Part:BBa_K801070

xanthosine methyltransferase CaXMT1-strep

This part encodes for xanthosine methyltransferase CaXMT1 from Coffea arabica followed by a strep-tag for purification and/or detection via western blot.

This part is based upon the mRNA sequences having been isolated out of coffea arabica by [http://www.ncbi.nlm.nih.gov/pubmed/12746542/ Uefuji et al.], 2003, and registered at [http://www.ncbi.nlm.nih.gov/nuccore/AB048794.1/ NCBI]. The sequence was modified in several ways, to make it iGEM compatible and improve the usage in general, respectively.

This part is characterized in Saccharomyces cerevisiae by cloning it into a yeast shuttle vector (pTUM104) and the expression was proved successfully by SDS-PAGE and western blot analysis.

Explanation on figure

Western blot of the crude extract of Saccharomyces cerevisiae INVSc1 transformed with pTUM104CaXMT1, pTUM104CaMXMT1 and pYES2_eGFP cultivated in selective expression Sc minimal medium lacking Uracil with 2% galactose as inducer. (from left to right: prestained protein ladder; eGFP (uninduced), CaMXMT1 (uninduced); CaXMT1 (uninduced); eGFP (20 h); CaMXMT1 (20 h); CaXMT1 (20 h); unstained protein ladder (previously stained with ponceau's reagent))

Note: Because of probable posttranslational modifications in yeast, the apparent weight of the protein differs from the theoretical weight. [http://2d.bjmu.edu.cn/show2d/Proteomics%20tools.asp ExPASy Proteomics tools] predicts the following modifications:

• acetylation at serine (second amino acid)
• O-GlcNAc modifikation at several positions
• phosphorylation at several positions

Background and principles

Text

Usage and Biology

The enzyme CaXMT1 (xanthosine N-methyltransferase 1 of coffea arabica) catalyses the first reaction step of the caffeine biosynthesis pathway and convertes the sustrate Xanthosine into 7-Methylxanthosine. It uses SAM als methyl-donor and is located in the cytoplasm of the plants. Furthermore it exists as homodimer, being also able to form heterodimers with the other enzymes of the caffeine pathway (see [http://www.brenda-enzymes.info Brenda]).

Modifications

• the 5' UTR and 3' UTR of the original sequences were removed
• the yeast consensus sequence for improved ribosome binding (TACACA) was added 5' of the start codon ATG
• according to N- end rule and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine), to optimize both, protein stability and mRNA translation. This decision was made after proofing the 3D- structure of the enzyme CaDXMT1. Due to the fact, that the first two residues of the amino acid sequence are not shown in the crystalized structure (probably because of high flexibility), we chose to exchange this amino acid, because it is probably not necessary for the uptake of the ligands ([http://www.uniprot.org/uniprot/Q9AVK0 uniprot] entry further shows, that it is not immediately involved in ligand binding). Because of the high similarity of the enzyme sequences, we also exchanged this amino acid.
• we added a c- terminal strep-tag for purification and detection
• the remaining coding sequence was extended with the standard RFC10 prefix and suffix, respectively
• at last we made an optimization of the coding sequences with respect to the codon usage and mRNA structures
• remove of all critical restriction sites (RFC10 and RFC25)

Note: Because of the yeast consensus sequence, this coding part does not start with ATG!

Cloning into pSB1C3

The cloning into pSB1C3 was proved by performing an analytical digest with XbaI and PstI.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. Plant Physiol, 132(1):372–80.