Abstract

The biosynthesis of L-tyrosine (L-Tyr) and L-phenylalanine (L-Phe) is directed by the interplay of three enzymes. Chorismate mutase (CM) catalyzes the rearrangement of chorismate to prephenate, which can be either converted to hydroxyphenylpyruvate by prephenate dehydrogenase (PD) or to phenylpyruvate by prephenate dehydratase (PDT). This work reports the first characterization of both the trifunctional PD-CM-PDT from the smallest hyperthermophilc archaeon (Nanoarchaeum equitans) and the bifunctional CM-PD from its host (the crenarchaeon Ignicoccus hospitalis). Hexa histidine-tagged proteins were expressed in Escherichia coli and purified by chromatography on Ni-NTA affinity resin. Both enzymes were highly thermally stable and exhibited maximal activity at 90°C. CM, PD and PDT activities were detected at temperatures consistent with enzymes from extreme thermophiles. Kinetic analysis revealed that unlike most PDs, the two archaeal enzymes were insensitive to regulation by L-Tyr and preferred NADP+ to NAD+ as a cofactor in the dehydrogenase reaction. N. equitans PDT was feedback inhibited by L-Phe (Ki = 0.8 μM) in a non-competitive fashion consistent with L-Phe’s combination at a site separate from that of prephenate. Gel filtration and analytical ultracentrifugation analysis of bifunctional CM-PD from I. hospitalis suggested that the enzyme is a native dimer. Limited proteolysis studies revealed that the enzyme is highly resistant to proteolysis but could be cleaved to yield a stable C-terminal PD domain. Mass spectrometry and mutagenesis studies confirmed that the PD domain of bifunctional I. hospitalis CM-PD could be independently isolated and expressed. Biochemical and biophysical characterization of this active truncated variant was performed and the results of solution studies were compared to those of the full-length protein and to information available from other PD enzymes. Guided by amino acid sequence alignment predictions and
by models based on the available crystal structures of bacterial homologues, eight variants containing site-specific replacements were generated in I. hospitalis CM-PD as attempts to alter cofactor selectivity and substrate and end-product binding. Those variant proteins were kinetically characterized in order to help define the role of active site residues in substrate/ inhibitor interactions. These are the first studies exploring the aromatic amino acid biosynthetic pathway from the two archaeal organisms, which provide efficient and stable catalysts as excellent candidates for applications in biotechnology.