An isolated bacterium, identified as a new Sphingomonas species, was demonstrated to contain a novel enzymatic pathway which acted on microcystin LR, the most common cyanobacterial cyclic peptide toxin. Degradation of microcystin LR was mediated by at least three intracellular hydrolytic enzymes. The use of classic protease inhibitors allowed (i) the classification of these enzymes into general protease families and (ii) the in vitro accumulation of otherwise transient microcystin LR degradation products. The initial site of hydrolytic cleavage of the parent cyclic peptide by an enzyme that we designate microcystinase is at the 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-deca-4,6-dienoic acid (Adda)-Arg peptide bond. Two intermediates of microcystin LR enzymatic degradation have been identified; one is linearized (acyclo-) microcystin LR, NH 2-Adda-Glu(iso)-methyldehydroalanine-Ala-Leu--methylaspartate-Arg-OH, and the other is the tetrapeptide NH 2-Adda-Glu(iso)-methyldehydroalanine-Ala-OH. The intermediate degradation products were less active than the parent cyclic peptide; the observed 50% inhibitory concentrations for crude chicken brain protein phosphatase were 0.6 nM for microcystin LR, 95 nM for linear LR, and 12 nM for the tetrapeptide. These linear peptides were nontoxic to mice at doses up to 250 g/kg. Ring opening of the potent hepatotoxin microcystin LR by bacterial microcystinase effectively renders the compound nontoxic by dramatically reducing the interaction with the target protein phosphatase.
A novel pathway for degradation of the cyanobacterial heptapeptide hepatotoxin microcystin LR was identified in a newly isolated Sphingomonas sp. (Bourne et al. 1996 Appl. Environ. Microbiol. 62: 4086-4094). We now report the cloning and molecular characterisation of four genes from this Sphingomonas sp. that exist on a 5.8-kb genomic fragment and encode the three hydrolytic enzymes involved in this pathway together with a putative oligopeptide transporter. The heterologously expressed degradation pathway proteins are enzymatically active. Microcystinase (MlrA), the first enzyme in the degradative pathway, is a 336-residue endopeptidase, which displays only low sequence identity with a hypothetical protein from Methanobacterium thermoautotrophicum. Inhibition of microcystinase by EDTA and 1,10-phenanthroline suggests that it is a metalloenzyme. The most likely residues that could potentially chelate an active-site transition metal ion are in the sequence HXXHXE, which would be unique for a metalloproteinase. Situated immediately downstream of mlrA with the same direction of transcription is a gene mlrD, whose conceptual translation (MlrD, 442 residues) shows significant sequence identity and similar potential transmembrane spanning regions to the PTR2 family of oligopeptide transporters. A gene mlrB is situated downstream of the mlrA and mlrD genes, but transcribed in the opposite direction. The gene encodes the enzyme MlrB (402 residues) which cleaves linear microcystin LR to a tetrapeptide degradation product. This enzyme belongs to the "penicillin-binding enzyme" family of active site serine hydrolases. The final gene in 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 significant sequence identity to a hypothetical protein from Streptomyces coelicolor. It is suspected to be a metallopeptidase based on inhibition by metal chelators. It is postulated on the basis of comparison with other microorganisms that the genes in this cluster may all be involved in cell wall peptidoglycan cycling and subsequently act fortuitously in hydrolysis of microcystin LR.
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