Evolution of Arginine Biosynthesis in the Bacterial Domain: Novel Gene-Enzyme Relationships from Psychrophilic Moritella Strains ( Vibrionaceae ) and Evolutionary Significance of N -α-Acetyl Ornithinase

Abstract

In the arginine biosynthetic pathway of the vast majority of prokaryotes, the formation of ornithine is catalyzed by an enzyme transferring the acetyl group of N-␣-acetylornithine to glutamate (ornithine acetyltransferase [OATase]) (argJ encoded). Only two exceptions had been reported-the Enterobacteriaceae and Myxococcus xanthus (members of the ␥ and ␦ groups of the class Proteobacteria, respectively)-in which ornithine is produced from N-␣-acetylornithine by a deacylase, acetylornithinase (AOase) (argE encoded). We have investigated the gene-enzyme relationship in the arginine regulons of two psychrophilic Moritella strains belonging to the Vibrionaceae, a family phylogenetically related to the Enterobacteriaceae. Most of the arg genes were found to be clustered in one continuous sequence divergently transcribed in two wings, argE and argCBFGH(A) ["H(A)" indicates that the argininosuccinase gene consists of a part homologous to known argH sequences and of a 3 extension able to complement an Escherichia coli mutant deficient in the argA gene, encoding N-␣-acetylglutamate synthetase, the first enzyme committed to the pathway]. Phylogenetic evidence suggests that this new clustering pattern arose in an ancestor common to Vibrionaceae and Enterobacteriaceae, where OATase was lost and replaced by a deacylase. The AOase and ornithine carbamoyltransferase of these psychrophilic strains both display distinctly cold-adapted activity profiles, providing the first cold-active examples of such enzymes.

Metabolic Enzymes from Psychrophilic Bacteria: Challenge of Adaptation to Low Temperatures in Ornithine Carbamoyltransferase from Moritella abyssi

“…In M. abyssi, the OTCaseencoding gene argF was found to be part of a large divergent operon of which the genes identified so far are clustered in the order ECBFGH(A), with argE constituting the left wing of the operon (59). Alignment of OTCase sequences by the Clustal program revealed that M. abyssi argF is clearly homologous to other OTCase genes.…”

“…Restriction enzymes and T4 ligase were purchased from Boehringer Mannheim and used according to the manufacturer's instructions. The argF gene of M. abyssi strain 2693 (57,59) was amplified by PCR with oligonucleotides 5Ј-GGAATTCCATATGGAAAATTT ATTATCAGTTAAAGATTTA-3Ј (start codon underlined) and 5Ј-CGGGATC CCTACTTTCTTAACTGTTTGTGTC-3Ј to produce NdeI and BamHI restriction sites at the ends of the fragment. The NdeI restriction site was designed to overlap the ATG start codon and the BamHI site after the TAA stop codon.…”

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

“…3). M. abyssi and M. profunda display an unusual structure for the argH gene that codes for the argininosuccinate lyase catalyzing the last step (EC 4.3.2.1) of arginine biosynthesis (82). The argH gene is extended by a 170-codon stretch able to complement an argA E. coli mutant.…”

“…The gene is present at the end of an argE/CBFGH(A) operon, where argE-coding for an acetylornithinase (AO)-and the rest of the cluster are expressed divergently, a pattern characteristic of many enteric bacteria. After the original report (82), the argH(A) gene was found in similar genetic contexts in closely related bacteria belonging to the Alteromonas-Vibrio group (81). Two of them-Pseudoalteromonas haloplanktis (46) and Idiomarina loihiensis (36)-do not harbor a genetically identifiable NAGS or OAT (Fig.…”

“…Evidence of substitutes for classical NAGS first came from genetic observations of Campylobacter jejuni, an ε-proteobacterium (35), and two marine psychrophilic ␥-Proteobacteria, Moritella abyssi and M. profunda (82).…”