S-Adenosyl Methionine Cofactor Modifications Enhance the Biocatalytic Repertoire of Small Molecule C-Alkylation

A tandem enzymatic strategy to enhance the scope of Calkylation of small molecules via the in situ formation of S-adenosyl methionine (SAM) cofactor analogues is described. A solventexposed channel present in the SAM-forming enzyme SalL tolerates 5'-chloro-5’-deoxyadenosine (ClDA) analogues modified at the 2position of the adenine nucleobase. Coupling SalL-catalyzed cofactor production with C-(m)ethyl transfer to coumarin substrates catalyzed by the methyltransferase (MTase) NovO forms C(m)ethylated coumarins in superior yield and greater substrate scope relative to that obtained using cofactors lacking nucleobase modifications. Establishing the molecular determinants which influence C-alkylation provides the basis to develop a late-stage enzymatic platform for the preparation of high value small molecule

An Aminocaprolactam Racemase from Ochrobactrum anthropi with Promiscuous Amino Acid Ester Racemase Activity

The kinetic resolution of amino acid esters (AAEs) is a useful synthetic strategy for the preparation of single enantiomer amino acids. The development of an enzymatic dynamic kinetic resolution (DKR) process for AAEs, which would give a theoretical yield of 100% of the enantiopure product, would require an amino acid ester racemase (AAER), however, no such enzyme has been described. We have identified low AAER activity of 15 U mg-1 in a homolog of a PLP-dependent α-amino ε-caprolactam racemase (ACLR) from Ochrobactrum anthropi. We have determined the structure of this enzyme, OaACLR, to a resolution of 1.87 Å and using structure-guided saturation mutagenesis, in combination with a colorimetric screen for AAER activity, we have identified a mutant, L293C, in which the promiscuous AAER activity of this enzyme towards L-phenylalanine methyl ester is improved 3.7 fold

Engineering enzymes with non-canonical active site functionality

The combination of computational enzyme design and laboratory evolution provides an attractive platform for the creation of protein catalysts with new function. To date, designed mechanisms have relied upon Nature’s alphabet of 20 genetically encoded amino acids, which greatly restricts the range of functionality which can be installed into enzyme active sites. Here, we have exploited engineered components of the cellular translation machinery to create a protein catalyst which operates via a non-canonical catalytic nucleophile. We have subsequently shown that powerful laboratory evolution protocols can be readily adapted to allow optimization of enzymes containing non-canonical active site functionality. Crystal structures obtained along the evolutionary trajectory highlight the origins of improved activity. Thus our approach merges beneficial features of organo- and biocatalysis, by combining the intrinsic reactivities and greater versatility of small molecule catalysts with the rate enhancements, reaction selectivities and evolvability of proteins. Please click Additional Files below to see the full abstract

The Broad Aryl Acid Specificity of the Amide Bond Synthetase McbA Suggests Potential for the Biocatalytic Synthesis of Amides

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