Characterization of two key enzymes for aromatic amino acid biosynthesis in symbiotic archaea.
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 a trifunctional PD-CM-PDT from the smallest hyperthermophilic archaeon Nanoarchaeum equitans and a bifunctional CM-PD from its host, the crenarchaeon Ignicoccus hospitalis. Hexa-histidine tagged proteins were expressed in Escherichia coli and purified by affinity chromatography. Specific activities determined for the trifunctional enzyme were 21, 80, and 30 U/mg for CM, PD, and PDT, respectively, and 47 and 21 U/mg for bifunctional CM and PD, respectively. Unlike most PDs, these two archaeal enzymes were insensitive to regulation by L-Tyr and preferred NADP(+) to NAD(+) as a cofactor. Both the enzymes were highly thermally stable and exhibited maximal activity at 90 °C. 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. Our results suggest that PD from the unique symbiotic archaeal pair encompass a distinct subfamily of prephenate dehydrogenases with regard to their regulation and co-substrate specificity.
Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke St. West, Montréal, QC, H4B 1R6, Canada.
Irina
I
Shlaifer
Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke St. West, Montréal, QC, H4B 1R6, Canada. Joanne.Turnbull@concordia.ca.
Joanne L
JL
Turnbull
D024322:Amino Acids, Aromatic / Q000096:biosynthesis; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002826:Chorismate Mutase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019620:Desulfurococcaceae / Q000201:enzymology* / Q000502:physiology; D004795:Enzyme Stability; D006358:Hot Temperature; D046588:Nanoarchaeota / Q000201:enzymology* / Q000502:physiology; D009602:Nitrosamines / Q000378:metabolism; D011302:Prephenate Dehydratase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011303:Prephenate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013559:Symbiosis
D024322:Amino Acids, Aromatic; D019843:Archaeal Proteins; D009602:Nitrosamines; C041197:N-nitroso-2-hydroxypropylamine; D011303:Prephenate Dehydrogenase; D011302:Prephenate Dehydratase; D002826:Chorismate Mutase
Aromatic amino acid biosynthesis; Chorismate mutase; Ignicoccus hospitalis; Nanoarchaeum equitans; Prephenate dehydratase; Prephenate dehydrogenase