Characterisation of a gene cluster involved in bacterial degradation of the cyanobacterial toxin microcystin LR

A novel pathway for degradation of the cyanobacterial heptapeptide hepatoto xin microcystin LR was identified in a newly isolated Sphingomonas sp. (Bou rne et al. 1996 Appl. Environ. Microbiol. 62: 4086-4094). We now report the cloning and molecular characterisation of four genes from this Sphingomona s sp. that exist on a 5.8-kb genomic fragment and encode the three hydrolyt ic enzymes involved in this pathway together with a putative oligopeptide t ransporter. The heterologously expressed degradation pathway proteins are e nzymatically active. Microcystinase (MirA), the first enzyme in the degrada tive pathway, is a 336-residue endopeptidase, which displays only low seque nce identity with a hypothetical protein from Methanobacterium thermoautotr ophicum. Inhibition of microcystinase by EDTA and 1,10-phenanthroline sugge sts that it is a metalloenzyme. The most likely residues that could potenti ally chelate an active-site transition metal ion are in the sequence HXXHXE , which would be unique for a metalloproteinase. Situated immediately downs tream of mlrA with the same direction of transcription is a gene mlrD, whos e conceptual translation (MlrD, 442 residues) shows significant sequence id entity and similar potential transmembrane spanning regions to the PTR2 fam ily of oligopeptide transporters. A gene mlrB is situated downstream of the mlrA and mlrD genes, but transcribed in the opposite direction. The gene e ncodes the enzyme MlrB (402 residues) which cleaves linear microcystin LR t o a tetrapeptide degradation product. This enzyme belongs to the "penicilli n-binding enzyme" family of active site serine hydrolases. The final gene i n the cluster mlrC, is located upstream of the mlrA gene and is transcribed in the opposite direction. It codes for MlrC (507 residues) which mediates further peptidolytic degradation of the tetrapeptide. This protein shows s ignificant sequence identity to a hypothetical protein from Streptomyces co elicolor. It is suspected to be a metallopeptidase based on inhibition by m etal chelators. It is postulated on the basis of comparison with other micr oorganisms that the genes in this cluster may all be involved in cell wall peptidoglycan cycling and subsequently act fortuitously in hydrolysis of mi crocystin LR. (C) 2001 by John Wiley & Sons, Inc.