Identifying reaction modules in metabolic pathways: bioinformatic deduction and experimental validation of a new putative route in purine catabolism.

BACKGROUND: Enzymes belonging to mechanistically diverse superfamilies often display similar catalytic mechanisms. We previously observed such an association in the case of the cyclic amidohydrolase superfamily whose members play a role in related steps of purine and pyrimidine metabolic pathways. To establish a possible link between enzyme homology and chemical similarity, we investigated further the neighbouring steps in the respective pathways. RESULTS: We identified that successive reactions of the purine and pyrimidine pathways display similar chemistry. These mechanistically-related reactions are often catalyzed by homologous enzymes. Detection of series of similar catalysis made by succeeding enzyme families suggested some modularity in the architecture of the central metabolism. Accordingly, we introduce the concept of a reaction module to define at least two successive steps catalyzed by homologous enzymes in pathways alignable by similar chemical reactions. Applying such a concept allowed us to propose new function for misannotated paralogues. In particular, we discovered a putative ureidoglycine carbamoyltransferase (UGTCase) activity. Finally, we present experimental data supporting the conclusion that this UGTCase is likely to be involved in a new route in purine catabolism. CONCLUSIONS: Using the reaction module concept should be of great value. It will help us to trace how the primordial promiscuous enzymes were assembled progressively in functional modules, as the present pathways diverged from ancestral pathways to give birth to the present-day mechanistically diversified superfamilies. In addition, the concept allows the determination of the actual function of misannotated proteins.

Functional characterization of two M42 aminopeptidases erroneously annotated as cellulases.

Several aminopeptidases of the M42 family have been described as tetrahedral-shaped dodecameric (TET) aminopeptidases. A current hypothesis suggests that these enzymes are involved, along with the tricorn peptidase, in degrading peptides produced by the proteasome. Yet the M42 family remains ill defined, as some members have been annotated as cellulases because of their homology with CelM, formerly described as an endoglucanase of Clostridium thermocellum. Here we describe the catalytic functions and substrate profiles CelM and of TmPep1050, the latter having been annotated as an endoglucanase of Thermotoga maritima. Both enzymes were shown to catalyze hydrolysis of nonpolar aliphatic L-amino acid-pNA substrates, the L-leucine derivative appearing as the best substrate. No significant endoglucanase activity was measured, either for TmPep1050 or CelM. Addition of cobalt ions enhanced the activity of both enzymes significantly, while both the chelating agent EDTA and bestatin, a specific inhibitor of metalloaminopeptidases, proved inhibitory. Our results strongly suggest that one should avoid annotating members of the M42 aminopeptidase family as cellulases. In an updated assessment of the distribution of M42 aminopeptidases, we found TET aminopeptidases to be distributed widely amongst archaea and bacteria. We additionally observed that several phyla lack both TET and tricorn. This suggests that other complexes may act downstream from the proteasome.

Overexpression, physicochemical characterization, and modeling of a hyperthermophilic pyrococcus furiosus type 2 IPP isomerase.

In the first step of this study, type 2 isopentenyl diphosphate isomerase (IDI2) from Pyrococcus furiosus (pf-IDI2), a hyperthermophilic microorganism, was cloned and overexpressed in E. coli. After purification, hyperthermophilic behavior of this protein was approached by means of enzymatic assays and thermal denaturation studies. Compared with the mesophilic Streptococcus pneumoniae IDI2, which unfolds and looses activity above 50 degrees C, pf-IDI2 is still folded and active at 80 degrees C. Molecular modeling was applied, in a parallel step, to understand the molecular basis of thermal stability. Comparison of IDI2 from S. pneumoniae, T. thermophilus, and P. furiosus suggested that additional charged residues present in the hyperthermophilic enzyme might contribute to its higher thermal stability. This could increase the number of salt bridges between monomers of IDI2 in P. furiosus enzyme and, hence, decrease flexibility of loops or N-terminal segment, thereby enhancing its thermal stability.

Crystal structure of T state aspartate carbamoyltransferase of the hyperthermophilic archaeon Sulfolobus acidocaldarius.

Aspartate carbamoyltransferase (ATCase) is a model enzyme for understanding allosteric effects. The dodecameric complex exists in two main states (T and R) that differ substantially in their quaternary structure and their affinity for various ligands. Many hypotheses have resulted from the structure of the Escherichia coli ATCase, but so far other crystal structures to test these have been lacking. Here, we present the tertiary and quaternary structure of the T state ATCase of the hyperthermophilic archaeon Sulfolobus acidocaldarius (SaATC(T)), determined by X-ray crystallography to 2.6A resolution. The quaternary structure differs from the E.coli ATCase, by having altered interfaces between the catalytic (C) and regulatory (R) subunits, and the presence of a novel C1-R2 type interface. Conformational differences in the 240 s loop region of the C chain and the C-terminal region of the R chain affect intersubunit and interdomain interfaces implicated previously in the allosteric behavior of E.coli ATCase. The allosteric-zinc binding domain interface is strengthened at the expense of a weakened R1-C4 type interface. The increased hydrophobicity of the C1-R1 type interface may stabilize the quaternary structure. Catalytic trimers of the S.acidocaldarius ATCase are unstable due to a drastic weakening of the C1-C2 interface. The hyperthermophilic ATCase presents an interesting example of how an allosteric enzyme can adapt to higher temperatures. The structural rearrangement of this thermophilic ATCase may well promote its thermal stability at the expense of changes in the allosteric behavior.

Dihydropteridine reductase as an alternative to dihydrofolate reductase for synthesis of tetrahydrofolate in Thermus thermophilus.

A strategy devised to isolate a gene coding for a dihydrofolate reductase from Thermus thermophilus DNA delivered only clones harboring instead a gene (the T. thermophilus dehydrogenase [DH(Tt)] gene) coding for a dihydropteridine reductase which displays considerable dihydrofolate reductase activity (about 20% of the activity detected with 6,7-dimethyl-7,8-dihydropterine in the quinonoid form as a substrate). DH(Tt) appears to account for the synthesis of tetrahydrofolate in this bacterium, since a classical dihydrofolate reductase gene could not be found in the recently determined genome nucleotide sequence (A. Henne, personal communication). The derived amino acid sequence displays most of the highly conserved cofactor and active-site residues present in enzymes of the short-chain dehydrogenase/reductase family. The enzyme has no pteridine-independent oxidoreductase activity, in contrast to Escherichia coli dihydropteridine reductase, and thus appears more similar to mammalian dihydropteridine reductases, which do not contain a flavin prosthetic group. We suggest that bifunctional dihydropteridine reductases may be responsible for the synthesis of tetrahydrofolate in other bacteria, as well as archaea, that have been reported to lack a classical dihydrofolate reductase but for which possible substitutes have not yet been identified.

Metabolic channeling of carbamoyl phosphate, a thermolabile intermediate: evidence for physical interaction between carbamate kinase-like carbamoyl-phosphate synthetase and ornithine carbamoyltransferase from the hyperthermophile Pyrococcus furiosus.

Two different approaches provided evidence for a physical interaction between the carbamate kinase-like carbamoyl-phosphate synthetase (CKase) and ornithine carbamoyltransferase (OTCase) from the hyperthermophilic archaeon Pyrococcus furiosus. Affinity electrophoresis indicated that CKase and OTCase associate into a multienzyme cluster. Further evidence for a biologically significant interaction between CKase and OTCase was obtained by co-immunoprecipitation combined with formaldehyde cross-linking experiments. These experiments support the hypothesis that CKase and OTCase form an efficient channeling cluster for carbamoyl phosphate, an extremely thermolabile and potentially toxic metabolic intermediate. Therefore, by physically interacting with each other, CKase and OTCase prevent the thermodenaturation of carbamoyl phosphate in the aqueous cytoplasmic environment.

Aspartate carbamoyltransferase from a psychrophilic deep-sea bacterium, Vibrio strain 2693: properties of the enzyme, genetic organization and synthesis in Escherichia coli.

The aspartate carbamoyltransferase (ATCase) genes of psychrophilic Vibrio strain 2693 were cloned by complementation in Escherichia coli and the enzyme was partly characterized. The genes constitute a pyrBI operon homologous to the cognate structure in E. coli where pyrB and pyrI respectively encode the catalytic and the regulatory chains of ATCase. The strong sequence similarities noted between Vibrio and E. coli ATCases include extensive conservation of residues involved in interactions between subunits, suggesting that the two enzymes have very similar tertiary and quaternary structures. Vibrio ATCase is, however, not activated by ATP and not synergistically inhibited by CTP and UTP. It is also much more thermolabile than E. coli ATCase. With respect to Pyrococcus abyssi and E. coli ATCases, Vibrio ATCase presents marked differences in composition which could be related to its psychrophilic character. The results of these structural and functional comparisons indicate that Vibrio 2693 ATCase is a suitable model for biochemical studies on structure-function relationships in a 'cold' allosteric enzyme. The operon is expressed from a promoter which is immediately followed by a pyrimidine-rich leader ORF terminating within a putative transcription attenuator. These genetic and enzymic data strengthen the evolutionary relationship already noted between Vibrionaceae and Enterobacteriaceae.

Genes and enzymes of the acetyl cycle of arginine biosynthesis in the extreme thermophilic bacterium Thermus thermophilus HB27.

An arginine biosynthetic gene cluster, argC-argJ, of the extreme thermophilic bacterium Thermus thermophilus HB27 was isolated by heterologous complementation of an Escherichia coli acetylornithinase mutant. The recombinant plasmid (pTHM1) conferred ornithine acetyltransferase activity to the E. coli host, implying that T. thermophilus uses the energetically more economic pathway for the deacetylation of acetylornithine. pTHM1 was, however, unable to complement an E. coli argA mutant and no acetylglutamate synthase activity could be detected in E. coli argA cells containing pTHM1. The T. thermophilus argJ-encoded enzyme is thus monofunctional and is unable to use acetyl-CoA to acetylate glutamate (contrary to the Bacillus stearothermophilus homologue). Alignment of several ornithine acetyltransferase amino acid sequences showed no obvious pattern that could account for this difference; however, the monofunctional enzymes proved to have shorter N-termini. Sequence analysis of the pTHM1 3.2 kb insert revealed the presence of the argC gene (encoding N-acetylglutamate-5-semialdehyde dehydrogenase) upstream of the argJ gene. Alignment of several N-acetylglutamate-5-semialdehyde dehydrogenase amino acid sequences allowed identification of two strongly conserved putative motifs for cofactor binding: a putative FAD-binding site and a motif reminiscent of the NADPH-binding fingerprint. The relationship between the amino acid content of both enzymes and thermostability is discussed and an effect of the GC content bias is indicated. Transcription of both the argC and argJ genes appeared to be vector-dependent. The argJ-encoded enzyme activity was twofold repressed by arginine in the native host and was inhibited by ornithine. Both upstream of the argC gene and downstream of the argJ gene an ORF with unknown function was found, indicating that the organization of the arginine biosynthetic genes in T. thermophilus is new.

Ammonia-dependent synthesis and metabolic channelling of carbamoyl phosphate in the hyperthermophilic archaeon Pyrococcus furiosus.

SUMMARYThe biosynthesis of carbamoyl phosphate (CP), a metabolic precursor of arginine and the pyrimidines was investigated in the hyperthermophilic archaeon Pyrococcus furiosus. The half-life of CP was found to be less than 2 s in the optimum temperature range of this organism (100-102 °C). The carbamoyl-phosphate synthase (CPSase) of P. furiosus uses ammonia as the nitrogen donor, and not glutamine like all micro-organisms investigated so far. The Mr of the enzyme, which is devoid of regulatory properties, is 70000, at variance with that of known CPSases. The possible significance of these findings with regard to hyperthermophilic nitrogen metabolism is discussed. Competition experiments with P. furiosus crude extracts indicated a marked preference of ornithine carbamoyltransferase (OTCase) for CP synthesized by CPSase rather than for CP added to the reaction mixture. In addition, the bisubstrate analogue -N-phosphonoacetyl-L-ornithine inhibits the formation of citrulline from bicarbonate, ammonia, ATP and ornithine much less than its synthesis from ornithine and CP in the presence of free OTCase. Such results suggest that, in vivo, CPSase and OTCase associate in a complex able to channel CP. Such a channelling may confer protection to CP, thus avoiding the accumulation of toxic amounts of cyanate arising from its decomposition as well as the waste of the two molecules of ATP required for its synthesis.