Putrescine N-methyltransferase: crystallisation experiments and site directed mutagenesis

BIRGIT DRÄGER
Martin-Luther-Universität Halle-Wittenberg
Institut für Pharmazeutische Biologie und Pharmakologie
Hoher Weg 8
D-06120 Halle (Saale)
birgit.draeger@pharmazie.uni-halle.de
http://ag-bioarznei.pharmazie.uni-halle.de

The amino acid sequence of putrescine N-methyltransferase (PMT, E.C. 2.1.1.53) is less similar to other methyltransferases than to spermidine synthase (SPDS, E.C. 2.5.1.16). It is hypothesised, that PMT has developed from SPDS during evolution. Within this process the cosubstrate binding changed from decarboxylated S-adenosylmethionine (dcSAM) to S-adenosylmethionine (SAM). SPDS transfers an aminopropyl residue from dcSAM to putrescine forming spermidine and 5´-methylthioadenosine, whereas PMT transfers a methyl group from SAM to putrescine forming N-methylputrescine and S-adenosylhomocysteine (SAH).

Recombinant PMT from Datura stramonium L. was chosen for crystallisation experiments. The enzyme was purified using Ni²⁺- chelate affinity chromatography and subsequent size exclusion chromatography (SEC). Activity of the fractions obtained was determined by a novel rapid colorimetric assay for SAM-dependent methyltransferases. The coupled assay is based on the conversion of SAH to homocysteine by 5´-methylthioadenosine nucleosidase (MTAN, EC 3.2.2.9) and S-ribosylhomocysteine lyase (LuxS, EC 4.4.1.21). Homocysteine is quantified using Ellman's reagent (5,5´-dithiobis-2-nitrobenzoic acid). The pooled and concentrated PMT fractions obtained by SEC were used for cyrstallisation experiments with commercially available screening kits. In the screening format, we reproducibly yielded crystals of rhombic shape, which grow during ten days at 13 °C in citrate buffer with 1 M Li₂SO₄ as precipitant. The crystals appeared to consist of protein due to their physical properties like temperature sensitivity and fragility. Yet, x-ray diffraction experiments yielded no or low diffraction. Further steps to enhance crystal size and regularity are in progress. SPDS of Datura stramonium L. was chosen for site directed mutagenesis in order to change SPDS activity into PMT activity. We suppose that cosubstrate specificity of SPDS for dcSAM is caused by sterical hindrance of the additional carboxyl group at the methionine residue of SAM. A computed model of Datura stramonium PMT based on the crystal structure of Bacillus subtilis SPDS was used to identify amino acids which may be responsible for this hindrance. Actually, these amino acids are used as targets for mutagenesis experiments.

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