Amide bond formation is one of the most important reactions in synthetic medicinal chemistry, however current methods rely on toxic or hazardous reagents and suffer from poor atom economy. In search of biocatalytic alternatives for amide bond synthesis, this work investigated a recently discovered class of ATP-dependent amide bond synthetases (ABSs), typified by McbA from Marinactinospora thermotolerans. A number of homologs, including AcABS from Actinoalloteichus cyanogriseus and ShABS from Streptalloteichus hindustanus, were identified and heterologously expressed in E. coli and purified. These homologs have demonstrated diverse activity, catalysing the formation of pharmaceutically relevant amides from β-carboline carboxylic acids and equimolar amine precursors. In addition to demonstrating an expanded substrate tolerance within families of parent acid and amine partners, these couplings can be achieved in high yield on a preparative scale. McbA showed good activity with much simpler acids, including indole-, naphthyl-, and benzoic acids, as well as simple aromatic and aliphatic amines, such as aniline, propargylamine and methylamine.
Crystallisation studies of McbA revealed a large 149° conformational change, around the hinge residue Q394, following formation of the AMP adenylate intermediate. These two conformations were annotated as ‘adenylation’ and ‘amidation’ conformers, mirroring the ‘adenylation’ and ‘thiolation’ conformations, respectively, of other adenylating enzymes such as the Carboxylic Acid Reductases (CARs) and CoA ligases. The acid binding site is comprised of hydrophobic residues, including F301 and L202, which form pi-interactions with the 1-acetyl-β-carboline carboxylic acid, and residue Y294 which may serve to limit the size of the active site. There are also interactions between the acetyl oxygen and pyridine nitrogen of the native acid with the main chain NH and C=O of residue G295. The limited number of interactions of active site residues with the native 1-acetyl-β-carboline carboxylic acid provide a possible explanation for the observed broad acid substrate tolerance. The nucleotide binding site is comprised of residues D377, R392 and R407, which form stabilising interactions with the ribose hydroxyls of ATP. Residue K483 has also been implicated in the stabilisation of the transition state on adenylate formation, through structural analysis and mutagenesis. McbA (K483A) mutant resulted in a 6-fold reduction in the Vmax and almost 5-fold increase in the KM compared to wt-McbA, using a fluorimetric assay to detect pyrophosphate release.
Docking studies with the 1-acetyl-β-carboline adenylate and 2-phenylethylamine revealed a hydrophobic amine binding site, comprised of residues including I197, L202, F241 and W246. The model placed the amine in close proximity to a catalytic aspartate residue, D201, proposed to act as a base in activating the amine prior to nucleophilic attack into the activated acyl adenylate. Mutagenesis of this aspartate to alanine, D201A, resulted in a significantly reduced conversion for the coupling of 1-acetyl-β-carboline acid and propargylamine, with wt-McbA and McbA (D201A) producing the amide product in 26% and 0.7% conversion, respectively, over 4 h. This provided evidence for the involvement of D201 in catalysis.
Substrate screens have highlighted application of these enzymes to the synthesis of a number of pharmaceutical molecules and, through the use of an ATP recycling system, this can be achieved using sub-stoichiometric concentrations of ATP. Two polyphosphate kinases, AjPPK2 and SmPPK2, were used to convert AMP to ADP and ADP to ATP respectively, using polyphosphoric acid as the phosphate source. This system was applied to the McbA-catalysed synthesis of the anti-depressant drug Moclobemide, achieving a conversion of 63%, highlighting these ABSs as promising biocatalysts for the sustainable synthesis of amide pharmaceuticals.
