Cyanobacterial residues

The ability to identify and quantify cyanobacterial toxins in animal and human clinical material following (suspected) intoxications or illnesses associated with contact with toxic cyanobacteria is an increasing requirement. The recoveries of anatoxin-a from animal stomach material and of microcystins from sheep rumen contents are relatively straightforward. However, the recovery of microcystin from liver and tissue samples cannot be expected to be complete without the application of proteolytic digestion and extraction procedures. This is likely because microcystins bind covalently to a cysteine residue in protein phosphatase. Unless an effective procedure is applied for the extraction of covalently bound microcystins (and nodiilarins), then a negative result in analysis cannot be taken to indicate the absence of toxins in clinical specimens. Furthermore, any positive result may be an underestimate of the true amount of microcystin in the material and would only represent free toxin, not bound to the protein phosphatases. Optimized procedures for the extraction of bound microcystins and nodiilarins from organ and tissue samples are needed. 

Fig. 5.1 Common cyanobacterial hepatotoxins. (a) Generalized structure of microcystin, a cyclic heptapeptide. Note that X and Z are L-amino acids. For example, microcystin-LR possesses lysine and arginine residues at X and Z, respectively, (b) Cylindrospermopsin, a hepatotoxic alkaloid from Cylindrospermopsis raceborskii...

Acetylcholinesterase inhibition has been widely used for pesticide detection [88-94], but less exploited than protein phosphatase inhibition for cyanobacterial toxin detection. Nevertheless, the anatoxin-a(s) has more inhibition power than most insecticides, as demonstrated by the higher inhibition rates [95]. In order to detect toxin concentrations smaller than usually, mutant enzymes with increased sensitivity were obtained by genetic engineering strategies residue replacement, deletion, insertion and combination of mutations. Modifications close to the active site, located at the bottom of a narrow gorge, made the entrance of the toxin easier and enhanced the sensitivity of the enzyme. 

While protein kinases are responsible for the phosphorylation of their substrates, protein phosphatases perform the opposite duty, removing phosphate groups from their substrates, thus countering the functional impact of the kinases. The two major types of protein phosphatases are the serine/threonine phosphatases and tyrosine phosphatases. Several natural compounds with potent serine/threonine phosphatase inhibitory activity have been identified, including the cyanobacterial metabolite microcystin [105,106]. This compound labels its targets via a Michael addition of a noncatalytic active site cysteine residue with an acceptor in the macrocyclic peptide backbone [107]. A fluorescent probe based on microcystin was synthesized by Shreder et al., and its use in Jurkat lysates identified two previously undescribed phosphatase targets of microcystin, PP-4 and PP-5 [108]. Whereas serine/threonine... 

Plant plastocyanins are synthesized in the cytosol as 160-170-ammo acid precursor polypeptides consisting of a 60-70-residue transit peptide followed by a 97 99-amino acid mature protein. The transit peptide imports the precursor plastocyanin molecule across the chloroplast envelope and thylakoid membranes to its final destination in the thylakoid lumen, where it shuttles electrons by accepting them from the membrane bound cytochrome / (cyt /) of the cyt b6/f complex and donating them to the photooxidized reaction center P700-I- of photosystem I. Cyanobacterial plastocyanins use an 30-amino acid leader seqnence for thylakoid membrane translocation. Currently, there are more than 100 plant and cyanobacterial plastocyanin sequences that are available either by direct protein sequencing or deduced from the nucleotide sequences of their genes. 

N-terminal amino acid analyses [159] of PC-645 and PE-545 revealed that the a-subunits have lost about 60 amino acid residues at their N-terminus compared with the cyanobacterial a-subunits. The chromophores are bound to Cys or Cys, respectively, homologous to Cys " in cyanobacterial and rhodophytan biliproteins... 

GAF domain tyrosine residue in cyanobacterial and plant phy- 58. tochromes. Biochem. 2005 44 15203-15215. 

We consider that all of the dolastatins will prove to be of cyanobacterial or other dietary origin. There are some important biosynthetic signatures, for exanqile, which suggest that dolastatin 15 (2) (Figure 1) is most probably derived from L majuscula. These signatures include the presence of the N,N-dimethylvaline residue, a valine-N-methylvaline-proline sequence also found in the microcolins (27, 28) (Figure 7) and the presence of a pyrrolidone ring presumably derived from condensation of phenylalanine and acetate. A... 

Dolastatin 18 (32) (Figure 7) also contains significant biosynthetic signatures that imply that it is of cyanobacterial origin. These include a 2,2-dimethyl-1,3-dicarbonyl moiety found in lyngbyastatin 1 (4a) and majusculamide C (6) and a dolaphenine residue found in symplostatin 1 (10) and barbamide (11). 

The aeruginosins are a class of cyanobacterial peptide incorporating two characteristic structural features an N-terminal hydroxyphenyllactic acid residue and the highly unusual amino acid 2-carboxy-6-hydroxy-octahydroindole (Choi). The structural diversity, synthesis, and biological activity of this structure class have recently been reviewed. The first example was aeruginosin 298-A (40), isolated from M. aeruginosa NIES-298. ° The structure was elucidated primarily by 2D NMR however, the absence of any NMR... 

The cyclamides are small cyclic peptides that characteristically contain multiple thiazole, thiazoline, oxazole, and oxazoline rings, which are derived from cysteine, serine, and threonine residues. Some of the first examples of this class to be described were the patellamides (53-55) from the tunicate Lissoclinum patella, although it was later determined they were produced by the symbiotic cyanobacterium Prochloron sp. The structures were solved by a combination of acid hydrolysis and GC analysis, coupled with 2D NMR. Smaller cyclic peptides from this class include the hexapeptides westiellamide (56) and microcyclamide (57) from M. aeruginosa Many members of the class possess cytotoxic properties, although their biological function or mechanism of action is not fully understood. In contrast to many cyanobacterial peptides, aside from the unusual heterocyclic residues, these peptides generally contain only ribosomal amino acids. 

Although similar in size to prokaryotic HAL (HAL from P. putida has 509 residues), cyanohacterial PALs are approximately 20% smaller than eukaryotic PALs (e.g., PAL from R. toruloides contains 716 residues per subunit) but, nevertheless, had significantly similar amino acid sequences to the latter. Relative comparison of the superimposed polypeptide hackhone chain of the structures of prokaryotic HAL and eukaryotic PAL with that of the cyanohacterial PAL thus established that the Anahaena variahilis homolog differs by virtue of approximately 1.4 A rmsd for 448 equivalent residues to parsley PAL (identity, 36%), 1.1 A rmsd for 463 equivalent residues to yeast PAL (identity, 34%), and 1.5 A rmsd for 450 equivalent residues to HAL from P. putida (identity, 29%), indicating that cyanobacterial PALs are more closely related structurally to yeast and plant PALs than prokaryotic HAL. Therefore, cyanobacterial PAL may represent an evolutionary intermediate from which eukaryotic PALs are derived. 

Cyanotoxin concentration is generally correlated to the cyanobacterial abundance and since the latter is depended on nntrient availability, it also correlates to nutrient concentrations. "" The correlations, however, can show a considerable residual scatter and thus the concentration of toxins can vary considerably among water bodies or different sampling sites within a water body despite similar cyanobacterial abundances. ... 

The 9 kDa protein is homologous to the 10 kDa protein of Svnecho-coccus PCC 6301 and also corresponds to the internal sequence of psaE product of higher plant (subunit iv ) [8], indicating that ca. 30 residues in N-terminal part of the higher plant protein are lacking in cyanobacterial protein. Similar lack has also been reported for Chlamydomonas protein [9]. This correspondence was overlooked in our previous paper [6]. 

The aligned N-terminal residues of CF II and CF I, as determined by automated Edman degradation by us [1,5], show that CFqII is homologous to cyanobacterial Fpb (13 identities and additional conservative replacements), and CFpI to cyanobacterial F b (15 and 16 identities and additional conservative replacements, cp. table 1). 

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