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

Abstract

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...

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

“…With the aim to identify the primary sequence of Neurospora crassa CAR, four literature known CAR enzyme sequences ( Ni CAR:5 Q6RKB1.1, Mm CAR:6 B2HN69, Sr CAR:8 WP_013138593.1 and At CAR:7 XP_001212808.1) were used as templates for a blastp search against the Gen‐Bank non‐redundant protein sequences of Neurospora crassa (taxid:5241). The hit with highest scores was a hypothetical protein with NCBI accession code XP_955820.1.…”

“…Carboxylate reductase (CAR) enzymes exhibit a broad substrate tolerance for the conversion of organic acids to the respective aldehydes 4. However, only few CAR enzyme sequences have been elucidated5, 6, 7, 8, 9, 10, 11, 12 and are available for biocatalysis to date, although Nature provides a great versatility of organisms with the capability to catalyze this reaction. [4,13–18] The reduction of salicylic acid,19 benzoic acid and derivatives20 as well as cinnamic acid and derivatives21 has been observed in the ascomycete Neurospora crassa ( Nc ).…”

GriC and GriD Constitute a Carboxylic Acid Reductase Involved in Grixazone Biosynthesis in Streptomyces griseus

“…GriD shows similarity to NAD(P)-dependent aldehyde dehydrogenases. The combination of GriC and GriD appears to constitute a reductase similar to the Nocardia CAR having the N-terminal AMP-binding domain and the C-terminal reductase domain [12,13]. The ATP-and NADPH-dependent reduction of a carboxylic acid to the corresponding aldehyde via an acyl-AMP intermediate has also been found for a long-chain fatty acid reductase complex of luminescence bacteria [18].…”

“…GriC showed sequence similarity to AMP-binding proteins, including the N-terminal AMP-binding domain of a CAR of Nocardia sp. [12,13]. The Nocardia CAR has a reductase domain in the C-terminal portion and catalyzes the reduction of aryl carboxylic acids to the corresponding aldehydes through acyl-AMP formation, followed by NADPH-dependent reduction of the intermediate [12,13].…”

“…A hypothetical pathway for reduction by GriC and GriD, on the basis of the sequence homology of GriC/GriD with the CAR of Nocardia [12], is also shown. GriG is nonessential for grixazone biosynthesis but apparently contributes the uptake of 3,4-AHBA excreted and/or leaked from the cells (this study).…”