Publications

• Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities.

  Akhtar M.K., Turner N.J., Jones P.R.

  Aliphatic hydrocarbons such as fatty alcohols and petroleum-derived alkanes have numerous applications in the chemical industry. In recent years, the renewable synthesis of aliphatic hydrocarbons has been made possible by engineering microbes to overaccumulate fatty acids. However, to generate end ... >> More

  Aliphatic hydrocarbons such as fatty alcohols and petroleum-derived alkanes have numerous applications in the chemical industry. In recent years, the renewable synthesis of aliphatic hydrocarbons has been made possible by engineering microbes to overaccumulate fatty acids. However, to generate end products with the desired physicochemical properties (e.g., fatty aldehydes, alkanes, and alcohols), further conversion of the fatty acid is necessary. A carboxylic acid reductase (CAR) from Mycobacterium marinum was found to convert a wide range of aliphatic fatty acids (C(6)-C(18)) into corresponding aldehydes. Together with the broad-substrate specificity of an aldehyde reductase or an aldehyde decarbonylase, the catalytic conversion of fatty acids to fatty alcohols (C(8)-C(16)) or fatty alkanes (C(7)-C(15)) was reconstituted in vitro. This concept was applied in vivo, in combination with a chain-length-specific thioesterase, to engineer Escherichia coli BL21(DE3) strains that were capable of synthesizing fatty alcohols and alkanes. A fatty alcohol titer exceeding 350 mg·L(-1) was obtained in minimal media supplemented with glucose. Moreover, by combining the CAR-dependent pathway with an exogenous fatty acid-generating lipase, natural oils (coconut oil, palm oil, and algal oil bodies) were enzymatically converted into fatty alcohols across a broad chain-length range (C(8)-C(18)). Together with complementing enzymes, the broad substrate specificity and kinetic characteristics of CAR opens the road for direct and tailored enzyme-catalyzed conversion of lipids into user-ready chemical commodities. << Less

  Proc. Natl. Acad. Sci. U.S.A. 110:87-92(2013) [PubMed] [EuropePMC]

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

  Li T., Rosazza J.P.

  An aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646 was purified 196-fold by a combination of Mono-Q, Reactive Green 19 agarose affinity, and hydroxyapatite chromatographies. The purified enzyme runs as a single band of 140 kDa on sodium dodecyl sulfate-polyacrylamide gel electropho ... >> More

  An aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646 was purified 196-fold by a combination of Mono-Q, Reactive Green 19 agarose affinity, and hydroxyapatite chromatographies. The purified enzyme runs as a single band of 140 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass was estimated to be 163 +/-3.8 kDa by gel filtration, indicating that this enzyme is a monomeric protein. The binding of the enzyme to Reactive Green 19 agarose was Mg2+ dependent. The binding capacity was estimated to be about 0.2 mg of Reactive Green agarose per ml in the presence of 10 mM MgCl2. This enzyme can catalyze the reduction of a wide range of aryl carboxylic acids, including substituted benzoic acids, phenyl-substituted aliphatic acids, heterocyclic carboxylic acids, and polyaromatic ring carboxylic acids, to produce the corresponding aldehydes. The Km values for benzoate, ATP, and NADPH were determined to be 645 +/-75, 29.3 +/- 3.1, and 57.3 +/-12.5 microM, respectively. The Vmax was determined to be 0.902 +/-0.04 micromol/min/mg of protein. Km values for (S)-(+)-alpha-methyl-4-(2-methylpropyl)-benzeneacetic acid (ibuprofen) and its (R)-(-) isomer were determined to be 155 +/- 18 and 34.5 +/-2.5 microM, respectively. The Vmax for the (S)-(+) and (R)-(-) isomers were 1.33 and 0.15 micromol/min/mg of protein, respectively. Anthranilic acid is a competitive inhibitor with benzoic acid as a substrate, with a Ki of 261 +/-30 microM. The N-terminal and internal amino acid sequences of a 76-kDa peptide from limited alpha-chymotrypsin digestion were determined. << Less

  J. Bacteriol. 179:3482-3487(1997) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Reduction of carboxylic acids by Nocardia aldehyde oxidoreductase requires a phosphopantetheinylated enzyme.

  Venkitasubramanian P., Daniels L., Rosazza J.P.

  Aldehyde oxidoreductase (carboxylic acid reductase (Car)) catalyzes the magnesium-, ATP-, and NADPH-dependent reduction of carboxylic acids to their corresponding aldehydes. Heterologous expression of the car gene in Escherichia coli afforded purified recombinant enzyme with a specific activity ne ... >> More

  Aldehyde oxidoreductase (carboxylic acid reductase (Car)) catalyzes the magnesium-, ATP-, and NADPH-dependent reduction of carboxylic acids to their corresponding aldehydes. Heterologous expression of the car gene in Escherichia coli afforded purified recombinant enzyme with a specific activity nearly 50-fold lower than that of purified native Nocardia sp. enzyme. The 5-fold increase in specific activity obtained by incubating purified recombinant Car with CoA and Nocardia cell-free extracts indicated that post-translational phosphopantetheinylation of Car is required for maximum enzyme activity. Nocardia phosphopantetheine transferase (PPTase) expressed in E. coli was isolated and characterized. When incubated with [(3)H]acetyl-CoA and Nocardia PPTase, the labeled acetylphosphopantetheine moiety was incorporated into recombinant Car. Coexpression of Nocardia Car and PPTase in E. coli gave a reductase with nearly 20-fold higher specific activity. Site-directed mutagenesis in which Ser(689) was replaced with Ala resulted in an inactive Car mutant. The results show that Car expressed in Escherichia coli is an apoenzyme that is converted to a holoenzyme by post-translational modification via phosphopantetheinylation. Doubly recombinant resting E. coli cells efficiently reduce vanillic acid to vanillin. << Less

  J. Biol. Chem. 282:478-485(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Nocardia sp. carboxylic acid reductase: cloning, expression, and characterization of a new aldehyde oxidoreductase family.

  He A., Li T., Daniels L., Fotheringham I., Rosazza J.P.

  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 benzoa ... >> More

  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. << Less

  Appl. Environ. Microbiol. 70:1874-1881(2004) [PubMed] [EuropePMC]