Structural insight into the reaction mechanism and evolution of cytokinin biosynthesis

Abstract

The phytohormone cytokinin regulates plant growth and development. This hormone is also synthesized by some phytopathogenic bacteria, such as Agrobacterium tumefaciens, and is as a key factor in the formation of plant tumors. The rate-limiting step of cytokinin biosynthesis is catalyzed by adenosine phosphateisopentenyltransferase (IPT). Agrobacterium IPT has a unique substrate specificity that enables it to increase trans-zeatin production by recruiting a metabolic intermediate of the host plant's biosynthetic pathway. Here, we show the crystal structures of Tzs, an IPT from A. tumefaciens, complexed with AMP and a prenyl-donor analogue, dimethylallyl S-thiodiphosphate. The structures reveal that the carbon-nitrogen-based prenylation proceeds by the SN2-reaction mechanism. Site-directed mutagenesis was used to determine the amino acid residues, Asp-173 and His-214, which are responsible for differences in prenyl-donor substrate specificity between plant and bacterial IPTs. IPT and the p loop-containing nucleoside triphosphate hydrolases likely evolved from a common ancestral protein. Despite structural similarities, IPT has evolved a distinct role in which the p loop transfers a prenyl moiety in cytokinin biosynthesis.Agrobacterium tumefaciens ͉ crystal structure ͉ isopentenyltransferase ͉ trans-zeatin ͉ p loop-containing nucleoside triphosphate hydrolases

Cytokinins - recent news and views of evolutionally old molecules

“…Binding affinity of nucleotides in the order of ATP > dATP~ADP > GTP > CTP > UTP was recently reported for HlIPT (Chu et al 2010). Crystallisation studies on Agrobacterium Tzs revealed that the carbon-nitrogen-based prenylation proceeds by the SN2-reaction mechanism (Sugawara et al 2008) and, together with structural studies of HlIPT (Chu et al 2010) and yeast tRNA-IPT (Zhou and Huang 2008), have given insight into substrate recognition.…”

“…In the virulence region of the Agrobacterium tumefaciens Ti-plasmid, which is not translocated to the plant cells, another gene encoding the enzyme of transzeatin synthesis (Tzs) is present, utilising HMBPP as a prenyl donor and AMP as the acceptor (Krall et al 2002). Recently, Tzs was crystallised and critical amino acid residues responsible for differences in the prenyl-donor substrate specificity of Tzs and plant IPTs were determined in its substrate binding pocket (Sugawara et al 2008). CK biosynthesis is also a virulence determinant of other gall-producing bacteria, and IPTs were identified and functionally proved in, for example, the collinear fas operons of Streptomyces turgidiscabies (Fas4; Joshi and Loria 2007) and Rhodococcus fascians (FasD; Pertry et al 2010).…”

Cytokinin Activity of cis-Zeatin and Phenotypic Alterations Induced by Overexpression of Putative cis-Zeatin-O-glucosyltransferase in Rice

“…Stable isotope-labeled tZRMP was synthesized from [10 13 C,5 15 N]AMP and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate with recombinant tZ secretion (Tzs) protein (Sugawara et al, 2008). Thus, cytokinins derived from this isotopelabeled tZRMP (labeled in the purine ring and ribosyl group; +15) have a molecular mass increased by 15 D. The tZRMP(+15) was dephosphorylated by alkaline phosphatase to form tZR(+15).…”

Agrobacterium tumefaciensTumor Morphology Root Plastid Localization and Preferential Usage of Hydroxylated Prenyl Donor Is Important for Efficient Gall Formation

“…The enzyme modifies cytokinin biosynthesis by recruiting HMBDP, an intermediate product of the methylerythritol phosphate pathway in plastids, to directly produce tZ riboside 59-monophosphate without CYP735A-mediated transhydroxylation Sakakibara et al, 2005). Despite the similar affinity of Tmr for HMBDP and DMAPP in vitro (Sakakibara et al, 2005;Sugawara et al, 2008), crown galls and Tmr-overexpressing transgenic plants almost exclusively contain tZ and its conjugates (tZ-type cytokinins; Morris, 1986;Faiss et al, 1997;Sakakibara et al, 2005), implying an unknown mechanism for preferential HMBDP usage by Tmr in vivo. In addition to tmr, nopaline-type Ti plasmids encode another IPT gene (tzs) within the virulence region, which is induced by plant phenolic compounds (John and Amasino, 1988).…”