Investigating the MIO-Dependent Aminomutase and Ammonia Lyase Superfamily

Abstract

The phenylalanine aminomutase (PAM) AdmH, from Pantoea agglomerans and the phenylalanine ammonia lyase (PAL) EncP, from the thermotolerant marine bacterium Streptomyces maritimus, exhibit significant sequence similarity yet fulfil distinct catalytic roles in secondary metabolism. AdmH catalyses the isomerisation of (2S)-alpha-phenylalanine to give (3S)-beta-phenylalanine required for the biosynthesis of the antibiotic andrimid, whilst EncP catalyses the elimination of ammonia from (2S)-alpha-phenylalanine to give the trans-cinnamate precursor for the biosynthesis of enterocin. Here we show that both enzymes are thermostable, thermophilic and exhibit bifunctionality with preferred ammonia lyase activity at elevated temperatures and aminomutase activity at lower temperatures. The observed thermal bifunctionality provides a rare example of how nature has evolved two very similar enzymes to catalyse differing reactions that are contingent on temperature - presumably as a consequence of the different environments inhabited by the mesophilic and thermotolerant bacterial hosts.The aminomutase and ammonia lyase activities of AdmH and EncP are shown to be dependent on the viscosity of the reaction medium, which indicates that both reactions are rate-limited by protein conformational changes. Kinetic isotope effects for the PAL reactions of AdmH and EncP were determined, yielding kH/kD values of between 2.0 and 2.5 for Vmax/KM. We postulate that the rate limiting C3 hydrogen abstraction is coupled to a conformational change in the inner loop which possesses the Tyr catalytic base. Such a conformational change would facilitate subsequent product release following elimination of ammonia from MIO.A model for the reaction mechanism is described in which ammonia lyase activity is favoured at higher temperatures where there is greater thermal energy to overcome the large energy barrier associated with disengaging the inner loop after abstraction of the C3 hydrogen. At lower temperatures when the PAM reaction predominates, there is presumably insufficient thermal energy to facilitate disengagement of the inner loop after C3 hydrogen abstraction. In this case the hydroxyl group of the Tyr base would protonate the C2 of trans-cinnamate, during conjugate addition of the amine-MIO. Electrostatic repulsion between the resulting Tyr phenolate anion and the carboxylate group of the beta-amino-MIO adduct could then favour disengagement of the inner loop which could also facilitate subsequent product release following elimination of ammonia from MIO.In contrast to the bacterial enzymes, the yeast PAL from Rhodotorula graminis does not exhibit temperature dependent bifunctionality; possessing only ammonia lyase activity with a rate limiting chemical step. We also demonstrate that the bacterial, and to a lesser extent the yeast enzyme, exhibit MIO-dependent (3S)-beta-phenylalanine ammonia lyase activity which disfavours the Friedel Crafts-type mechanism.The aminomutase reaction sees catalysis of cheap and readily available alpha-amino acids into relatively more valuable corresponding β-amino acids. Furthermore, in the presence of high ammonia concentrations, aminomutases catalyse the reversible amination of trans-cinnamate to give a combination of alpha- and β-amino acids. Since neither cofactor recycling nor other additives are needed, there is promising potential for both reactions to be used in industrial processes. Here, we explore the biocatalytic properties of the MIO-dependent AdmH and EncP enzymes. We optimise enzyme catalysed ammonia addition and use protein engineering via rational design in an attempt to alter or broaden the enzymes' substrate selectivity. Focused protein engineering around the aromatic ring binding site decreases / abolishes enzyme activity, most likely as a consequence of a sensitive hydrogen-bonding network. In addition, we develop a novel coupled assay which has the advantages of being rapid, simple to use and which has th

Date of Award	1 Aug 2012
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Jason Micklefield (Supervisor)