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

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.

Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin LR.

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, NH2-Adda-Glu(iso)-methyldehydroalanine-Ala-Leu-beta-methylas partate-Arg-OH, and the other is the tetrapeptide NH2-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 micrograms/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.

Characterisation of genes encoding a nucleoside monophosphate kinase and a L35 ribosomal protein from Babesia bovis.

We have sequenced a region of the Babesia bovis nuclear genome that encodes a L35 ribosomal protein homologue (bl35) and a putative nucleoside monophosphate kinase (bnmk) that is most similar to the adenylate kinase of gram-positive bacteria and the mitochondrial form of adenylate kinase in eukaryotes. BNMK appears to be unique in that it is the first eukaryotic family member to feature a putative zinc-binding domain. bnmk and bl35 are closely linked and transcribed from opposite DNA strands. Examination of the gene structures indicate that the coding regions contain small intervening sequences that obey the GT-AG rule of eukaryotic spliceosomal introns. The single intron separates the bl35 initiation codon from the remainder of the coding region and the 6-exon bnmk gene does not appear to be differentially spliced. Both genes utilise multiple polyadenylation sites and the canonical mammalian polyadenylation signal AATAAA is absent from their 3' untranslated regions. Primer extension analyses reveal that the bnmk gene utilises a cluster of transcription start points, one of which is used most frequently. The bnmk mRNA 5' end does not appear to be cis- or trans-spliced. We report here the first evidence of intronic sequences, as well as heterogeneous 5' and 3' ends for mRNA of a member of the Babesia genus.

Degradation of the cyanobacterial hepatotoxin microcystin by aquatic bacteria.

Bacterial degradation of the cyanobacterial cyclic peptide hepatotoxin microcystin was confirmed in natural waters and by isolated laboratory strains. Degradation of 1 mg L-1 microcystin LR typically began 2-8 days after addition to surface water samples. At concentrations greater than 1 mg L-1 there was an initial slow removal of microcystin LR, rather than a distinct lag (or conditioning) phase, before rapid degradation commenced. The lag phase was absent upon re-addition of microcystin LR to the water. Both single strains and mixed bacterial cultures capable of degrading microcystin LR were isolated from surface water samples. One single strain isolated was a gram-negative rod and appeared to be a Pseudomonas sp., although standard taxonomic tests have given inconclusive results. Degradative activity was mostly intracellular and equally active against microcystin LR and RR, but not against nodularin.

Cloning and sequencing of a jack bean urease-encoding cDNA.

A cDNA which encodes the entire amino acid (aa) sequence of the mature jack bean urease has been cloned in Escherichia coli from a library prepared from the mRNA of developing jack beans. It was necessary to use reverse transcriptase in the cDNA was obtained in the form of two contiguous DNA fragments, each of which was completely sequenced. The conceptual translation of the nt sequence gave an 840-aa sequence which was identical to the directly determined sequence except for one conservative aa substitution (Takashima et al., Eur. J. Biochem. 175 (1988) 151-165). These data constitute the first report on the cloning and sequence of the cDNA encoding a urease from any higher plant.

Jack bean urease (EC 3.5.1.5). V. On the mechanism of action of urease on urea, formamide, acetamide, N-methylurea, and related compounds.

Acetamide and N-methylurea have been shown for the first time to be substrates for jack bean urease. In the enzymatic hydrolysis of urea, formamide, acetamide, and N-methylurea at pH 7.0 and 38 degrees C, kcat has the values 5870, 85, 0.55, and 0.075 s-1, respectively. The urease-catalyzed hydrolysis of all these substrates involves the active-site nickel ion(s). Enzymatic hydrolysis of the following compounds could not be detected: phenyl formate, p-nitroformanilide, trifluoroacetamide, p-nitrophenyl carbamate, thiourea, and O-methylisouronium ion. In the enzymatic hydrolysis of urea, the pH dependence of kcat between pH 3.4 and 7.8 indicates that at least two prototropic forms are active. Enzymatic hydrolysis of urea in the presence of methanol gave no detectable methyl carbamate. A mechanism of action for urease is proposed which involves initially an O-bonded complex between urea and an active-site Ni2+ ion and subsequently an O-bonded carbamato-enzyme intermediate.

Jack bean urease (EC 3.5.1.5). IV. The molecular size and the mechanism of inhibition by hydroxamic acids. Spectrophotometric titration of enzymes with reversible inhibitors.

Kinetic, spectral, and other studies establish that hydroxamic acids bind reversibly to active-site nickel ion in jack bean urease. Equilibrium ultracentrifugation studies establish that the molecular weight of native urease is 590 000 +/- 30 000 while that of the subunit formed in 6 M guanidinium chloride in the presence of beta-mercaptoethanol is approximately 95 000. Essentially the same subunit molecular weight (approximately 93 000) is found by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, subsequent to denaturation in a guanidinium chloride - beta-mercaptoethanol system at various temperatures. Coupled with an equivalent weight of 96 600 for binding of the inhibitors acetohydroxamic acid and phosphoramidate, these results establish securely that urease is a hexamer with one active site per 96 600-dalton subunit. Consistent values for the equivalent weight are obtained by a routine spectrophotometric titration of the active site of freshly prepared urease with trans-cinnamoylhydroxamic acid. General equations are derived which describe spectrophotometric titrations of binding sites of any enzyme with a reversible inhibitor. These equations allow the evaluation of the difference spectrum of the protein-inhibitor complex even when the binding sites cannot readily be saturated with the inhibitor or vice versa.

Jack been urease (EC 3.5.1.5). II. The relationship between nickel, enzymatic activity, and the "abnormal" ultraviolet spectrum. The nickel content of jack beans.

At low pH, EDTA promotes the loss of the tightly bound nickel ions from jack bean urease. The specific activity of soluble enzyme after partial EDTA-promoted inactivation is a linear function of the nickel content. The results are consistent with the presence of 2.0 nickel ions per 97 000-dalton subunit in pure urease. The time scale for loss of enzymatic activity and nickel under these conditions is similar to that for loss of the "abnormal" tail absorption in the ultraviolet and visible absorption spectrum of urease (including the shoulder at approximately 420 nm). This indicates that nickel in urease is essential for enzymatic activity and establishes that the metal ions are in part responsible for the tail absorption in the ultraviolet spectrum of urease. After partial inactivation in the presence of EDTA either at low pH or in 2.5 M guanidinium chloride at neutral pH, urease did not regain activity in the presence of Ni2+. As yet apourease has not been produced reversibly. Jack bean seeds grown hydroponically without added nickel were low in both urease activity and nickel (10 and 6%, respectively, of parent seeds). Several other metal ions were readily available. This result suggests that metal ions other than nickel cannot substitute for nickel in the formation of normally active urease.

Jack bean urease (EC 3.5.1.5). III. The involvement of active-site nickel ion in inhibition by beta-mercaptoethanol, phosphoramidate, and fluoride.

Interaction of beta-mercaptoethanol with urease produces large, rapid and fully reversible spectral changes in that part of the electronic absorption spectrum which is associated with the tightly bound nickel ions. The spectrophotometrically determined value of the dissociation constant of the beta-mercaptoethanol-urease complex (0.9 +/- 0.05 mM at pH 7.12 and 25 degrees C) is in agreement with the Ki (0.72 +/- 0.26 mM) for beta-mercaptoethanol acting as a competitive inhibitor in the hydrolysis of urea. This constitutes direct evidence that the nickel in jack bean urease is at the active site. Inhibition of urease by phosphoramidate is slowly achieved and slowly reversed, and upon reactivation of the isolated phosphoramidate-urease complex, phosphoramidate is regenerated in good yield. Spectrophotometric experiments indicate that phosphoramidate binds to nickel ion in urease. Competition with beta-mercaptoethanol was used to determine a dissociation constant (1.23 +/- 0.10 mM at pH 7.12 and 25 degrees C) for a fluoride-evidence is presented which indicates that in the presence of urea, a ternary complex (fluoride-urea-urease) is formed.

Jack bean urease (EC 3.5.1.5). I. A simple dry ashing procedure for the microdetermination of trace metals in proteins. The nickel content of urease.

A simple and inexpensive procedure for determination of microgram quantities of metal ions in proteins is described and tested with nickel and iron. The method involves (a) dry ashing in an oxygen atmosphere at 450-460 degrees C in Pyrex vessels, (b) conversion of the metal oxides or other compounds to readily soluble species, and (c) spectrophotometric analysis. An improved procedure for the direct spectrophotometric determination of nickel using dimethylglyoxime is accurate to +/- 2% or better with samples of 1-5 microgram of nickel. These techniques were used to determine that the nickel content of freshly prepared jack bean urease in 2.00 +/- 0.12 g-at./96 600 g protein. The corresponds to 2.0 nickel ions per subunit. This result was confirmed by atomic absorption analysis, which also showed that calcium, manganese, cobalt, and iron are not present in significant amounts in urease.

Metal ions in enzymes using ammonia or amides.

In an attempt to understand the role of nickel in jack bean urease (1), we turned to a variety of other enzymes important in the utilization, production, or transfer of ammonia. We found several, including the L-histidine and L-phenylalanine ammonialyases and some enzymes that utilize glutamine or ammonia in amidotransferase reactions, all of which show evidence for the involvement of as yet unreported transition metal ions in their mechanism of action. We support the view that catalysis by metalloenzymes may be a reflection of the chemistry of the metal ion itself as a Lewis acid, and that perhaps too much emphasis has been placed on supposed special characteristics (such as strains, "entasis") of the enzyme-metal ion association. In this context, we have discussed the mechanism of catalysis of hydrolysis of specific substrates by carboxypeptidase A, and have returned to urease to examine the role of nickel in its mechanism of action.