In this review we examine the fascinating array of microbial and enzymatic transformations of ferulic acid. Ferulic acid is an extremely abundant, preformed phenolic aromatic chemical found widely in nature. Ferulic acid is viewed as a commodity scale, renewable chemical feedstock for biocatalytic conversion to other useful aromatic chemicals. Most attention is focused on bioconversions of ferulic acid itself. Topics covered include cinnamoyl side-chain cleavage; nonoxidative decarboxylation; mechanistic details of styrene formation; purification and characterization of ferulic acid decarboxylase; conversion of ferulic acid to vanillin; O-demethylation; and reduction reactions. Biotransformations of vinylguaiacol are discussed, and selected biotransformations of vanillic acid including oxidative and nonoxidative decarboxylation are surveyed. Finally, enzymatic oxidative dimerization and polymerization reactions are reviewed.

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Flavonoids are among the most ubiquitous phenolic compounds found in nature. These compounds have diverse physiological and pharmacological activities such as estrogenic, antitumor, antimicrobial, antiallergic, and anti-inflammatory effects. They are well-known antioxidants and metal ion-chelators. In the present review, biotransformations of numerous flavonoids catalyzed mainly by microbes and few plant enzymes are described in four different flavonoid classes, viz., chalcones, isoflavones, catechins, and flavones. Both phase I (oxidative) and phase II (conjugative) biotransformations representing a variety of reactions including condensation, cyclization, hydroxylation, dehydroxylation, alkylation, O-dealkylation, halogenation, dehydrogenation, double-bond reduction, carbonyl reduction, glycosylation, sulfation, dimerization, or different types of ring degradations are elaborated here. In some cases, the observed microbial transformations mimic mammalian and/or plant metabolism. This review recognizes Norman Farnsworth, who through his fascination and hard work in pharmacognosy has fostered the excitement of discovery by numerous students and faculty far and beyond the halls of the University of Illinois at Chicago. It is with grateful thanks for these efforts that we dedicate this review to him.

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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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...

Horseradish peroxidase (HRP; EC 1.11.1.7) catalyzed the H(2)O(2)-dependent oxidative coupling of (+)-catechin 1 to form three different biphenyl C-C dimers 2-4, whereas Rhus vernicifera laccase catalyzed the formation of two new catechin-hydroquinone adducts 5 and 6. Spectroscopic evidence showed that HRP dimers were linked through position 8 of the A-ring of one catechin moiety to C-5' of ring B in 2 and 4 and to C-2 of ring C in 3. The unusual catechin dicarboxylic acid dimer 4 was obtained by ortho cleavage of the E-ring. Hydroquinone served as both a shuttle oxidant and a reactant by coupling at C-2' and C-5' of the catechin B-ring during laccase oxidations. HRP and laccase oxidation products were compared to D,L-alpha-tocopherol and (+)-catechin for their abilities to inhibit iron-induced lipid peroxidation in rat brain homogenates and Fe(3+)-ADP/NADPH in rat liver microsomes, as measured by the intensity of thiobarbituric acid reactive substance. All metabolites exhibited anti-lipid peroxidation with IC(50) values approximately 2-8 times higher than those of standard compounds. Characteristic reaction products may prove to be novel markers for (+)-catechin antioxidant reactions in living systems.