The biocatalytic synthesis of amides from carboxylic acids and primary amines in aqueous media can be achieved using the ATP-dependent amide bond synthetase McbA, via an adenylate intermediate, using only 1.5 equiv of the amine nucleophile. Following earlier studies that characterized the broad carboxylic acid specificity of McbA, we now show that, in addition to the natural amine substrate 2phenylethylamine, a range of simple aliphatic amines, including methylamine, butylamine, and hexylamine, and propargylamine are coupled efficiently to the native carboxylic acid substrate 1-acetyl-9Hβ-carboline-3-carboxylic acid by the enzyme, to give amide products with up to >99% conversion. The structure of wild-type McbA in its amidation conformation, coupled with modeling and mutational studies, reveal an amine access tunnel and a possible role for residue D201 in amine activation. Amide couplings were slower with anilines and alicyclic secondary amines such as pyrrolidine and piperidine. The broader substrate specificity of McbA was exploited in the synthesis of the monoamine oxidase A inhibitor moclobemide, through the reaction of 4-chlorobenzoic acid with 1.5 equiv of 4-(2aminoethyl)morpholine, and utilizing polyphosphate kinases SmPPK and AjPPK in the presence of polyphosphoric acid and 0.1 equiv of ATP, required for recycling of the cofactor.
Cytochrome P450 CYP153A M.aq from Marinobacter aquaeolei serves as a model enzyme for the terminal (ω-) hydroxylation of medium- to long-chain fatty acids. We have engineered this enzyme using different mutagenesis approaches based on structure-sequence-alignments within the 3DM database and crystal structures of CYP153A M.aq and a homologue CYP153A P.sp . Applying these focused mutagenesis strategies and site-directed saturation mutagenesis, we created a variant that ω-hydroxylates octanoic acid. The M.aqRLT variant exhibited 151-fold improved catalytic efficiency and showed strongly improved substrate binding (25-fold reduced K m compared to the wild type). We then used molecular dynamics simulations to gain deeper insights into the dynamics of the protein. We found the tunnel modifications and the two loop regions showing greatly reduced flexibility in the engineered variant were the main features responsible for stabilizing the enzyme–substrate complex and enhancing the catalytic efficiency. Additionally, we showed that a previously known fatty acid anchor (Q129R) interacts significantly with the ligand to hold it in the reactive position, thereby boosting the activity of the variant M.aqRLT toward octanoic acid. The study demonstrates the significant effects of both substrate stabilization and the impact of enzyme flexibility on catalytic efficiency. These results could guide the future engineering of enzymes with deeply buried active sites to increase or even establish activities toward yet unknown types of substrates.
Amide bond formation is one of the most important reactions in pharmaceutical synthetic chemistry. The development of sustainable methods for amide bond formation, including those that are catalyzed by enzymes, is therefore of significant interest. The ATP‐dependent amide bond synthetase (ABS) enzyme McbA, from Marinactinospora thermotolerans, catalyzes the formation of amides as part of the biosynthetic pathway towards the marinacarboline secondary metabolites. The reaction proceeds via an adenylate intermediate, with both adenylation and amidation steps catalyzed within one active site. In this study, McbA was applied to the synthesis of pharmaceutical‐type amides from a range of aryl carboxylic acids with partner amines provided at 1–5 molar equivalents. The structure of McbA revealed the structural determinants of aryl acid substrate tolerance and differences in conformation associated with the two half reactions catalyzed. The catalytic performance of McbA, coupled with the structure, suggest that this and other ABS enzymes may be engineered for applications in the sustainable synthesis of pharmaceutically relevant (chiral) amides.
Cytochrome P450 monooxygenases (P450s) play crucial roles in the cell metabolism and provide an unsurpassed diversity of catalysed reactions. Here, we report the identification and biochemical characterization of two P450s from Arthrobacter sp., a Gram-positive organism known to degrade the opium alkaloid papaverine. Combining phylogenetic and genomic analysis suggested physiological roles for P450s in metabolism and revealed potential gene clusters with redox partners facilitating the reconstitution of the P450 activities in vitro. CYP1232F1 catalyses the para demethylation of 3,4-dimethoxyphenylacetic acid to homovanillic acid while CYP1232A24 continues demethylation to 3,4-dihydroxyphenylacetic acid. Interestingly, the latter enzyme is also able to perform both demethylation steps with preference for the meta position. The crystal structure of CYP1232A24, which shares only 29% identity to previous published structures of P450s helped to rationalize the preferred demethylation specificity for the meta position and also the broader substrate specificity profile. In addition to the detailed characterization of the two P450s using their physiological redox partners, we report the construction of a highly active whole-cell Escherichia coli biocatalyst expressing CYP1232A24, which formed up to 1.77 g l−1 3,4-dihydroxyphenylacetic acid. Our results revealed the P450s’ role in the metabolic pathway of papaverine enabling further investigation and application of these biocatalysts.
Amide bond formation is one of the most important reactions in pharmaceutical synthetic chemistry. The development of sustainable methods for amide bond formation, including those that are catalyzed by enzymes, is therefore of significant interest. The ATP‐dependent amide bond synthetase (ABS) enzyme McbA, from Marinactinospora thermotolerans, catalyzes the formation of amides as part of the biosynthetic pathway towards the marinacarboline secondary metabolites. The reaction proceeds via an adenylate intermediate, with both adenylation and amidation steps catalyzed within one active site. In this study, McbA was applied to the synthesis of pharmaceutical‐type amides from a range of aryl carboxylic acids with partner amines provided at 1–5 molar equivalents. The structure of McbA revealed the structural determinants of aryl acid substrate tolerance and differences in conformation associated with the two half reactions catalyzed. The catalytic performance of McbA, coupled with the structure, suggest that this and other ABS enzymes may be engineered for applications in the sustainable synthesis of pharmaceutically relevant (chiral) amides.
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