillus niger
0.1388	-	salicylate	pH 6.0, 45°C, recombinant enzyme	Aspergillus niger

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
salicylate + NADH + 2 H+ + O2	Aspergillus niger	-	catechol + NAD+ + H2O + CO2	-	?
salicylate + NADH + 2 H+ + O2	Aspergillus niger NRRL3	-	catechol + NAD+ + H2O + CO2	-	?

Organism

Organism	UniProt	Comment	Textmining
Aspergillus niger	-	-	-
Aspergillus niger NRRL3	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography	Aspergillus niger

Source Tissue

Source Tissue	Comment	Organism	Textmining
additional information	transcriptome analysis	Aspergillus niger	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
2,3-dihydroxybenzoate + NADH + 2 H+ + O2	82% activity compared to salicylate	Aspergillus niger	pyrogallol + NAD+ + H2O + CO2	-	?
2,3-dihydroxybenzoate + NADH + 2 H+ + O2	82% activity compared to salicylate	Aspergillus niger NRRL3	pyrogallol + NAD+ + H2O + CO2	-	?
4-aminosalicylate + NADH + 2 H+ + O2	72% activity compared to salicylate	Aspergillus niger	4-aminocatechol + NAD+ + H2O + CO2	-	?
4-aminosalicylate + NADH + 2 H+ + O2	72% activity compared to salicylate	Aspergillus niger NRRL3	4-aminocatechol + NAD+ + H2O + CO2	-	?
gentisate + NADH + 2 H+ + O2	93% activity compared to salicylate	Aspergillus niger	hydroxyquinol + NAD+ + H2O + CO2	-	?
additional information	incubation of salicylic acid by a combination of cell extracts from the shyA with the crcA or the hqdA overexpression strains results in the formation of cis,cis-muconic acid, demonstrating that the combination of these enzymes is sufficient for this biochemical conversion. The conversion is highly efficient. No activity with protocatechuate, vanillate, syringate, 3-hydroxybenzoate, 4-hydroxybenzoate, and benzoate. ShyA shows activity on o-hydroxylated benzoic acids, such as 4-aminosalicylic acid, 2,3-dihydroxybenzoic acid, and gentisic acid, but not on benzoic acid derivatives that are not o-hydroxylated. The enzyme also exhibits NADH oxidase activity besides the more rapid hydroxylase activity	Aspergillus niger	?	-	-
additional information	incubation of salicylic acid by a combination of cell extracts from the shyA with the crcA or the hqdA overexpression strains results in the formation of cis,cis-muconic acid, demonstrating that the combination of these enzymes is sufficient for this biochemical conversion. The conversion is highly efficient. No activity with protocatechuate, vanillate, syringate, 3-hydroxybenzoate, 4-hydroxybenzoate, and benzoate. ShyA shows activity on o-hydroxylated benzoic acids, such as 4-aminosalicylic acid, 2,3-dihydroxybenzoic acid, and gentisic acid, but not on benzoic acid derivatives that are not o-hydroxylated. The enzyme also exhibits NADH oxidase activity besides the more rapid hydroxylase activity	Aspergillus niger NRRL3	?	-	-
salicylate + NADH + 2 H+ + O2	-	Aspergillus niger	catechol + NAD+ + H2O + CO2	-	?
salicylate + NADH + 2 H+ + O2	best substrate, almost complete conversion	Aspergillus niger	catechol + NAD+ + H2O + CO2	-	?
salicylate + NADH + 2 H+ + O2	-	Aspergillus niger NRRL3	catechol + NAD+ + H2O + CO2	-	?
salicylate + NADH + 2 H+ + O2	best substrate, almost complete conversion	Aspergillus niger NRRL3	catechol + NAD+ + H2O + CO2	-	?

Subunits

Subunits	Comment	Organism
?	x * 49200, recombinant His-tagged enzyme, SDS-PAGE	Aspergillus niger

Synonyms

Synonyms	Comment	Organism
NRRL3_9723	-	Aspergillus niger
salicylate hydroxylase	-	Aspergillus niger
salicylic acid hydroxylase	-	Aspergillus niger
ShyA	-	Aspergillus niger

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	in vivo assay at	Aspergillus niger
45	50	purified recombinant His-tagged enzyme	Aspergillus niger

Temperature Range [°C]

Temperature Minimum [°C]	Temperature Maximum [°C]	Comment	Organism
20	65	activity range, profile overview	Aspergillus niger

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
80	-	purified recombinant enzyme, pH 6.0, 10 min inactivation	Aspergillus niger

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6	-	recombinant His-tagged enzyme	Aspergillus niger

pH Range

pH Minimum	pH Maximum	Comment	Organism
4.5	8.5	activity range, profile overview	Aspergillus niger

Cofactor

Cofactor	Comment	Organism	Structure
FAD	required	Aspergillus niger
NADH	-	Aspergillus niger

Expression

Organism	Comment	Expression
Aspergillus niger	the enzyme is highly induced by salicylic acid	up

General Information

General Information	Comment	Organism
malfunction	deletion of shyA, dhbA, and crcA in Aspergillus niger results in reduced growth on salicylic acid, 2,3-dihydroxybenzoic acid, and catechol, respectively, confirming their in vivo roles	Aspergillus niger
metabolism	in the filamentous fungus Aspergillus niger, two salicylic acid metabolic pathways have been suggested. The first pathway converts salicylic acid to catechol by a salicylate hydroxylase (ShyA). In the second pathway, salicylic acid is 3-hydroxylated to 2,3-dihydroxybenzoic acid, followed by decarboxylation to catechol by 2,3-dihydroxybenzoate decarboxylase (DhbA). ShyA, DhbA, and CrcA are involved in the fungal salicylic acid pathway, overview. The recombinant ShyA and CrcA together can efficiently convert salicylic acid into cis,cis-muconic acid through catechol as an intermediate. NRRL3_43 is suggested to be a salicylic acid hydroxylase-like enzyme	Aspergillus niger