Relationships between structure, function and stability for pyridoxal 5′‐phosphate‐dependent starch phosphorylase from Corynebacterium callunae as revealed by reversible cofactor dissociation studies

Abstract

Using 0.4 M imidazole citrate buffer (pH 7.5) containing 0.1 mM L-cysteine, homodimeric starch phosphorylase from Corynebacterium calluane (CcStP) was dissociated into native-like folded subunits concomitant with release of pyridoxal 5¢-phosphate and loss of activity. The inactivation rate of CcStP under resolution conditions at 30°C was, respectively, four-and threefold reduced in two mutants, Arg234fiAla and Arg242fiAla, previously shown to cause thermostabilization of CcStP [Griessler, R., Schwarz, A., Mucha, J. & Nidetzky, B. (2003) Eur. J. Biochem. 270, 2126-2136. The proportion of original enzyme activity restored upon the reconstitution of wild-type and mutant apo-phosphorylases with pyridoxal 5¢-phosphate was increased up to 4.5-fold by added phosphate. The effect on recovery of activity displayed a saturatable dependence on the phosphate concentration and results from interactions with the oxyanion that are specific to the quarternary state. Arg234fiAla and Arg242fiAla mutants showed, respectively, eight-and > 20-fold decreased apparent affinities for phosphate (K app ), compared to the wild-type (K app 6 mM). When reconstituted next to each other in solution, apoprotomers of CcStP and Escherichia coli maltodextrin phosphorylase did not detectably associate to hybrid dimers, indicating that structural complementarity among the different subunits was lacking. Pyridoxal-reconstituted CcStP was inactive but 60% and 5% of wild-type activity could be rescued at pH 7.5 by phosphate (3 mM) and phosphite (5 mM), respectively. pH effects on catalytic rates were different for the native enzyme and pyridoxal-phosphorylase bound to phosphate and could reflect the differences in pK a values for the cofactor 5¢-phosphate and the exogenous oxyanion.Keywords: apo-phosphorylase; a-glucan; glycogen; maltodextrin; pyridoxal 5¢-phosphate. Structure-function relationship studies of a-glucan phosphorylases (GP) have a rich history in biochemical literature. It is well established that pyridoxal 5¢-phosphate (PLP) is the essential cofactor in all known GPs [1]. PLP is bound via a Schiff base between its aldehyde group and a conserved lysine side chain in the active site [1,2]. The 5¢-phosphate group is a main catalytic component of PLP and is required for GP activity [2]. The functional oligomeric state of GP is dimeric [3][4][5]. It has been shown that dissociation of the subunits under localized denaturing conditions exposes PLP to solvent. PLP is released from the enzyme and the activity is lost [6][7][8]. Apo-phosphorylase can be reconstituted, either with PLP or a range of structural analogues thereof [2,9,10]. Whereas restoration of enzyme activity upon the apofiholo conversion is determined by cofactor structure, the process of dimerization is relatively indiscriminate in respect to structural modifications of PLP. Induction of structural complementarity of the interacting subunits such that they are able to recognize each other and associate to dimers is correlated with enzyme-cofactor bond formation [5,9]. In a thoro...

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

“…T28A: 5'-ACCTCGCT GCT GATCGCAAG-3' (forward primer), 5'-AGAACTTGCGATC AGC AGCGAG-3' (reverse primer); K31A: 5'-CTACTGATCGC GCG TTCTGGACTG-3' (forward primer), 5'-CAGTCCAGAA CGC GCGATCAGTAG-3' (reverse primer); R141A: 5'-TGGTCTGCTCTAC GCC TTCGGTC-3' (forward primer), 5'- AGACCGAA GGC GTA-GAGCAGACC-3' (reverse primer); S174A: 5'-TCGTGCA GCC GACCAGTTG-3' (forward primer), 5'-TGGTC GGC TGCACGACGAATAG-3' (reverse primer). Plasmid vectors harbouring sequence-proven inserts (VBC Genomics) were transformed into E. coli JM109, and recipient strains were grown for recombinant protein production as reported previously [11]. Protein purification followed a published protocol [19] except that no heat treatment was used.…”

“…All known GlgP enzymes are naturally active as dimers of two identical PLP-containing subunits [7-9]. Dimeric structure formation results in marked stabilization of the otherwise chemically labile protein-cofactor bond such that PLP is not detectably dissociable from native phosphorylase dimers [7,10,11]. GlgP enzymes in which activity is under control of covalent phosphorylation and/or allosteric effectors respond to regulatory signals through extensive rearrangements of their intersubunit contacts [5,12,13].…”

“…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.…”

Starch phosphorylase: Role in starch metabolism and biotechnological applications

“…The stabilization effects of StP upon ligand (phosphate ion) binding is outstanding and led to a series of investigations on manipulating its stability via conformational engineering. Griessler et al (2004) have probed the forces that stabilize its quaternary structure.…”

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.…”