title: Biochemical characterization of TyrA enzymes from Ignicoccus hospitalis and Haemophilus influenzae: A comparative study of the bifunctional and monofunctional dehydrogenase forms.
abstract: Biosynthesis of l-tyrosine (l-Tyr) is directed by the interplay of two enzymes. Chorismate mutase (CM) catalyzes the rearrangement of chorismate to prephenate, which is then converted to hydroxyphenylpyruvate by prephenate dehydrogenase (PD). This work reports the first characterization of the independently expressed PD domain of bifunctional CM-PD from the crenarchaeon Ignicoccus hospitalis and the first functional studies of both full-length CM-PD and the PD domain from the bacterium Haemophilus influenzae. All proteins were hexa-histidine tagged, expressed in Escherichia coli and purified. Expression and purification of I. hospitalis CM-PD generated a degradation product identified as a PD fragment lacking the protein's first 80 residues, Δ80CM-PD. A comparable stable PD domain could also be generated by limited tryptic digestion of this bifunctional enzyme. Thus, Δ80CM-PD constructs were prepared in both organisms. CM-PD and Δ80CM-PD from both organisms were dimeric and displayed the predicted enzymatic activities and thermal stabilities in accord with their hyperthermophilic and mesophilic origins. In contrast with H. influenzae PD activity which was NAD+-specific and displayed >75% inhibition with 50μM l-Tyr, I. hospitalis PD demonstrated dual cofactor specificity with a preference for NADP+and an insensitivity to l-Tyr. These properties are consistent with a model of the I. hospitalis PD domain based on the previously reported structure of the H. influenzae homolog. Our results highlight the similarities and differences between the archaeal and bacterial TyrA proteins and reveal that the PD activity of both prokaryotes can be successfully mapped to a functionally independent unit.
authors.affiliation: Department of Chemistry and Biochemistry and the Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke St. West, Montréal, Québec H4B 1R6, Canada.
authors.forename: Irina
authors.initials: I
authors.lastname: Shlaifer
authors.affiliation: Department of Chemistry and Biochemistry and the Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke St. West, Montréal, Québec H4B 1R6, Canada.
authors.forename: Peter Kojo
authors.initials: PK
authors.lastname: Quashie
authors.affiliation: Department of Chemistry and Biochemistry and the Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke St. West, Montréal, Québec H4B 1R6, Canada.
authors.forename: Hyun Young
authors.initials: HY
authors.lastname: Kim
authors.affiliation: Department of Chemistry and Biochemistry and the Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke St. West, Montréal, Québec H4B 1R6, Canada. Electronic address: Joanne.Turnbull@concordia.ca.
authors.forename: Joanne L
authors.initials: JL
authors.lastname: Turnbull
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D002826:Chorismate Mutase / Q000378:metabolism; D019620:Desulfurococcaceae / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D006193:Haemophilus influenzae / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D011303:Prephenate Dehydrogenase / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D009097:Multienzyme Complexes; C076398:TyrA protein, Bacteria; D009243:NAD; D014443:Tyrosine; D006639:Histidine; D009249:NADP; D011303:Prephenate Dehydrogenase; D002826:Chorismate Mutase
keywords: Aromatic amino acid biosynthesis; Chorismate mutase; Haemophilus influenzae; Ignicoccus hospitalis; Prephenate dehydrogenase; TyrA