Purification and Characterisation of an α‐Glucan Phosphorylase from the Thermophilic Bacterium Thermus thermophilus

Abstract

An a-glucan phosphorylase has been purified 4500-fold from the thermophilic bacteria Thermus thermophilus. In contrast to other bacterial phosphorylases the thermophilic enzyme seems neither to be inducible by maltose nor repressed by glucose. 7: tliernzophilus phosphorylase shares major properties with known mesophilic phosphorylases such as pyridoxal 5'-phosphate content (1 M pyridoxal-PIM subunit), subunit molecular mass (about 90 kDa) and inhibitor constants. The optimum temperature of 7: therniophilus phosphorylase was observed at 70°C in the pH range 5. 5-6.5. While at 25°C the subunit composition of the thermophilic enzyme is an octameric form, the preferential form at the optimum temperature of 70°C seems to be a dimer. Most remarkably, in the direction of synthesis and degradation the limiting size of the oligosaccharide substrate is shorter by one glucose residue than the minimum size of substrate degraded by other a-glucan phosphorylases. Maltotetraose and glycogen are degraded with rates similar to that observed with maltoheptaose (V,,,;,, = 18 U/mg). Correspondingly, maltotriose functions ,as primer in the synthesis direction. Differences in fluorescence and absorption spectra of the cofactor and the failure of arsenatc acting as a substrate indicate that the active site structure of 7: thermophilus phosphorylase differs from that of known rr-glucan phosphorylases.Keywnrds: glycogen phosphorylase ; thermophilic : Therrnus ; pyridoxal 5'-phosphate.Pyridoxal-5'-phosphate(pyridoxal-P)-dependent a-glucanphosphorylases catalyse the first step in the mobilisation of storage polysaccharides, resulting in the energy-conserving release of a-u-glucose 1 -phosphate (Glc-I 2'). All known a-glucanphosphorylases share the absolute requirement for pyridoxal-P linked to the c-amino group of a conserved lysine residue. Studies with pyridoxal-P derivatives, 31P-NMR and glycosylic substrate analogs r'evealed, that the close contact of the cofactor phosphate to the substrate phosphate is essential for activity. The non-hydrolytic cleavage in an aqueous environment requires complex interactions between cofactor, substrates and enzyme protein and the exclusion of water. In contrast to most other vitamin-B6-dependent enzymes, catalysis in phophorylases is dependent on the ionisable phosphate residue of the enzymebound pyridoxal-P (for a review see [I -31).Beside sharing pyridoxal-P as cofactor, a-glucan-phosphorylases from various sources have some features in common, such as their kinetic mechanism and their subunit molecular masses around 90 kDa. The amino acid sequence of domains involved i n substrate binding and catalysis is highly conserved (92 -100 %) among all known a-glucan-phosphorylases. Partial or complete sequeince divergence characterises those regions of phosphorylases that were believed to participate in individual responses to polysaccharide binding, allosteric effectors or protein phosphorylation. All known bacterial phosphorylases are devoid of allosteric or covalent regulation but subject to transcript...

Developing and Engineering Enzymes for Manufacturing Amylose

“…The minimum primer for glucan synthesis reaction and the minimum effective substrate for phosphorylation of the enzyme from T. aquaticus are maltotriose and maltotetraose, respectively. These values corresponded well to those of the enzyme from Thermus thermophilus, 29) but are one unit shorter than those of potato enzyme.…”

Molecular and Physiological Role of the Trehalose-Hydrolyzing α-Glucosidase from Thermus thermophilus HB27

“…Many of the mal genes from E. coli, including the global regulator malT and those encoding MalQ, MalP, and MalZ, have homologues in the genome of strain HB27 (4,12). In strain HB8, the 4-␣-glucanotransferase (MalQ) (TTC0897 in HB27) had very low activity with maltose, the maltodextrin glucosidase (MalZ) (TTC1283 in HB27) has been found to hydrolyze sucrose and maltose, and the maltodextrin phosphorylase (MalP) (TTC0808 in HB27) hydrolyzed maltooligosaccharides and glycogen but not maltose (3,17,30). In Bacillus species, maltose can be taken up and hydrolyzed to glucose and glucose-1-phosphate by a maltose phosphorylase, which is absent from strain HB27 (14).…”

Synthesis of Non-natural Oligosaccharides by ^|^alpha;-Glucan Phosphorylase-Catalyzed Enzymatic Glycosylations Using Analogue Substrates of ^|^alpha;-D-Glucose 1-Phosphate

“…The smallest maltooligosaccharides typically accepted for the phosphorolysis and glucosylation by α-glucan phosphorylase isolated from plants such as potato are maltopentaose (Glc 5 ) and maltotetraose (Glc 4 ), respectively. On the other hand, it was reported that the smallest substrates accepted by α-glucan phosphorylase isolated from thermophilic bacterial sources (thermostable α-glucan phosphorylase) for the former and latter reactions were Glc 4 and maltotriose (Glc 3 ), respectively (20)(21)(22). These observations indicate that α-glucan phosphorylases exhibit different recognition behaviors for the substrates depending on their sources.…”