Biochemical characterization and preliminary X-ray crystallographic analysis of cyanobacterial α-glucan phosphorylases

Abstract

Several cyanobacterial species, including Crocosphaera subtropica ATCC 51142, accumulate cyanobacterial starch instead of glycogen, although nearly all cyanobacteria accumulate glycogen. The glycogen-producing Synechococcus elongatus PCC 7942 possesses one α-glucan phosphorylase (Pho) isozyme, whereas strain 51142 has three Pho isozymes. Based on their primary structures, these enzymes belong to glycosyl transferase (GT) family 35, with the cyanobacterial GT35-type Phos further subdivided into types I–III. In this study, to elucidate the significance of the coexistence of multiple GT35-type Pho isozymes, those from strain 51142 (type I, cce_1629; type II, cce_1603 and cce_5186) and strain 7942 (type I, Synpcc7942_0244) were overexpressed in Escherichia coli and biochemically characterized. All isozymes catalysed the phosphorolysis and reverse phosphorolysis reactions. The type I isozyme from a cyanobacterial starch-producing strain (cce_1629) differed in substrate specificity and specific activity compared with the others. The behaviour towards the effectors (AMP and ATP) of the type I and type II isozymes differed from each other. These findings enhance our understanding of the roles of cyanobacterial Pho isozymes in α-glucan metabolism. Furthermore, recombinant cce_1603 was crystallized using the hanging-drop vapour-diffusion method. Crystals were obtained at 293 K in the presence of 10 mM maltoheptaose, 45%(w/v) PEG 400, 0.1 M Tris–HCl pH 8.0, 0.2 M lithium sulfate. The crystals belonged to space group R32 (hexagonal setting) with unit-cell parameters a = b = 267.23, c = 204.43 Å, and diffracted to beyond 2.70 Å resolution. Matthews coefficient calculations indicated the presence of two molecules in the asymmetric unit. Structural determination is currently under way. The crystal structure of cce_1603 will aid in the understanding of the structural basis of cyanobacterial GT35-type Pho isozymes.