Characterization of a key trifunctional enzyme for aromatic amino acid biosynthesis in Archaeoglobus fulgidus.
In the aromatic amino acid biosynthesis pathway, chorismate presents a branch point intermediate that is converted to tryptophan, phenylalanine (Phe), and tyrosine (Tyr). In bacteria, three enzymes catalyze the conversion of chorismate to hydroxyphenylpyruvate or pyruvate. The enzymes, chorismate mutase (CM), prephenate dehydratase (PDT), and prephenate dehydrogenase (PDHG) are either present as distinct proteins or fusions combining two activities. Gene locus AF0227 of Archaeoglobus fulgidus is predicted to encode a fusion protein, AroQ, containing all three enzymatic domains. This work describes the first characterization of a trifunctional AroQ. The A. fulgidus aroQ gene was cloned and overexpressed in Escherichia coli. The recombinant protein purified as a homohexamer with specific activities of 10, 0.51, and 50 U/mg for CM, PDT, and PDHG, respectively. Tyr at 0.5 mM concentration inhibited PDHG activity by 50%, while at 1 mM PDT was activated by 70%. Phe at 5 muM inhibited PDT activity by 66% without affecting the activity of PDHG. A fusion of CM, PDT, and PDHG domains is evident in the genome of only one other organism sequenced to date, that of the hyperthermophilic archaeon, Nanoarchaeum equitans. Such fusions of contiguous activities in a biosynthetic pathway may constitute a primitive strategy for the efficient processing of labile metabolites.
Lim
Sierin
S
Biomedical Engineering Interdepartmental Program, 5121 Engineering V, University of California, Los Angeles, CA 90095, USA.
Springstead
James R
JR
Yu
Marcella
M
Bartkowski
Wojciech
W
Schröder
Imke
I
Monbouquette
Harold G
HG
D024322:Amino Acids, Aromatic / Q000096:biosynthesis*; D019611:Archaeoglobus fulgidus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002853:Chromatography, Liquid; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D013058:Mass Spectrometry; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D013816:Thermodynamics
D024322:Amino Acids, Aromatic; D017931:DNA Primers