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STRUCTURE-FUNCTION RELATIONSHIPS IN ALLOSTERIC CARBAMOYLTRANSFERASES FROM MESOPHILES AND EXTREME THERMOPHILES

Objective


Understanding at the molecular level the conformational transitions underlying catalysis and the transmission of regulatory signals in enzymes is a major goal in fundamental science whereas, in biotechnology, such knowledge is required to lay out the principles of a rational protein design. This question is investigated in a family of proteins, including enzymes from highly thermophilic bacteria, a potential source of thermostable biocatalysts.

The aim of the project was to characterize intrachain and interchain interactions involved in the regulatory behaviour of a model allosteric enzyme of known 3-dimensional structure: aspartate carbamoyltransferase (ATCase) of Escherichia coli, a protein consisting of distinct catalytic and regulatory subunits. ATCase catalyzes the carbamoylation of aspartate by carbamoylphosphate (CP). The enzyme is inhibited by cytosine triphosphate (CTP) and activated by adenosine triphosphate (ATP). It consists of 2 catalytic (C) trimers held together by 3 dimers of regulatory (R) subunit which is itself composed of an effector binding domain and a Zinc binding domain. CTP and ATP bind competitively to the same R site. Each R chain interacts with 2 C chains belonging to different C trimers, giving rise to the so-called R1-C1 and R1-C4 interfaces.

In order to investigate the interchain contacts involved in the cooperative interactions between the catalytic sites, a series of modified forms of the enzyme was prepared by site directed mutagenesis. The results obtained show that the R1-C4 interaction is essential for the establishment of the enzyme conformation having low affinity for aspartate (T state), and consequently for the existence of cooperactivity between the catalytic sites. From similar experiments, it appears that the contact between the carboxyl terminal region of the R chain and the 240s loop of the C chain (R1-C4 interaction) is essential for the transmission of the CTP regulatory signal. None of the modifications made in the R1-C4 interface altered the sensitivity of the enzyme to the activator ATP suggesting that the effect of this nucleotide involves the R1-C1 type of interface. These results are in agreement with the previously proposed interpretation that CTP and ATP do not simply act in inverse ways on the same equilibrium. The behaviour of a remarkable mutant with a single substitution at the interface between the 2 domains of the E chain, where the effect of ATP is reversed without affecting the CTP response, also supports this view.

Site directed mutagenesis was used to check the catalytic role of residue C his 134, which is in a position to interact with the carbonyl group of CP, and was a candidate for bearing a group whose deprotonation is involved in catalytic efficiency. The results establish that C his 134 is indeed the residue interacting with the carbonyl group of CP but that it is not directly involved in the catalytic mechanism.

Understanding at the molecular level the conformational transitions underlying catalysis and the transmission of regulatory signals in enzymes is a major goal in fundamental science whereas, in biotechnology, such knowledge is required to lay out the principles of a rational protein design. This question is investigated in a family of proteins, including enzymes from highly thermophilic bacteria, a potential source of thermostable biocatalysts.

The aim of the project was to determine the structure of a highly regulated carbamoyltransferase consisting of only 1 type of subunit, the catabolic ornithine carbamoyltransferase (OTCase) of Pseudomonas aeruginosa and to identify residues and domains involved in the allosteric behaviour of this protein (Prof Haas, Zuerich).The catabolic OTCase of Pseudomonas aeruginosa is unable to promote the anabolic reaction because of a pronounced sigmoidal carbamoylphosphate (CP) saturation curve and a high CP concentration for half maximal velocity. The structural basis for this kinetic specialisation was examined. THe OTCase lost most of its homotropic cooperativity and gained anabolic activity when residue glutamic acid (Glu) 106, near the CP binding site, was replaced by alanine (Ala) or glycine (Gly). When the corresponding Glu NH2 residue of Escherichia coli anabolic OTCase was exchanged for Glu, no cooperativity resulted, however. Thus, in catabolic OTCase other features in addition to Glu106 are important for sigmoidal CP saturation and such a sequence was identified in the carboxyl terminal part. Indeed when the 9 carboxyl terminal residues of catabolic OTCase were replaced, using an in vivo gene fusion technique, by the homologous 8 amino acids from anabolic E. coli OTcase, the homotropic cooperativity was markedly reduced. This gene fusion method should be generally useful for directed enzyme evolution.

Understanding the structure function relationships in this and other carbamoyltransferases requires 3-dimensional stru ctural determination. With this purpose in mind both the wild type and a muted form (without sigmoidal kinetics) of the enzyme were crystallised. 1 crystal form was used to solve the symmetry of the oligomer. Starting with the data collected on an area detector, a self rotation function was computed. This function shows clearly an assembly with cubic 23 (T) symmetry. Taking the molecular weight into account, the enzyme is therefore a dodecamer. A molecular model was constructed using molecular replacement methods. The model will be useful for solving the 3-dimensional structure of this and other carbamoyltransferases such as those from thermophilic bacteria.

Understanding at the molecular level the conformational transitions underlying catalysis and the transmission of regulatory signals in enzymes is a major goal in fundamental science whereas, in biotechnology, such knowledge is required to lay out the principles of a rational protein design. This question is investigated in a family of proteins, including enzymes from highly thermophilic bacteria, a potential source of thermostable biocatalysts.

The aim of the project was to investigate enzymatic carbamoylation in extreme thermophiles with the objective of undertaking a long term study of structure function relationships in thermophilic carbamoyltransferases.
The eubacteria Thermus aquaticus and Thermotoga maritima were found to recycle the acetyl group of the arginine precursors and Sulfolobus solfataricus to use a linear pathway. In Pyrococcus furiosus (obtained, as Ta. maritima, by courtesy of Prof K Stetter, Regensburg) only the last 3 enzymes of the pathway could be detected.

Ornithine carbamoyltransferase (OTCase) from T. aquaticus resembles Escherichia coli OTCase in terms of size, structure (trimeric) and kinetics; it exhibits a reversible cold denaturation. Thermostability is high and still considerably increased by the presence of ornithine and/or phosphate. Aspartate carbamoyltransferase (ATCase) presents kinetic and regulatory features reminiscent of the E. coli equivalent. In Thermotoga, the most thermophilic eubacterium ever described, OTCase kinetically resembles the Thermus enzyme; the thermostability varies with the concentration but even in dilute extracts it can be protected efficiently by ornithine and/or phosphate. ATCase presents an allosteric behaviour and is very stable. In Sulfolobus, ATCase is devoid of regulatory properties and extremely stable (half life at 90C is several hours). OTCase is much larger and more complex than in mesophiles or in Thermus; isoenzymes exist, which appear to be built by the association of 2 types of subuni ts. In P. furiosus also, OTCase is a large enzyme (molecular weight of 380 kD), built up of identical subunits with a molecular weight of about 37.5 kD. Heat resistance is very high and still increased by ornithine and/or phosphate.

The genes for ATCase from T. aquaticus and Ta. maritima have been cloned by complementing appropriate E. coli auxotrophs.

Critical interchain and intrachain contacts involved inthe transmission of allosteric effects in Escherichia coli aspartate carbamoyltransferase (ATCase) have been identified. Adenosine triphosphate (ATP) and cytidine triphosphate (CTP) have been shown to exert their effects by different pathways even though they bind to the same regulatory site. Progress has been made in understanding substrate binding. Critical residues and domains involved in the allosteric behaviour of Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase (OTCase) have been identified. The enzyme (wild type and regulatory mutant form) has been crystallized and shown to be a dodecamer. An in vivo gene fusion technique has been devised to study directed enzyme evolution. The pathway of arginine biosynthesis in several thermophilic eubacteria and archaebacteria has been clarified. OTCases and ATCases have been purified and partially characterized from Thermus aquaticus, Thermotoga maritima, Sulfolobus solfataricus and Pyrococcus furiosus. Several instances of extreme heat stability (in particular in the presence of substrate and/or products) have been brought to light. The genes for Thermus aquaticus and Thermotoga maritima ATCases have been cloned.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

VRIJE UNIVERSITEIT BRUSSEL
Address
Pleinlaan 2
Brussels
Belgium