Two Proteins with Ornithine Acetyltransferase Activity Show Different Functions in Streptomyces clavuligerus: Oat2 Modulates Clavulanic Acid Biosynthesis in Response to Arginine

Abstract

The oat2 gene, located in the clavulanic acid gene cluster in Streptomyces clavuligerus, is similar to argJ, which encodes N-acetylornithine:glutamic acid acetyltransferase activity. Purified proteins obtained by expression in Escherichia coli of the argJ and oat2 genes of S. clavuligerus posses N-acetyltransferase activity. The kinetics and substrate specificities of both proteins are very similar. Deletion of the oat2 gene did not affect the total N-acetylornithine transferase activity and slightly reduced the formation of clavulanic acid under standard culture conditions. However, the oat2 mutant produced more clavulanic acid than the parental strain in cultures supplemented with high levels (above 1 mM) of arginine. The purified S. clavuligerus ArgR protein bound the arginine box in the oat2 promoter, and the expression of oat2 was higher in mutants with a disruption in argR (arginine-deregulated), confirming that the Arg boxes of oat2 are functional in vivo. Our results suggest that the Oat2 protein or one of its reaction products has a regulatory role that modulates clavulanic acid biosynthesis in response to high arginine concentrations.Streptomyces clavuligerus produces clavulanic acid (CA), an important ␤-lactamase inhibitor synthesized by the condensation of arginine (23,27), and glyceraldehyde-3-phosphate to form carboxyethylarginine (12, 18). In wild-type strains, arginine is a limiting precursor for clavulanic acid biosynthesis (23). Studies of the arginine gene cluster have been highly interesting as a way of understanding the channeling of this precursor into clavulanic acid (20,21).The presence of a cyclic arginine pathway in Streptomyces species has been reported previously (20). The S. clavuligerus arginine gene cluster contains an argJ gene encoding a protein of 39,733 Da, similar to other ArgJ proteins, as deduced from the genome sequences of Streptomyces coelicolor (74.9% identical amino acids), Mycobacterium tuberculosis (47.8% identity), and Corynebacterium glutamicum (40.7% identity). ArgJ recycles the acetyl group from N-acetylornithine to L-glutamic acid in the first step of the pathway. The argJ gene complements an Escherichia coli XSD2 mutant lacking N-acetylornithinase activity, indicating that the ornithine N-acetyltransferase (OAT) activity encoded by argJ is able to complement the N-acetylornithinase activity encoded by the argE gene of the lineal arginine pathway in E. coli.In the clavulanic acid gene cluster of S. clavuligerus, the oat2 gene (initially described as ORF6), located downstream of the pah gene, encodes a protein of 41,607 Da with a high similarity to ornithine acetyltransferases (9). ArgJ and Oat2 share 31.1% amino acid identity over the entire sequence. When oat2 was subcloned and transformed into E. coli XSD2, the argE mutation of this strain was complemented, indicating that the oat2 promoter was expressed in E. coli; moreover, the transformants showed a strong OAT activity but no ornithinase activity (20). A putative ARG box, which is characteristic of genes tha...

Induction of Holomycin Production and Complex Metabolic Changes by the argR Mutation in Streptomyces clavuligerus NP1

“…However, the role of Oat2 in CA biosynthesis is still not well understood. At high arginine concentrations (above 1 mM), the Oat2 protein or one of its reaction products may negatively affect CA biosynthesis (12).…”

“…The complexity of CA-clavam biosynthesis is also reflected at the level of regulation, and at least 6 regulatory genes (ccaR [45,52], claR [43], cvm7P [59], and orf21 to orf23 [25,55]) were identified among the three gene clusters. Intricate cross-regulation between the arginine and CA (12,51), cephamycin C and CA (44,45), and CA and holomycin (11) pathways were also reported. In the case of arginine-CA cross-regulation, the oat genes (homologous to the arginine biosynthetic gene argJ, encoding ornithine acetyltransferase) were found in both the CA (oat2) and the paralog (oat1) clusters, and the oat2 mutant showed interesting changes in CA production levels, depending on the arginine concentrations, indicating it plays a role in controlling the flux between arginine and CA (12).…”

“…To increase the arginine supply in vivo, one strategy is to inactivate the key repressor gene of arginine biosynthesis, argR. argR disruption in wild-type S. clavuligerus ATCC 27064 resulted in derepression of the transcription of arginine biosynthetic genes and oat2 (12,49,50) and lower production of CA (P. Liras et al, personal communication). However, little is known about the effects of argR mutation on the overall cellular metabolism.…”

Surprising Arginine Biosynthesis: a Reappraisal of the Enzymology and Evolution of the Pathway in Microorganisms

“…The X-ray crystal structure of a monofunctional OAT of Streptomyces clavuligerus involved in clavulanic acid biosynthesis (19), rather than primary metabolism (23), has been determined, but there is presently no in silico clue allowing us to distinguish monofunctional from bifunctional OATs. The phylogenetic tree (Fig.…”

“…This alignment was then used to reconstruct a phylogenetic tree with Phyml, a fast and efficient program based on a maximum-likelihood analysis of a distance tree (33). The tree was rooted with the two paralogues of ArgJ that are present in Streptomyces clavuligerus (19). The root is marked by a black oval.…”