title: Inhibition of Arabidopsis chloroplast β-amylase BAM3 by maltotriose suggests a mechanism for the control of transitory leaf starch mobilisation.
abstract: Starch breakdown in leaves at night is tightly matched to the duration of the dark period, but the mechanism by which this regulation is achieved is unknown. In Arabidopsis chloroplasts, β-amylase BAM3 hydrolyses transitory starch, producing maltose and residual maltotriose. The aim of the current research was to investigate the regulatory and kinetic properties of BAM3. The BAM3 protein was expressed in Escherichia coli and first assayed using a model substrate. Enzyme activity was stimulated by treatment with dithiothreitol and was increased 40% by 2-10 μM Ca2+ but did not require Mg2+. In order to investigate substrate specificity and possible regulatory effects of glucans, we developed a GC-MS method to assay reaction products. BAM3 readily hydrolysed maltohexaose with a Km of 1.7 mM and Kcat of 4300 s-1 but activity was 3.4-fold lower with maltopentaose and was negligible with maltotetraose. With maltohexaose or amylopectin as substrates and using [UL-13C12]maltose in an isotopic dilution method, we discovered that BAM3 activity is inhibited by maltotriose at physiological (mM) concentrations, but not by maltose. In contrast, the extracellular β-amylase of barley is only weakly inhibited by maltotriose. Our results may explain the impaired starch breakdown in maltotriose-accumulating mutants such as dpe1 which lacks the chloroplast disproportionating enzyme (DPE1) metabolising maltotriose to glucose. We hypothesise that the rate of starch breakdown in leaves can be regulated by inhibition of BAM3 by maltotriose, the concentration of which is determined by DPE, which is in turn influenced by the stromal concentration of glucose. Since the plastid glucose transporter pGlcT catalyses facilitated diffusion between stroma and cytosol, changes in consumption of glucose in the cytosol are expected to lead to concomitant changes in plastid glucose and maltotriose, and hence compensatory changes in BAM3 activity.
authors.affiliation: School of Plant Biology, The University of Western Australia, Western Australia, Australia.
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.affiliation: Centre for Energy, Faculty of Engineering, Computing and Mathematics, The University of Western Australia, Western Australia, Australia.
authors.forename: Wenxu
authors.initials: W
authors.lastname: Zhou
authors.affiliation: School of Plant Biology, The University of Western Australia, Western Australia, Australia.
authors.forename: Perigio
authors.initials: P
authors.lastname: Francisco
authors.affiliation: School of Plant Biology, The University of Western Australia, Western Australia, Australia.
authors.forename: Russell
authors.initials: R
authors.lastname: Wong
authors.affiliation: Centre for Energy, Faculty of Engineering, Computing and Mathematics, The University of Western Australia, Western Australia, Australia.
authors.forename: Dongke
authors.initials: D
authors.lastname: Zhang
authors.affiliation: School of Biological Sciences, University of Tasmania, Tasmania, Australia.
authors.forename: Steven M
authors.identifier: http://orcid.org/0000-0001-5661-9994
authors.initials: SM
authors.lastname: Smith
mesh_terms: D017360:Arabidopsis; D029681:Arabidopsis Proteins / Q000378:metabolism*; D002736:Chloroplasts / Q000187:drug effects* / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli; D008401:Gas Chromatography-Mass Spectrometry; D018515:Plant Leaves / Q000187:drug effects / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013213:Starch / Q000378:metabolism*; D014312:Trisaccharides / Q000494:pharmacology*; D001614:beta-Amylase / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D014312:Trisaccharides; C008317:maltotriose; D013213:Starch; C581880:BAM3 protein, Arabidopsis; D017346:Protein Serine-Threonine Kinases; D001614:beta-Amylase