Microcystins ADDA side chain

A gas chromatographic (GC) method has been described in the literature. GC is based on the oxidation of microcystins which splits the Adda side chain to produce 3-methoxy-2-metlyl-4-pheitylbu-tyric acid (MMPB), which is then determined, either by GC or GC/MS (as its methyl ester) (Sano 1992 Kaya and Sano 1999) or by HPLC/fluoiescence detection (after conversion to a fluorescent derivative) (Sano 1992). GC/MS has been used to monitor microcystins in Japanese lakes (Tanaka 1993) and in sediments (Tsuji 2001). A similar method was developed by Harada (1996), but in this case the MMPB was determined directly without derivatization using GC/MS or LC/MS. The results of this approach ate given in terms of total toxin concentration, which then can be expressed in terms of microcystin-LR. However, individual toxins ate not determined and consequently it is not possible to produce a result in terms of microcystin-LR toxicity equivalents. This procedure cannot therefore be used to monitor water samples in relation to the proposed guideline. 

As well portrayed in Fig. 9, in our distance geometry-derived stmctures of microcystine-LR the Adda side-chain retains sufficient conformational space for relatively large movements despite the restricted rotational freedom imparted by the double bonds. In family A the Adda side-chain is bent over the ring structure and in family B it is directed away from the ring, an effect even more pronounced in family C. The latter position as well as the relative flexibility of the terminal portion of the side-chain compares well with the microcystin-LR structure determined by Trogen et al (110) in DMSO and in DMSO/water. Moreover, this hydrophobic side-chain is at some distance from the sequentially adjacent Arg side-chain. 

Microcystins have a cyclic heptapeptide structure (Figure 31.1). More than 80 naturally occurring structural variants have been described (Welker and Von Dohren, 2006). Six amino acids, including four nonprotein amino acids and two protein amino acids, form a ring structure. One nonprotein amino acid, referred to as ADDA (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca 4,6-dienoic acid), forms a side chain. The ADDA side chain... 

Yuan, M., Namikoshi, M., Otsuki, A., and Sivonen, K. 1998. Effect of amino acid side-chain on fragmentation of cyclic peptide ions differences of electrospray ionization collision-induced decomposition mass spectra of toxic heptapeptide microcystins containing ADM Adda instead of Adda. EurMass Spectrom 4 287-298. 

Figure 3.6. A microcystin containing 2-amino-2-butenoic add (Dhb), isolated from Oscillatoria agardhii. It also contains Adda, D-Ala, d-G1u, D-Asp and two L-Arg residues. It is, strictly speaking, an isopeptide, since the aspartic and glutamic add residues are linked through their side-chain carboxy groups.

From structure-function studies it is known that esterification of the a-carboxyl group of D-iJo-Glu of microcystin generates untoxic compounds, probably as a result of the lack of inhibition of the protein phosphatases (34,53) and that the methyl ester of okadaic acid is inactive as well (86). Correspondingly, this carboxylic function seems to play an essential role. In order to account for the almost identical potency of microcystins and okadaic acid, we have proposed a location of the carboxyl function of the two diverse compounds at the same position and a wrapping of the okadaic polyether chain around part of the microcystin ring and alignment of its hydrophobic tail with the Adda residue side-chain (108). 

Finally, microcystin LY has a significantly larger stalk due to the parallel array of the Adda and Tyr side chains in a hydrophobic collapse-type interaction. If the size of this hydrophobic finger in the enzyme-bound state is conditioned by the size of the binding cleft of the protein phosphatases, dismption of the hydrophobic interaction between the Adda and Tyr side-chain is required for binding. The resulting large... 

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