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Characterization of a key trifunctional enzyme for aromatic amino acid biosynthesis in Archaeoglobus fulgidus

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Abstract

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 μM 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.

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Acknowledgments

We thank Chi-Hee Kim for the construction of plasmid pIS85. This work was supported by the National Science Foundation (BES-9911718) and the US Department of Commerce/NIST (Cooperative Agreement 70NANBOH0064).

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Author notes

  1. Sierin Lim

    Present address: Bioengineering Division, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, 637457, Singapore

Authors and Affiliations

  1. Biomedical Engineering Interdepartmental Program, 5121 Engineering V, University of California, Los Angeles, CA, 90095, USA

    Sierin Lim, Imke Schröder & Harold G. Monbouquette

  2. Chemical and Biomolecular Engineering Department, 5531 Boelter Hall, University of California, Los Angeles, CA, 90095, USA

    James R. Springstead, Marcella Yu & Harold G. Monbouquette

  3. Microbiology, Immunology, and Molecular Genetics Department, 1602 Molecular Sciences Building, University of California, Los Angeles, CA, 90095, USA

    Wojciech Bartkowski & Imke Schröder

Authors
  1. Sierin Lim
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  2. James R. Springstead
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  3. Marcella Yu
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  4. Wojciech Bartkowski
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  5. Imke Schröder
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  6. Harold G. Monbouquette
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Correspondence to Harold G. Monbouquette.

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Communicated by L. Huang.

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Lim, S., Springstead, J.R., Yu, M. et al. Characterization of a key trifunctional enzyme for aromatic amino acid biosynthesis in Archaeoglobus fulgidus . Extremophiles 13, 191–198 (2009). https://doi.org/10.1007/s00792-008-0209-z

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  • DOI: https://doi.org/10.1007/s00792-008-0209-z

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