Application of amine transaminases (ATAs) came into focus as efficient biocatalysts for the preparation of optically pure amines which represent highly valuable key intermediates or products in the pharmaceutical, chemical, and agricultural sectors. It is an attractive alternative to transition-metal catalysed asymmetric synthesis to reduce cost and inadequate stereo selectivity. However, their catalytic efficiency towards bulky ketone substrates is greatly limited by steric hindrance and therefore presents a great challenge for industrial synthetic applications. However, for application as suitable biocatalysts for stereoselective amination towards bulky ketone substrates, extensive protein engineering of ATAs is required. ATAs are enzymes, difficult to engineer because of the unique structural architecture of the active site that limits bulkier substrates.
The most prominent example of such a successful ATA engineering resulted from a collaboration between Merck & Co. and Codexis which led to the realisation of an industrial process for the enzymatic asymmetric synthesis of the antidiabetic drug (R)-sitagliptin with >99.95% optical purity. Prior to this, access to (R)-sitagliptin was via enamine formation followed by asymmetric hydrogenation at high pressure using a rhodium-based chiral catalyst which rendered lower stereo selectivity and a rhodium contaminated product stream. The biocatalytic approach using the engineered variant ATA-117 is a prime example of green chemistry including overall waste reduction, lower energy consumption and avoidance of hazardous heavy metal catalyst waste treatment.
In our study we have used (S)-selective ATAs to expand the substrate scope towards bulky ketones using a novel Quantum mechanics (QM) based engineering framework. The framework predicts hotspots by analyzing the E-S molecular dynamics (MD) and QM simulations using novel methods developed in-house.
The enzyme penicillin G acylase (PGA; EC 188.8.131.52) is a heterodimeric protein consisting of a small subunit and a large subunit, which are formed by the processing of a single polypeptide precursor. PGA belongs to the structural superfamily of N-terminal nucleophile hydrolases that share a common fold around the active site bearing a catalytic serine, cysteine, or threonine at the N-terminal position. Functionally, PGA acts on the side chains of penicillin G, cephalosporin G, and other related antibiotics to produce antibiotic intermediates such as 6-amino penicillanic acid (6-APA) and 7-amino des- acetoxy cephalosporanic acid (7-ADCA), leaving behind phenyl acetic acid (PAA) as a common by-product. PGA can be used to produce valuable backbone chemicals, such as, 6-aminopenicillanic acid (6-APA), and 7-amino des- acetoxy cephalosporanic acid (7-ADCA), respectively, for the synthesis of semi-synthetic penicillins and cephalosporins. Penicillin G acylase (PGA) gained a unique position among enzymes used by pharmaceutical industry for production of β-lactam antibiotics. Kinetically controlled enzymatic syntheses of cephalosporins of novel generations in which PGA catalyzes coupling of activated acyl donor with nucleophile belong among the latest large-scale applications. Contrary to rather specific roles of other enzymes involved in β-lactam biocatalyses, the PGA seems to have the greatest potential. β-lactams represent the most important group of antibiotics comprising 65 % of the world antibiotic market explains such a tremendous and continuous interest in this enzyme. Indeed, the annual consumption of PGA has recently been estimated to range from 10 to 30 million tons.