Prokaryotic Expression And Secretory Character Of Oxalate Decarboxylase From A. Tumefaciens C58

Oxalate decarboxylases (OXDCs) (EC4.1.1.2) are enzymes catalyzing the conversion of oxalate to formate and CO₂. These enzymes, which have been found in fungi and bacteria, belong to a functionally diverse protein superfamily known as the cupin that are characterized by their conserved motifs, most recently defined as G(X)₅HXH(X)₁₁G and G(X)₅P(X)₄H(X)₃N constituting a conserved six-strandedβ-barrel fold. Due to the presence of a duplication of this domain, OXDCs are members of the bicupin subclass and are thus thought to contain twoβ-barrels each comprising sixβ-strands.The best-characterized OXDCs are enzymes that have a wood-rotting fungal and bacterial origin. The fungal OXDCs are secreted enzymes, and a secretion signal has been found in the Flammulina velutipes oxalate decarboxylase that can mediate the secretion of heterologous proteins into the medium and periplasmic space in Schizosaccharomyces pombes. It is believed that oxalate synthesized by fungi contribute to lignin degradation, nutrient availability, pathogenesis, and competition. The oxalate-degrading enzymes secreted by these organisms are likely to be involved in pH regulation, and to help reduce toxicity caused by excess accumulations of oxalic acid in the microenvironment. In contrast, most bacterial OXDCs are localized in the cytosol. A decarboxylative phosphorylation mechanism has been described in the gram-negative bacterium Oxalobacter formigenes, in which the antiporting of oxalate and formate are coupled to oxalate decarboxylation by oxalyl-CoA decarboxylase, thereby generating a proton-motive gradient that drives ATP synthsis. It is possible that the bacterial Cytosolic OXDCs have a similar function in energy metabolism as oxalyl-CoA decarboxylase in O. formigenes.In A. tumfaciens C58 a locus for a putative oxalate decarboxylase (here denoted as AtuOXDC) is present. It contains a 1248 bp open reading frame (ORF) for a 415-amino acid polypeptide with a predicated molecular mass of 44.7 kDa. AtuOXDC has two conserved cupin domains shared by members of the cupin superfamily. The N- and C-terminus cupin domain was constituted of 120 tol75 and 301 to 367 amino acid residues respectively. The inter-motif region (IMR) was involved of 20 amino acid residues.Interestingly, analysis of the amino acids revealed a prominent hydrophobic segment of 23 amino acid residues, near the N-terminus of the AtuOXDC amino acid sequence, with a length characteristic of a secretion signal sequence. The most likely cleavage position is between position 27 and 28: AGA-AS. Multiple alignment and phylogeny tree indicate that AtuOXDC and putative OXDCs from Bradyrhizobium japonicum and Burkholderiashare were clustered into a subclass due to the more identity. In addition, the subclass was phylogeneticlly near with OXDCs from fungi.To confirm that the AtuOXDC does indeed encode an oxalate decarboxylase, the truncated AtuOXDC lacking the putative signal portion was expressed in E. coli, and its enzymatic activity was determined. Analysis of the sonicated cell extracts showed that the expression products were present either in the supernatants as soluble protein or in the pellets as inclusion bodies. Using Ni-NTA affinity chromatography, the homologous AtuOXDC was purified. The purified truncated AtuOXDC showed enzymatic activity at 6.567μmol formate/min·mg.A full-length version (AtuFOXDC) and a signal-peptideless version (AtuTOXDC) were cloned into the pTrc200 plasmid which can function as an expression vector in both E. coli and A. tumefaciens. The results of expression support the prediction that a secretion signal is present in the AtuOXDC protein and the protein was targeted in the periplasm of A. tumefaciens C58.When the -RR- motif of the AtuOXDC signal peptide was replaced with -KK- using site-directed mutagenesis, the expression of resulting mutant version (AtuMOXDC) of the full-length AtuOXDC protein in A. tumefaciens strain C58 and subsequent immune-blotting analysis showed that OXDC translocation of the mutant version was completely blocked, implying that the AtuOXDC protein is translocated by the TAT pathway.