Preparation of Unnatural Amino Acids with Ammonia-Lyases and 2,3-Aminomutases
Ammonia-lyases catalyze a wide range of processes leading to α,β-unsaturated compounds by elimination of ammonia. In this chapter, ammonia-lyases are reviewed with major emphasis on their synthetic applications in stereoselective preparation of unnatural amino acids. Besides the synthesis of various unnatural α-amino acids with the aid of phenylalanine ammonia-lyases (PALs) utilizing the 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) prosthetic groups, the biotransformations leading to various unnatural β-amino acids with phenylalanine 2,3-aminomutases using the same catalytic MIO prosthetic group are discussed. Cloning, production, purification, and biotransformation protocols for PAL are described in detail.
Key wordsPhenylalanine ammonia-lyase Phenylalanine 2,3-aminomutase Recombinant enzyme Biotransformation Unnatural amino acid
This research was supported by the Hungarian National Office for Research and Technology (NKFP-07-A2 FLOWREAC), by the Hungarian National Science Foundation (OTKA, K68229, and CK78646), and by Howard Hughes Medical Institutes #55000342. This work is also related to the scientific program of “Development of quality-oriented and harmonized R+D+I strategy and functional model at BME” project (TÁMOP-4.2.1/B-09/1/KMR-2010-0002), supported by the New Hungary Plan. The financial support to Csaba Paizs from the Romanian Ministry of Education and Research (CNCSIS No. 527/2461) is gratefully acknowledged. We thank János Rétey (Institute of Organic Chemistry, Karlsruhe Institute of Technology, Germany), G. E. Schulz, and M. Baedeker (University of Freiburg, Germany) for providing us the Petroselinum crispum PAL expression system, András Holczinger (Department of Applied Biotechnology and Food Science, Budapest University of Technology and Economics, Budapest, Hungary) for providing us the gene of Photorhabdus luminescens PAL in pBAD-24 vector, and also M. Stieger (Hoffmann-La Roche, Basel, Switzerland) for vector pREP4-GroESL carrying the HSP-60 system.
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