Formation and Reduction of Intermediate Acyladenylate by Aryl‐Aldehyde

Gross, Georg G.

doi:10.1111/j.1432-1033.1972.tb02569.x

Cited by 39 publications

(22 citation statements)

References 18 publications

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“…The classification of this enzyme as aryl‐aldehyde:NADP oxidoreductase21 was based on its substrate scope which appeared to be restricted to compounds with aromatic ring structures. Aliphatic acids and amino acids were not reduced by this enzyme 23. The carboxylate reduction is dependent on ATP, NADPH and magnesium ions (Scheme 1), leading eventually to its classification as an E.C.…”

Section: Introductionmentioning

confidence: 95%

Selective Enzymatic Transformation to Aldehydes in vivo by Fungal Carboxylate Reductase from Neurospora crassa

et al. 2016

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The enzymatic reduction of carboxylic acids is in its infancy with only a handful of biocatalysts available to this end. We have increased the spectrum of carboxylate‐reducing enzymes (CARs) with the sequence of a fungal CAR from Neurospora crassa OR74A (NcCAR). NcCAR was efficiently expressed in E. coli using an autoinduction protocol at low temperature. It was purified and characterized in vitro, revealing a broad substrate acceptance, a pH optimum at pH 5.5–6.0, a T m of 45 °C and inhibition by the co‐product pyrophosphate which can be alleviated by the addition of pyrophosphatase. The synthetic utility of NcCAR was demonstrated in a whole‐cell biotransformation using the Escherichia coli K‐12 MG1655 RARE strain in order to suppress overreduction to undesired alcohol. The fragrance compound piperonal was prepared from piperonylic acid (30 mM) on gram scale in 92 % isolated yield in >98% purity. This corresponds to a productivity of 1.5 g/L/h.

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Section: Introductionmentioning

confidence: 95%

Selective Enzymatic Transformation to Aldehydes in vivo by Fungal Carboxylate Reductase from Neurospora crassa

et al. 2016

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“…Further work showed that this enzyme catalyzed the initial reaction between an aromatic acid and ATP to form an acyl-AMP intermediate which was subsequently reduced by NADPH to form the aldehyde ( Fig. 1) (12). This enzyme was named as an aryl aldehyde oxidoreductase (EC 1.2.1.30).…”

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confidence: 99%

“…This aryl aldehyde oxidoreductase can also catalyze the reduction of benzoyl-AMP by NADPH (19). The Nocardia enzyme was purified, and it was proposed that it reduced benzoate by the same mechanism as the Neurospora enzyme (12).…”

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Purification, characterization, and properties of an aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646

Rosazza

1997

J Bacteriol

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Microbial reductions of aromatic carboxylic acids, usually to their corresponding alcohols, have been observed with wholecell biotransformations by a number of microorganisms, including Actinomyces (17), Clostridium thermoaceticum (31), Aspergillus niger (2, 24), Corynespora melonis (2), Coriolus (2), Neurospora (3), Glomerella cingulata (29, 30), Gloeosporium laeticolor (30), and Nocardia (8, 18). In all reported cases, substrates contained an aromatic moiety, although not all directly attached to the carboxyl groups being reduced to the corresponding alcohols. When the carbon next to the carboxyl group was chiral, the reduction displayed different degrees of enantioselectivity (8,29,30). The enantioselective step of this reduction has never been studied. Gross and Zenk found that in Neurospora crassa, an aldehyde intermediate was formed as a carboxylic acid was reduced to an alcohol (13,14). The enzyme reducing aryl carboxylic acids to aldehydes was purified and identified as a monomeric protein with an apparent molecular mass of 120,000 Da (13). The reduction required ATP, Mg 2ϩ , and NADPH as cofactors (13). Further work showed that this enzyme catalyzed the initial reaction between an aromatic acid and ATP to form an acyl-AMP intermediate which was subsequently reduced by NADPH to form the aldehyde (Fig. 1) (12). This enzyme was named as an aryl aldehyde oxidoreductase (EC 1.2.1.30).Kato et al. showed that Nocardia asteroides JCM 3016 could transform benzoic acid to benzyl alcohol (18). The enzyme from this organism reduces benzoic acid to benzaldehyde with ATP, NADPH, and Mg 2ϩ as cofactors. The K m values for benzoic acid, NADPH, and ATP were 260, 6, and 23 nM, respectively. A number of mono-and multisubstituted benzoic acids can be reduced by the enzyme. This aryl aldehyde oxidoreductase can also catalyze the reduction of benzoyl-AMP by NADPH (19). The Nocardia enzyme was purified, and it was proposed that it reduced benzoate by the same mechanism as the Neurospora enzyme (12).In our laboratory, a strain of Nocardia (NRRL 5646) was found to efficiently reduce aryl carboxylic acids to give aldehydes and alcohols. With this organism, the substrate specificity for carboxylic acids appears to be significantly different than that for the enzyme reported by Kato et al. (19). Whole-cell reductions with Nocardia sp. strain NRRL 5646 were highly enantioselective with ibuprofen isomers as substrates (8). We now report a rapid purification method, the characterization of the enzyme, the enantioselectivity of the enzyme with two isomers of ibuprofen, and the range of reductions of a series of aryl carboxylic acid substrates. MATERIALS AND METHODS Materials.A Mono-Q anion exchanger, hydroxyapatite, and molecular weight standards for gel electrophoresis were purchased from Bio-Rad (Hercules, Calif.). Polyvinylidene difluoride membranes were from Applied Biosystems (Foster City, Calif.). (S)-(ϩ)-␣-Methyl-4-(2-methylpropyl)-benzeneacetic acid (ibuprofen) was from Aldrich (Milwaukee, Wis.). The (R)-(Ϫ) isomer of ibuprofe...

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“…Chemical methods for carboxylic acid reductions are limited, and they usually require prior derivatization and product deblocking with reactants containing competing functional groups. Biocatalytic reductions of carboxylic acids are attractive because the substrates are water soluble, blocking chemistry is not necessary, reductions are enantiospecific, and the scope of the reaction is very broad (24, 32).Although microbial reductions of carboxylic acids, usually producing the acids' corresponding aldehydes or alcohols, have been observed with whole-cell reactions of bacteria and fungi (3,4,6,8,20,22,24,25,30,(36)(37)(38)40), enzymatic reductions of carboxylic acids are relatively new and unexploited biocatalytic reactions of great potential value in organic synthesis (12).Aldehyde oxidoreductases, also known as carboxylic acid reductases (CAR), require ATP, Mg 2ϩ , and NADPH as cofactors (16,17,18,21,24). The reduction is a stepwise process involving initial binding of both ATP and the carboxylic acid to the enzyme in order to form mixed 5Ј-adenylic acid-carbonyl anhydride intermediates (9,15,25,27,39) that are subsequently reduced by hydride delivery from NADPH to form aldehyde products (16,25) (Fig.…”

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Nocardia sp. Carboxylic Acid Reductase: Cloning, Expression, and Characterization of a New Aldehyde Oxidoreductase Family

He¹,

Li²,

Daniels

et al. 2004

Appl Environ Microbiol

107

100

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We have cloned, sequenced, and expressed the gene for a unique ATP-and NADPH-dependent carboxylic acid reductase (CAR) from a Nocardia species that reduces carboxylic acids to their corresponding aldehydes. Recombinant CAR containing an N-terminal histidine affinity tag had K m values for benzoate, ATP, and NADPH that were similar to those for natural CAR, and recombinant CAR reduced benzoic, vanillic, and ferulic acids to their corresponding aldehydes. car is the first example of a new gene family encoding oxidoreductases with remote acyl adenylation and reductase sites.Aromatic, aliphatic, and alicyclic aldehydes and alcohols are useful intermediates in the chemical, pharmaceutical, and food industries. Chemical methods for carboxylic acid reductions are limited, and they usually require prior derivatization and product deblocking with reactants containing competing functional groups. Biocatalytic reductions of carboxylic acids are attractive because the substrates are water soluble, blocking chemistry is not necessary, reductions are enantiospecific, and the scope of the reaction is very broad (24, 32).Although microbial reductions of carboxylic acids, usually producing the acids' corresponding aldehydes or alcohols, have been observed with whole-cell reactions of bacteria and fungi (3,4,6,8,20,22,24,25,30,(36)(37)(38)40), enzymatic reductions of carboxylic acids are relatively new and unexploited biocatalytic reactions of great potential value in organic synthesis (12).Aldehyde oxidoreductases, also known as carboxylic acid reductases (CAR), require ATP, Mg 2ϩ , and NADPH as cofactors (16,17,18,21,24). The reduction is a stepwise process involving initial binding of both ATP and the carboxylic acid to the enzyme in order to form mixed 5Ј-adenylic acid-carbonyl anhydride intermediates (9,15,25,27,39) that are subsequently reduced by hydride delivery from NADPH to form aldehyde products (16,25) (Fig. 1). Aromatic CARs have been purified to homogeneity only from Neurospora (17) and Nocardia (21, 24) species. Although N-terminal and internal amino acid sequences were reported for our Nocardia enzyme (24), sequences have never been determined for any gene coding for CARs. CAR from Nocardia sp. strain NRRL 5646 has an extremely wide substrate range, and it enantiospecifically reduces carboxylic acids (8,24,32). We report here the cloning and expression of the first CAR gene, car, and the use of cloned enzyme in vitro and in vivo to reduce carboxylic acid substrates.Strains, plasmids, media, and growth. Bacteria and plasmids used in this study are listed in Table 1. Nocardia sp. strain NRRL 5646 (14) was grown at 30°C in Luria-Bertani (LB) medium containing 0.05% Tween 80 (vol/vol [liquid medium only]). With Escherichia coli as the recombinant host for pHAT-based vectors, cells were grown at 37°C on solid or in liquid LB medium. Ampicillin (100 g/ml) was incorporated into LB medium to select for recombinants, and isopropyl-␤-D-thiogalactopyranoside (IPTG) (1 mM) and/or 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranosi...

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Formation and Reduction of Intermediate Acyladenylate by Aryl‐Aldehyde

Cited by 39 publications

References 18 publications

Selective Enzymatic Transformation to Aldehydes in vivo by Fungal Carboxylate Reductase from Neurospora crassa

Selective Enzymatic Transformation to Aldehydes in vivo by Fungal Carboxylate Reductase from Neurospora crassa

Purification, characterization, and properties of an aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646

Nocardia sp. Carboxylic Acid Reductase: Cloning, Expression, and Characterization of a New Aldehyde Oxidoreductase Family

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