New experimental approaches for investigating interactions between Pyrococcus furiosus carbamate kinase and carbamoyltransferases, enzymes involved in the channeling of thermolabile carbamoyl phosphate.

Jan Massant,Nicolas Glansdorff

doi:10.1155/2005/865962

Abstract

A somewhat neglected but essential aspect of the molecular physiology of hyperthermophiles is the protection of thermolabile metabolites and coenzymes. An example is carbamoyl phosphate (CP), a precursor of pyrimidines and arginine, which is an extremely labile and potentially toxic intermediate. The first evidence for a biologically significant interaction between carbamate kinase (CK) and ornithine carbamoyltransferase (OTC) from Pyrococcus furiosus was provided by affinity electrophoresis and co-immunoprecipitation in combination with cross-linking (Massant et al. 2002). Using the yeast two-hybrid system, Hummel-Dreyer chromatography and isothermal titration calorimetry, we obtained additional concrete evidence for an interaction between CK and OTC, the first evidence for an interaction between CK and aspartate carbamoyltransferase (ATC) and an estimate of the binding constant between CK and ATC. The physical interaction between CK and OTC or ATC may prevent thermodenaturation of CP in the aqueous cytoplasmic environment. Here we emphasize the importance of developing experimental approaches to investigate the mechanism of thermal protection of metabolic intermediates by metabolic channeling and the molecular basis of transient protein-protein interactions in the physiology of hyperthermophiles.

Highlights

Most low molecular weight metabolites and coenzymes found in thermophiles are identical to those found in mesophiles, yet many are extremely thermolabile, and in some cases, their degradation products are toxic to the cell
Isotopic competition experiments with cell-free extracts of P. furiosus showed a marked preference of ornithine carbamoyltransferase (OTC) for Carbamoyl phosphate (CP) synthesized by carbamate kinase (CK) rather than for CP added to the reaction mixture (Legrain et al 1995)
The two-hybrid system (Fields and Song 1989, Chien et al 1991, Fields and Sternglanz 1994) relies on the modular nature of the yeast GAL4 protein, which consists of a DNA-binding domain (BD) and a transcriptional activation domain (AD)

Summary

Introduction

Most low molecular weight metabolites and coenzymes found in thermophiles are identical to those found in mesophiles, yet many are extremely thermolabile, and in some cases, their degradation products are toxic to the cell. Isotopic competition experiments with cell-free extracts of P. furiosus showed a marked preference of ornithine carbamoyltransferase (OTC) for CP synthesized by carbamate kinase (CK) rather than for CP added to the reaction mixture (Legrain et al 1995). These results suggested that CK and OTC formed a channeling complex to protect CP from decomposition. Similar results indicated the existence of channeling of CP to both OTC and aspartate carbamoyltransferase (ATC) in Thermus ZO5 (Van de Casteele et al 1997)

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Discussion

Conclusion