Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae

Abstract

Glycogen phosphorylases (GPs) constitute a family of widely spread catabolic a1,4-glucosyltransferases that are active as dimers of two identical, pyridoxal 5¢-phosphatecontaining subunits. In GP from Corynebacterium callunae, physiological concentrations of phosphate are required to inhibit dissociation of protomers and cause a 100-fold increase in kinetic stability of the functional quarternary structure. To examine interactions involved in this large stabilization, we have cloned and sequenced the coding gene and have expressed fully active C. callunae GP in Escherichia coli. By comparing multiple sequence alignment to structurefunction assignments for regulated and nonregulated GPs that are stable in the absence of phosphate, we have scrutinized the primary structure of C. callunae enzyme for sequence changes possibly related to phosphate-dependent dimer stability. Location of Arg234, Arg236, and Arg242 within the predicted subunit-to-subunit contact region made these residues primary candidates for site-directed mutagenesis. Individual Arg fi Ala mutants were purified and characterized using time-dependent denaturation assays in urea and at 45°C. R234A and R242A are enzymatically active dimers and in the absence of added phosphate, they display a sixfold and fourfold greater kinetic stability of quarternary interactions than the wild-type, respectively. The stabilization by 10 mM of phosphate was, however, up to 20-fold greater in the wild-type than in the two mutants. The replacement of Arg236 by Ala was functionally silent under all conditions tested. Arg234 and Arg242 thus partially destabilize the C. callunae GP dimer structure, and phosphate binding causes a change of their tertiary or quartenary contacts, likely by an allosteric mechanism, which contributes to a reduced protomer dissociation rate.Keywords: interface; oxyanion; phosphate; stabilization; subunit dissociation.Glycogen phosphorylases (GPs) catalyse degradation of glycogen and structurally related reserve polysaccharides in the cytosol to provide energy via the branch point metabolite a-D-glucose-1-phosphate. All known GPs are functional homodimers composed of % 90-kDa subunits and require pyridoxal 5¢-phosphate (PLP) cofactor for activity [1][2][3][4][5][6][7]. Although a very low basal activity may be present in the holoenzyme protomer, quarternary interactions clearly determine physiological levels of phosphorylase activity and are a prerequisite for the regulatory properties of eukaryotic GPs [8][9][10]. Forces that stabilize the dimer structure of GP are therefore essential to optimal enzyme function under physiological boundary conditions. GPs are a/b proteins that display a twodomain fold in which the N-terminal domain and the C-terminal domain are separated by a catalytic site cleft. The structural elements that comprise the subunit-subunit interface are located in the N-terminal domain. The dimer contact regions of regulated and nonregulated GPs share structural similiarity overall, but differ on the molecular level [3][4][5][6][7...

Orthophosphate binding at the dimer interface of Corynebacterium callunae starch phosphorylase: mutational analysis of its role for activity and stability of the enzyme

“…Considering results of previous studies delineating the disruptive effect of individual site-directed substitutions of Arg 234 and Arg 242 by Ala (R234A, R242A) on orthophosphate-dependent stability of Cc GlgP [15], we here selected Arg 141 from the P-site for mutational analysis. Figure 1A shows that Arg 242 has indirect interactions with orthophosphate via the co-ordinating Glu 235 .…”

“…The plasmid pQE 30-GlgP harbouring the gene encoding wild-type Cc GlgP fused to an N-terminal metal affinity peptide (RGSHHHHHHGSA) [15] was used as template for site-directed mutagenesis. Mutations were introduced by employing a modified two-stage PCR protocol [18] in which the following pairs of oligonucleotide primers (Invitrogen) were used with mismatched codons underlined.…”

“…Phosphorylase activity was measured with a continuous coupled enzyme assay described elsewhere [15]. The Bio-Rad dye binding assay referenced against BSA was used for determination of protein concentrations.…”

“…Occupancy of the P-site was thought to result in substantially stabilized intersubunit contacts. Results of mutagenesis experiments located residues responsible for orthophosphate-dependent stability in the region of the so-called TOWER helix, a conserved secondary structural element of the dimer interface in GlgP enzymes [11,15,16]. The crystal structure of Cc GlgP at 1.9 Å resolution (PDB code: 2C4M) now reveals that not one but three orthophosphate sites are dispersed across the dimer contact region of the enzyme.…”

The α-Glucan Phosphorylase MalP of Corynebacterium glutamicum Is Subject to Transcriptional Regulation and Competitive Inhibition by ADP-Glucose

“…Therefore, we investigate in this communication also the effects of the deletion of glgA and glgC on malP transcription and MalP activity. The well-characterized ␣-glucan phosphorylase CcStP from Corynebacterium callunae was described as a starch phosphorylase based on its substrate preference for the branched glucose-polymer starch over linear maltodextrins (13,(46)(47)(48)(49)(50)(51)(52). Based on the close relatedness of C. glutamicum MalP to CcStP, which probably acts as the glycogen phosphorylase in C. callunae, we also analyzed the substrate spectrum of C. glutamicum MalP as well as control of MalP activity by ATP, ADP, AMP, and ADP-glucose.…”