Anne Boyen

Genes and enzymes of the acetyl cycle of arginine biosynthesis in Corynebacterium glutamicum: enzyme evolution in the early steps of the arginine pathway

A cluster of arginine biosynthetic genes of Corynebacterium glutamicum ATCC 13032, comprising argJ, argB and argD as well as part of argC and argF, has been cloned by heterologous complementation of an Escherichia coli argE mutant. The gene order has been established as argCJBDF by sequencing the entire 4.4 kb cloned DNA fragment. The C. glutamicum argB gene can be transcribed in E. coli cells from an internal promoter located in the coding part of the preceding argJ gene, whereas transcription of the argJ gene appears vector-dependent. Expression of the corynebacterial argB gene is repressed by arginine in the native host but not in recombinant E. coli cells. Feedback inhibition of the corresponding N-acetylglutamate kinase activity was observed both in cell extracts of C. glutamicum and in recombinant E. coli argB auxotrophic strains. Extracts of E. coli cells carrying cloned corynebacterial DNA display an ornithine acetyltransferase activity (encoded by argJ) which alleviates the acetylornithinase (encoded by argE) deficiency of the enterobacterial host. In contrast to Bacillus stearothermophilus ornithine acetyltransferase which also exhibits acetylglutamate synthase activity, C. glutamicum ornithine acetyltransferase appears monofunctional. ArgA and ArgB proteins from different sources share highly significant similarities. The evolutionary implications of these data are discussed.

Nucleotide sequence of Escherichia coli argB and argC genes: comparison of N-acetylglutamate kinase and N-acetylglutamate-γ-semialdehyde dehydrogenase with homologous and analogous enzymes

The Escherichia coli argB and argC gene products are functionally analogous to kinases and dehydrogenases of other pathways, which by their successive action also achieve the conversion of a carboxylate into an aldehyde function. This raises the question of possible evolutionary relationship within each of these sets of enzymes. We have therefore undertaken the nucleotide sequence analysis of the argB and argC genes and compared the derived amino acid sequences with the known sequences of analogous enzymes active in the proline and homoserine biosynthetic pathways and in glycolysis. No significant amino acid sequence similarity pointing to the existence of a common ancestor could be detected. Comparison of the amino acid sequence of the argB and argC gene products with the polypeptide deduced from the Saccharomyces cerevisiae ARG5,6 gene sequence (C. Boonchird, F. Messenguy and E. Dubois, in preparation) allowed the unambiguous localization of the corresponding domains in yeast.

The evolutionary history of carbamoyltransferases: A complex set of paralogous genes was already present in the last universal common ancestor.

Abstract. Forty-four sequences of ornithine carbamoyltransferases (OTCases) and 33 sequences of aspartate carbamoyltransferases (ATCases) representing the three domains of life were multiply aligned and a phylogenetic tree was inferred from this multiple alignment. The global topology of the composite rooted tree (each enzyme family being used as an outgroup to root the other one) suggests that present-day genes are derived from paralogous ancestral genes which were already of the same size and argues against a mechanism of fusion of independent modules. A closer observation of the detailed topology shows that this tree could not be used to assess the actual order of organismal descent. Indeed, this tree displays a complex topology for many prokaryotic sequences, with polyphyly for Bacteria in both enzyme trees and for the Archaea in the OTCase tree. Moreover, representatives of the two prokaryotic Domains are found to be interspersed in various combinations in both enzyme trees. This complexity may be explained by assuming the occurrence of two subfamilies in the OTCase tree (OTC α and OTC β) and two other ones in the ATCase tree (ATC I and ATC II). These subfamilies could have arisen from duplication and selective losses of some differentiated copies during the successive speciations. We suggest that Archaea and Eukaryotes share a common ancestor in which the ancestral copies giving the present-day ATC II/OTC β combinations were present, whereas Bacteria comprise two classes: one containing the ATC II/OTC α combination and the other harboring the ATC I/OTC β combination. Moreover, multiple horizontal gene transfers could have occurred rather recently amongst prokaryotes. Whichever the actual history of carbamoyltransferases, our data suggest that the last common ancestor to all extant life possessed differentiated copies of genes coding for both carbamoyltransferases, indicating it as a rather sophisticated organism.

Escherichia coli and Saccharomyces cerevisiae acetylornithine aminotransferases: evolutionary relationship with ornithine aminotransferases

Genes argD and ARG8, encoding the acetylornithine aminotransferase (ACOAT) subunit in Escherichia coli and Saccharomyces cerevisiae, respectively, have been cloned and sequenced. The deduced amino acid sequences show substantial similarity. Moreover, they resemble ornithine aminotransferase (OAT) sequences (i.e., those from yeast, rat and man); the observed similarities are statistically significant, indicating that the enzymes are homologous. However, in contrast to OATs, which appear to be substrate (i.e., ornithine)-specific, S. cerevisiae ACOAT transaminates ornithine about as efficiently as E. coli does. The evolutionary relationship between ACOATs and OATs is discussed in terms of substrate ambiguity.

Acetylornithine deacetylase, succinyldiaminopimelate desuccinylase and carboxypeptidase G2 are evolutionarily related.

The nucleotide (nt) sequence of the Escherichia coli argE gene, encoding the acetylornithine deacetylase (AO) subunit, has been established and corresponds to a 43-kDa (M(r) 42,320) polypeptide. The enzyme has been purified to near homogeneity and it appears to be a dimer consisting of two 43-kDa subunits. The amino acid sequence deduced from the nt sequence was compared to that of the subunit of E. coli succinyldiaminopimelate desuccinylase (the dapE gene product involved in the diaminopimelate pathway for lysine biosynthesis), since both enzymes share functional and biochemical features. Significant similarity covering the entire sequence allows us to infer a common origin for both deacylases. This homology extends to the Pseudomonas sp. G2 carboxypeptidase (G2CP); this or a functionally related enzyme may be responsible for the minor AO activity found in organisms relying on ornithine acetyltransferase for ornithine biosynthesis.

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.

Involvement of arginine in in vitro repression of transcription of arginine genes C, B and H in Escherichia coli K 12.

Abstract The combination of L-arginine and partially purified arginine repressor (the argR gene product) represses transcription of argCBH in vitro .

Enhancement of translation efficiency in Escherichia coli by mutations in a proximal domain of messenger RNA

Abstract The effect of two mutations in Escherichia coli located 5′ to the Shine-Dalgarno sequence of argE messenger RNA suggests that maximal translation efficiency may depend on the composition of proximal sequences not necessarily involved in secondary structures.

Heteroduplex analysis of regulatory mutations and of insertions (IS1, IS2, IS5) in the bipolar argECBH operon of Escherichia coli

SummarySeveral insertions have been characterized by heteroduplex mapping in the argECBH bipolar operon. They include 1. an IS1 element in argB (mutation argB5) in orientation II with respect to argH. The insertion is strongly polar; the residual argH expression is due mainly to transcription events initiated at the control region, between argE and argC. In terms of transcription polarity, the insertion appears as proximal to the secondary promotor (P2) present in argB.2. An IS5 element in argB (mutation argB2), distal to P2, exerting an extremely strong polar effect on argH. IS5 is shown to be capable of inaccurate excision at a frequency of about 10-8 the number of cells plated; no rigorously correct excision could be observed on a total of more than 1012 cells.3. Two IS2 elements inserted in opposite orientations in the control region of a strain bearing a deletion of the argE promotor.The present results together with evidence presented in the accompanying paper by Boyen et al. (1978) show that IS2 can exert a polar effect when it is inserted downstream from an active promotor in orientation I or II; IS2 however promotes low levels of transcription into an adjacent gene lacking a functional promotor. The significance of these results is discussed in the accompanying paper by Boyen et al. (1978).

Parameters of gene expression in the bipolar argECBH operon of E. coli K12

SummaryThe pattern of divergent transcription of the argECBH cluster of genes previously demonstrated by the hybridization of RNA to the separated strand of a ϕ80 drag transducing phage, is confirmed with the DNA of a set of different λdarg phages.The accurate determination of argE and argCBH m-RNA levels in different steady states of expression of the arg regulon supports the following conclusions: 1.The ratio between maximal (derepressed) and minimal (repressed) rates of expression is lower when it is expressed in terms of % hybridizable RNA than in terms of enzyme specific activities. The discrepancy is about 3 fold. Thus in conditions of repression, the cell produces relatively more unused m-RNA than in derepression. Different interpretations of this phenomenon appear possible:a)the messenger RNA molecules synthesized in repressed cells could be degraded more rapidly or translated less efficiently than in derepressed cells.b)an untranslated segment of the RNA could account for a larger part of the RNA detected in repression than in derepression. These interpretations are not mutually exclusive.2.The discrepancy observed between the amplitudes of variation of argE and argCBH expression, expressed in terms of enzyme specific activities, is, in fact, determined at the level of DNA transcription. This provides direct evidence for the occurrence of differential transcription effectiveness in a regulon exhibiting a correlative but not strictly coordinated pattern of enzyme synthesis. This also supports our earlier suggestion regarding the possible complexity of the internal operator region situated between argE and C.