Literature summary for 1.2.1.30 extracted from

Kramer, L.; Le, X.; Hankore, E.D.; Wilson, M.A.; Guo, J.; Niu, W.
Engineering and characterization of hybrid carboxylic acid reductases (2019), J. Biotechnol., 304, 52-56 .

Protein Variants

Protein Variants	Comment	Organism
E697Q	site-directed mutagenesis, the mutant enzyme retains 48.8% of wild-type activity with benzoate substrate	Mycobacterium avium
additional information	hybrid enzymes that contain domains from four bacterial CARs and one fungal CAR are constructed based on domain boundaries that are defined using a combination of bioinformatics and structural analysis. Hybrid CARs are characterized in both steady-state and transient kinetics studies using aromatic and straight-chain (C3-C5) carboxylate substrates. Kinetic data support that the inter-domain interactions play an important role in the function of both wild-type and hybrid CARs and further lead to the hypothesis that reduction is the rate-determining step in CAR catalysis. Analysis of CAR catalysis and rationale for hybrid CAR engineering, overview. Combination of Mycobacterium avium domains with domains (R, A, and P) from CARs derived from fungus Neurospora crassa (Nc), resulting in hybrid enzymes: Mav(A)-Nc(PR), Mav(AP)-Nc(R), Nc(A)-Mav(PR), and Nc(AP)-Mav(R), kinetic analysis with dicarboxylate and hydroxyacid substrates, domain dynamics of hybrid CARs. Analysis of substrate specificity of recombinant hybrid mutant enzymes	Neurospora crassa
additional information	hybrid enzymes that contain domains from four bacterial CARs and one fungal CAR are constructed based on domain boundaries that are defined using a combination of bioinformatics and structural analysis. Hybrid CARs are characterized in both steady-state and transient kinetics studies using aromatic and straight-chain (C3-C5) carboxylate substrates. Kinetic data support that the inter-domain interactions play an important role in the function of both wild-type and hybrid CARs and further lead to the hypothesis that reduction is the rate-determining step in CAR catalysis. Analysis of CAR catalysis and rationale for hybrid CAR engineering, overview. Combination of Mycobacterium avium domains with domains (R, A, and P) from CARs derived from Kutzneria albida (Ka) resulting in hybrid enzymes: Mav(A)-Ka(PR), Mav(AP)-Ka(R), Ka(A)-Mav(PR), and Ka(AP)-Mav(R), kinetic analysis with dicarboxylate and hydroxyacid substrates, domain dynamics of hybrid CARs. Analysis of substrate specificity of recombinant hybrid mutant enzymes	Kutzneria albida
additional information	hybrid enzymes that contain domains from four bacterial CARs and one fungal CAR are constructed based on domain boundaries that are defined using a combination of bioinformatics and structural analysis. Hybrid CARs are characterized in both steady-state and transient kinetics studies using aromatic and straight-chain (C3-C5) carboxylate substrates. Kinetic data support that the inter-domain interactions play an important role in the function of both wild-type and hybrid CARs and further lead to the hypothesis that reduction is the rate-determining step in CAR catalysis. Analysis of CAR catalysis and rationale for hybrid CAR engineering, overview. Combination of Mycobacterium avium domains with domains (R, A, and P) from CARs derived from Mycobacterium marinum (Mm) resulting in hybrid enzymes: Mav(A)-Mm(PR), Mav(AP)-Mm(R), Mm(A)-Mav(PR), and Mm(AP)-Mav(R), kinetic analysis with dicarboxylate and hydroxyacid substrates, domain dynamics of hybrid CARs. Analysis of substrate specificity of recombinant hybrid mutant enzymes	Mycobacterium marinum
additional information	hybrid enzymes that contain domains from four bacterial CARs and one fungal CAR are constructed based on domain boundaries that are defined using a combination of bioinformatics and structural analysis. Hybrid CARs are characterized in both steady-state and transient kinetics studies using aromatic and straight-chain (C3-C5) carboxylate substrates. Kinetic data support that the inter-domain interactions play an important role in the function of both wild-type and hybrid CARs and further lead to the hypothesis that reduction is the rate-determining step in CAR catalysis. Analysis of CAR catalysis and rationale for hybrid CAR engineering, overview. Combination of Mycobacterium avium domains with domains (R, A, and P) from CARs derived from Mycobacterium marinum (Mm), Kutzneria albida (Ka), and Nocardia iowensis (Ni), and one fungal strain, Neurospora crassa (Nc), resulting in hybrid enzymes: Mav(A)-Mm(PR), Mav(AP)-Mm(R), Mm(A)-Mav(PR), Mm(AP)-Mav(R), Mav(A)-Ka(PR), Mav(AP)-Ka(R), Ka(A)-Mav(PR), Ka(AP)-Mav(R), Mav(A)-Ni(PR), Mav(AP)-Ni(R), Ni(A)-Mav(PR), and Ni(AP)-Mav(R), kinetic analysis with dicarboxylate and hydroxyacid substrates, domain dynamics of hybrid CARs, overview. When mutations Q637E and E697Q are introduced into hybrid CAR, Mm(A)-Mav(PR), reduction in activities is also observed, albeit to a less extent. Analysis of substrate specificity of recombinant hybrid mutant enzymes	Mycobacterium avium
additional information	hybrid enzymes that contain domains from four bacterial CARs and one fungal CAR are constructed based on domain boundaries that are defined using a combination of bioinformatics and structural analysis. Hybrid CARs are characterized in both steady-state and transient kinetics studies using aromatic and straight-chain (C3-C5) carboxylate substrates. Kinetic data support that the inter-domain interactions play an important role in the function of both wild-type and hybrid CARs and further lead to the hypothesis that reduction is the rate-determining step in CAR catalysis. Analysis of CAR catalysis and rationale for hybrid CAR engineering, overview. Combination of Mycobacterium avium domains with domains (R, A, and P) from CARs derived from Nocardia iowensis (Ni) resulting in hybrid enzymes: Mav(A)-Ni(PR), Mav(AP)-Ni(R), Ni(A)-Mav(PR), and Ni(AP)-Mav(R), kinetic analysis with dicarboxylate and hydroxyacid substrates, domain dynamics of hybrid CARs. Analysis of substrate specificity of recombinant hybrid mutant enzymes	Nocardia iowensis
Q637E	site-directed mutagenesis, the mutant enzyme retains 47.1% of wild-type activity with benzoate substrate	Mycobacterium avium

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	stopped-flow measurements, steady-state (kcat) and single-turnover (kobs) kinetics of wild-type and hybrid mutant enzymes, comparisons, overview	Neurospora crassa
additional information	-	additional information	stopped-flow measurements, steady-state (kcat) and single-turnover (kobs) kinetics of wild-type and hybrid mutant enzymes, comparisons, overview	Mycobacterium avium
additional information	-	additional information	stopped-flow measurements, steady-state (kcat) and single-turnover (kobs) kinetics of wild-type and hybrid mutant enzymes, comparisons, overview	Kutzneria albida
additional information	-	additional information	stopped-flow measurements, steady-state (kcat) and single-turnover (kobs) kinetics of wild-type and hybrid mutant enzymes, comparisons, overview	Nocardia iowensis
additional information	-	additional information	stopped-flow measurements, steady-state (kcat) and single-turnover (kobs) kinetics of wild-type and hybrid mutant enzymes, comparisons, overview	Mycobacterium marinum

Metals/Ions

Metals/Ions	Comment	Organism
Mg2+	required	Neurospora crassa
Mg2+	required	Mycobacterium avium
Mg2+	required	Kutzneria albida
Mg2+	required	Nocardia iowensis
Mg2+	required	Mycobacterium marinum

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
benzoate + NADPH + H+ + ATP	Neurospora crassa	-	benzaldehyde + NADP+ + AMP + diphosphate	-	ir
benzoate + NADPH + H+ + ATP	Mycobacterium avium	-	benzaldehyde + NADP+ + AMP + diphosphate	-	ir
benzoate + NADPH + H+ + ATP	Kutzneria albida	-	benzaldehyde + NADP+ + AMP + diphosphate	-	ir
benzoate + NADPH + H+ + ATP	Nocardia iowensis	-	benzaldehyde + NADP+ + AMP + diphosphate	-	ir
benzoate + NADPH + H+ + ATP	Mycobacterium marinum	-	benzaldehyde + NADP+ + AMP + diphosphate	-	ir
benzoate + NADPH + H+ + ATP	Mycobacterium marinum ATCC BAA-535	-	benzaldehyde + NADP+ + AMP + diphosphate	-	ir
vanillate + NADPH + H+ + ATP	Neurospora crassa	-	vanillin + NADP+ + AMP + diphosphate	-	ir
vanillate + NADPH + H+ + ATP	Mycobacterium avium	-	vanillin + NADP+ + AMP + diphosphate	-	ir
vanillate + NADPH + H+ + ATP	Kutzneria albida	-	vanillin + NADP+ + AMP + diphosphate	-	ir
vanillate + NADPH + H+ + ATP	Nocardia iowensis	-	vanillin + NADP+ + AMP + diphosphate	-	ir
vanillate + NADPH + H+ + ATP	Mycobacterium marinum	-	vanillin + NADP+ + AMP + diphosphate	-	ir
vanillate + NADPH + H+ + ATP	Mycobacterium marinum ATCC BAA-535	-	vanillin + NADP+ + AMP + diphosphate	-	ir

Organism

Organism	UniProt	Comment	Textmining
Kutzneria albida	-	-	-
Mycobacterium avium	-	-	-
Mycobacterium marinum	B2HN69	-	-
Mycobacterium marinum ATCC BAA-535	B2HN69	-	-
Neurospora crassa	-	-	-
Nocardia iowensis	-	-	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
3-hydroxypropionate + NADPH + H+ + ATP	-	Kutzneria albida	3-hydroxypropanal + NADP+ + AMP + diphosphate	-	ir
3-hydroxypropionate + NADPH + H+ + ATP	-	Nocardia iowensis	3-hydroxypropanal + NADP+ + AMP + diphosphate	-	ir
3-hydroxypropionate + NADPH + H+ + ATP	-	Mycobacterium marinum	3-hydroxypropanal + NADP+ + AMP + diphosphate	-	ir
3-hydroxypropionate + NADPH + H+ + ATP	-	Mycobacterium marinum ATCC BAA-535	3-hydroxypropanal + NADP+ + AMP + diphosphate	-	ir
3-hydroxypropionate + NADPH + H+ + ATP	-	Mycobacterium avium	? + NADP+ + AMP + diphosphate	-	ir
4-hydroxybutyrate + NADPH + H+ + ATP	-	Kutzneria albida	4-hydroxybutanal + NADP+ + AMP + diphosphate	-	ir
4-hydroxybutyrate + NADPH + H+ + ATP	-	Nocardia iowensis	4-hydroxybutanal + NADP+ + AMP + diphosphate	-	ir
4-hydroxybutyrate + NADPH + H+ + ATP	-	Mycobacterium marinum	4-hydroxybutanal + NADP+ + AMP + diphosphate	-	ir
4-hydroxybutyrate + NADPH + H+ + ATP	-	Mycobacterium marinum ATCC BAA-535	4-hydroxybutanal + NADP+ + AMP + diphosphate	-	ir
4-hydroxybutyrate + NADPH + H+ + ATP	-	Mycobacterium avium	? + NADP+ + AMP + diphosphate	-	ir
5-hydroxypentanoate + NADPH + H+ + ATP	-	Kutzneria albida	5-hydroxypentanal + NADP+ + AMP + diphosphate	-	ir
5-hydroxypentanoate + NADPH + H+ + ATP	-	Nocardia iowensis	5-hydroxypentanal + NADP+ + AMP + diphosphate	-	ir
5-hydroxypentanoate + NADPH + H+ + ATP	-	Mycobacterium marinum	5-hydroxypentanal + NADP+ + AMP + diphosphate	-	ir
5-hydroxypentanoate + NADPH + H+ + ATP	-	Mycobacterium avium	? + NADP+ + AMP + diphosphate	-	ir
benzoate + NADPH + H+ + ATP	-	Neurospora crassa	benzaldehyde + NADP+ + AMP + diphosphate	-	ir
benzoate + NADPH + H+ + ATP	-	Mycobacterium avium	benzaldehyde + NADP+ + AMP + diphosphate	-	ir
benzoate + NADPH + H+ + ATP	-	Kutzneria albida	benzaldehyde + NADP+ + AMP + diphosphate	-	ir
benzoate + NADPH + H+ + ATP	-	Nocardia iowensis	benzaldehyde + NADP+ + AMP + diphosphate	-	ir
benzoate + NADPH + H+ + ATP	-	Mycobacterium marinum	benzaldehyde + NADP+ + AMP + diphosphate	-	ir
benzoate + NADPH + H+ + ATP	-	Mycobacterium marinum ATCC BAA-535	benzaldehyde + NADP+ + AMP + diphosphate	-	ir
glutarate + NADPH + H+ + ATP	-	Kutzneria albida	5-oxopentanoate + 1,5-pentanedial + NADP+ + AMP + diphosphate	-	ir
glutarate + NADPH + H+ + ATP	-	Nocardia iowensis	5-oxopentanoate + 1,5-pentanedial + NADP+ + AMP + diphosphate	-	ir
glutarate + NADPH + H+ + ATP	-	Mycobacterium marinum	5-oxopentanoate + 1,5-pentanedial + NADP+ + AMP + diphosphate	-	ir
glutarate + NADPH + H+ + ATP	-	Mycobacterium avium	? + NADP+ + AMP + diphosphate	-	ir
malonate + NADPH + H+ + ATP	low activity	Kutzneria albida	3-oxopropanoate + 1,3-propanedial + NADP+ + AMP + diphosphate	-	ir
malonate + NADPH + H+ + ATP	low activity	Nocardia iowensis	3-oxopropanoate + 1,3-propanedial + NADP+ + AMP + diphosphate	-	ir
malonate + NADPH + H+ + ATP	low activity	Mycobacterium marinum	3-oxopropanoate + 1,3-propanedial + NADP+ + AMP + diphosphate	-	ir
malonate + NADPH + H+ + ATP	-	Mycobacterium avium	? + NADP+ + AMP + diphosphate	-	ir
additional information	no activity of the wild-type enzyme with succinate. Substrate specificity of recombinant hybrid mutant enzymes, overview	Kutzneria albida	?	-	-
additional information	substrate specificity of recombinant hybrid mutant enzymes, overview	Neurospora crassa	?	-	-
additional information	substrate specificity of recombinant hybrid mutant enzymes, overview	Mycobacterium avium	?	-	-
additional information	substrate specificity of recombinant hybrid mutant enzymes, overview	Nocardia iowensis	?	-	-
additional information	substrate specificity of recombinant hybrid mutant enzymes, overview	Mycobacterium marinum	?	-	-
additional information	substrate specificity of recombinant hybrid mutant enzymes, overview	Mycobacterium marinum ATCC BAA-535	?	-	-
succinate + NADPH + H+ + ATP	-	Nocardia iowensis	4-oxobutanoate + 1,4-butanedial + NADP+ + AMP + diphosphate	-	ir
succinate + NADPH + H+ + ATP	-	Mycobacterium marinum	4-oxobutanoate + 1,4-butanedial + NADP+ + AMP + diphosphate	-	ir
succinate + NADPH + H+ + ATP	-	Mycobacterium avium	? + NADP+ + AMP + diphosphate	-	ir
vanillate + NADPH + H+ + ATP	-	Neurospora crassa	vanillin + NADP+ + AMP + diphosphate	-	ir
vanillate + NADPH + H+ + ATP	-	Mycobacterium avium	vanillin + NADP+ + AMP + diphosphate	-	ir
vanillate + NADPH + H+ + ATP	-	Kutzneria albida	vanillin + NADP+ + AMP + diphosphate	-	ir
vanillate + NADPH + H+ + ATP	-	Nocardia iowensis	vanillin + NADP+ + AMP + diphosphate	-	ir
vanillate + NADPH + H+ + ATP	-	Mycobacterium marinum	vanillin + NADP+ + AMP + diphosphate	-	ir
vanillate + NADPH + H+ + ATP	-	Mycobacterium marinum ATCC BAA-535	vanillin + NADP+ + AMP + diphosphate	-	ir

Synonyms

Synonyms	Comment	Organism
CAR	-	Neurospora crassa
CAR	-	Mycobacterium avium
CAR	-	Kutzneria albida
CAR	-	Nocardia iowensis
CAR	-	Mycobacterium marinum
Carboxylic acid reductase	-	Neurospora crassa
Carboxylic acid reductase	-	Mycobacterium avium
Carboxylic acid reductase	-	Kutzneria albida
Carboxylic acid reductase	-	Nocardia iowensis
Carboxylic acid reductase	-	Mycobacterium marinum
kaCAR	-	Kutzneria albida
maCAR	-	Mycobacterium marinum
mmCAR	-	Mycobacterium avium
NcCAR	-	Neurospora crassa
niCAR	-	Nocardia iowensis

Cofactor

Cofactor	Comment	Organism
ATP	-	Neurospora crassa
ATP	-	Mycobacterium avium
ATP	-	Kutzneria albida
ATP	-	Nocardia iowensis
ATP	-	Mycobacterium marinum
NADPH	-	Neurospora crassa
NADPH	-	Mycobacterium avium
NADPH	-	Kutzneria albida
NADPH	-	Nocardia iowensis
NADPH	-	Mycobacterium marinum

General Information

General Information	Comment	Organism
physiological function	carboxylic acid reductases (CARs) are valuable biocatalysts due to their ability to reduce a broad range of carboxylate substrates into the corresponding aldehyde products. CARs are multi-domain enzymes with separate catalytic domains for the adenylation and the subsequent reduction of substrates. Inter-domain dynamics are crucial for the catalytic activities of CARs	Neurospora crassa
physiological function	carboxylic acid reductases (CARs) are valuable biocatalysts due to their ability to reduce a broad range of carboxylate substrates into the corresponding aldehyde products. CARs are multi-domain enzymes with separate catalytic domains for the adenylation and the subsequent reduction of substrates. Inter-domain dynamics are crucial for the catalytic activities of CARs	Mycobacterium avium
physiological function	carboxylic acid reductases (CARs) are valuable biocatalysts due to their ability to reduce a broad range of carboxylate substrates into the corresponding aldehyde products. CARs are multi-domain enzymes with separate catalytic domains for the adenylation and the subsequent reduction of substrates. Inter-domain dynamics are crucial for the catalytic activities of CARs	Kutzneria albida
physiological function	carboxylic acid reductases (CARs) are valuable biocatalysts due to their ability to reduce a broad range of carboxylate substrates into the corresponding aldehyde products. CARs are multi-domain enzymes with separate catalytic domains for the adenylation and the subsequent reduction of substrates. Inter-domain dynamics are crucial for the catalytic activities of CARs	Nocardia iowensis
physiological function	carboxylic acid reductases (CARs) are valuable biocatalysts due to their ability to reduce a broad range of carboxylate substrates into the corresponding aldehyde products. CARs are multi-domain enzymes with separate catalytic domains for the adenylation and the subsequent reduction of substrates. Inter-domain dynamics are crucial for the catalytic activities of CARs	Mycobacterium marinum