Enzymatic formation of unnatural cytokinin analogs by adenylate isopentenyltransferase from mulberry.
A cDNA encoding adenylate isopentenyltransferase (AIPT) was cloned from young leaves of mulberry (Morus alba) by a homology-based RT-PCR. A recombinant enzyme expressed in Escherichia coli catalyzed prenyl transfer from DMAPP to the N6 amino group of ADP and ATP, respectively, while AMP was a poor substrate of the enzyme. Interestingly, M. alba AIPT also accepted dADP, dATP, CDP, and GDP as the prenyl acceptors, and IPP, HMBPP, and GPP as the prenyl donors, to produce a series of cytokinin analogs. In particular, it was remarkable that the enzyme accepted HMBPP to produce trans-zeatin riboside phosphates, which suggested that trans-zeatin may be also produced from adenosine phosphates and HMBPP. Finally, alanine-scanning mutagenesis of conserved D49, Y54, F93, F120, Y153, F157, W159, Y170, Y217, and Q255, resulted in significant loss of enzyme activity except Y170A, confirming the functional and structural importance of the residues.
Abe
Ikuro
I
School of Pharmaceutical Sciences, The COE 21 Program, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan. abei@ys7.u-shizuoka-ken.ac.jp
Tanaka
Hideya
H
Abe
Tsuyoshi
T
Noguchi
Hiroshi
H
D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003583:Cytokinins / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D031625:Morus / Q000201:enzymology* / Q000235:genetics; D016296:Mutagenesis; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D003583:Cytokinins; D009711:Nucleotides; D010940:Plant Proteins; D011994:Recombinant Proteins; D019883:Alkyl and Aryl Transferases; C024274:adenylate isopentenyltransferase