Myrosinases aphid myrosinase

BRIDGES, M, JONES, A.M.E., BONES, A.M., HODGSON, C., COLE, R, BARTLET, E WALLSGROVE, R., KARAPAPA, V.K., WATTS, N., ROSSITER, J.T., Spatial organization of the glucosinolate-myrosinase system in Brassica specialist aphids is similar to that of the host plant., Proc. R. Soc. Lond. Ser. B-Biol. Sci, 2002, 269, 187-191. 

Mechanistic Differences Between Plant and Aphid Myrosinases. 130... 

We have recently reported the first purification of an insect myrosinase and its partial characterization from the cabbage aphid (Brevicoryne brassicae).9 Of all aphids examined for myrosinase,10 only the crucifer specialists, B. brassicae and Lipaphis erysimi, possessed activity although some other polyphagous aphids such as Myzus persicae can also feed on crucifers. 

The native molecular mass of aphid myrosinase, estimated from gel filtration, is approximately 97 kDa, while the molecular mass of the denatured and reduced protein is 53 kDa, estimated from SDS PAGE, and the subunit by MALDI-TOF mass spectrometry 54 kDa 500 Da. Thus, aphid myrosinase appears to be a dimeric protein, with identical subunits. The isoelectric points (pi) of aphid myrosinase are 4.90 and 4.95, the latter isoform being the most abundant. 

Western blots have shown that the antibody raised to aphid myrosinase (Wye Q) was highly specific to a single band in crude extracts of B. brassicae by SDS PAGE analysis. Wye Q did not cross react with proteins (also using Western blotting techniques) from Sinapis alba and did not show a reaction to proteins from other Brassica pests tested (data not shown). Anti-plant myrosinase antibodies did not cross-react with B. brassicae proteins, and anti-aphid myrosinase does not cross react with plant myrosinase. The results of the Western blots are summarized in Table 6.1. 

Table 6.1 Summary of the results of Western blots with anti-plant-myrosinase antibodies and the anti-aphid myrosinase antibody. Wye Q was raised against aphid myrosinase. Wye E, D, and DCJ were all raised against plant myrosinases. + indicates a positive reaction, - indicates a negative reaction. indicates that this combination was not tested.

By using aphid myrosinase purified previously, the cDNA sequence of aphid myrosinase was obtained.12 Five peptides were sequenced and used to design degenerate PCR primers. The full cDNA sequence was obtained using both degenerate and specific primers with Rapid Amplification of cDNA Ends. The GenBank accession number for this sequence is AAL25999. 

MECHANISTIC DIFFERENCES BETWEEN PLANT AND APHID MYROSINASES... 

We have previously developed a protein homology model based on the sequence of aphid myrosinase and templates from plant myrosinase and cyanogenic P-glucosidase.12 These enzymes all adopt the (p/a)8-barrel structure typical of this... 

Figure 6.2 Active sites of plant and aphid myrosinases.

The residues acting as proton donor and nucleophile in the hydrolysis of glucosinolates by aphid myrosinase have been identified as Glu 167 and Glu 374, respectively (Fig. 6.2). The equivalent residues in plant myrosinase are Gin 187 and Glu 409 (Fig. 6.2). A generalized mechanism for the aphid myrosinase is shown in Fig. 6.3 and for the plant myrosinase in Fig. 6.4. 

Figure 6.3 A generalized mechanism for aphid myrosinase hydrolysis of glucosinolates.

Recognition of the sulphate group of glucosinolates is probably mediated by residues Arg 194 and Arg 259 within a positive pocket in plant myrosinase. In aphid myrosinase, Lys 173 and Val 228 are similarly positioned, and it is possible that Lys 173, but not Val 228, may play a similar role (Fig. 6.2). In addition, the basic residue Arg 312 is located in the active site and may contribute to recognition of the sulphate group. Amino acid residues that play a part within the active site of plant myrosinase are shown in Fig. 6.5, while potential residues involved in aphid myrosinase are shown in Fig. 6.6. 

Figure 6.6 Amino acid residues associated with the active site of aphid myrosinase.

Rossiter, J.T., Jones, A.M. and Bones, A.M. (2003) A novel myrosinase-glucosinolate defense system in cruciferous specialist aphids, in Recent Advances in Phytochemistry Integrative Phytochemistry From Ethnobotany to Molecular Ecology (ed. J.T. Romeo). Pergamon, Oxford pp. 127-42. 

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