Bacterial α-glucan phosphorylases (original) (raw)
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1999
Phosphorylases are key enzymes of carbohydrate metabolism. Structural studies have provided explanations for almost all features of control and substrate recognition of phosphorylase but one question remains unanswered. How does phosphorylase recognize and cleave an oligosaccharide substrate? To answer this question we turned to the Escherichia coli maltodextrin phosphorylase (MalP), a non-regulatory phosphorylase that shares similar kinetic and catalytic properties with the mammalian glycogen phosphorylase. The crystal structures of three MalP-oligosaccharide complexes are reported: the binary complex of MalP with the natural substrate, maltopentaose (G5); the binary complex with the thio-oligosaccharide, 4-S-α-D-glucopyranosyl-4-thiomaltotetraose (GSG4), both at 2.9 Å resolution; and the 2.1 Å resolution ternary complex of MalP with thio-oligosaccharide and phosphate (GSG4-P). The results show a pentasaccharide bound across the catalytic site of MalP with sugars occupying sub-sites-1 to ⍣4. Binding of GSG4 is identical to the natural pentasaccharide, indicating that the inactive thio compound is a close mimic of the natural substrate. The ternary MalP-GSG4-P complex shows the phosphate group poised to attack the glycosidic bond and promote phosphorolysis. In all three complexes the pentasaccharide exhibits an altered conformation across sub-sites-1 and ⍣1, the site of catalysis, from the preferred conformation for α(1-4)-linked glucosyl polymers.
PLANT PHYSIOLOGY, 2006
Transitory starch of leaves is broken down hydrolytically, making maltose the predominant form of carbon exported from chloroplasts at night. Maltose metabolism in the cytoplasm of Escherichia coli requires amylomaltase (MalQ) and maltodextrin phosphorylase (MalP). Possible orthologs of MalQ and MalP in the cytosol of Arabidopsis (Arabidopsis thaliana) were proposed as disproportionating enzyme (DPE2, At2g40840) and α-glucan phosphorylase (AtPHS2, At3g46970). In this article, we measured the activities of recombinant DPE2 and AtPHS2 proteins with various substrates; we show that maltose and a highly branched, soluble heteroglycan (SHG) are excellent substrates for DPE2 and propose that a SHG is the in vivo substrate for DPE2 and AtPHS2. In E. coli, MalQ and MalP preferentially use smaller maltodextrins (G3–G7) and we suggest that MalQ and DPE2 have similar, but nonidentical, roles in maltose metabolism. To study this, we complemented a MalQ− E. coli strain with DPE2 and found that ...
Proteins: Structure, Function, and Bioinformatics, 2019
Glycoside phosphorylases (GPs) with specificity for β-(1 ! 3)-gluco-oligosaccharides are potential candidate biocatalysts for oligosaccharide synthesis. GPs with this linkage specificity are found in two families thus far-glycoside hydrolase family 94 (GH94) and the recently discovered glycoside hydrolase family 149 (GH149). Previously, we reported a crystallographic study of a GH94 laminaribiose phosphorylase with specificity for disaccharides, providing insight into the enzyme's ability to recognize its' sugar substrate/product. In contrast to GH94, characterized GH149 enzymes were shown to have more flexible chain length specificity, with preference for substrate/product with higher degree of polymerization. In order to advance understanding of the specificity of GH149 enzymes, we herein solved X-ray crystallographic structures of GH149 enzyme Pro_7066 in the absence of substrate and in complex with laminarihexaose (G6). The overall domain organization of Pro_7066 is very similar to that of GH94 family enzymes. However, two additional domains flanking its catalytic domain were found only in the GH149 enzyme. Unexpectedly, the G6 complex structure revealed an oligosaccharide surface binding site remote from the catalytic site, which, we suggest, may be associated with substrate targeting. As such, this study reports the first structure of a GH149 phosphorylase enzyme acting on β-(1 ! 3)-gluco-oligosaccharides and identifies structural elements that may be involved in defining the specificity of the GH149 enzymes.
FEBS Journal, 2009
Abbreviations ABC, ATP-binding cassette; GH, glycoside hydrolase family; HPAEC-PAD, high-performance ion-exchange chromatography equipped with a pulsed amperometric detector; MalE, maltodextrin-binding protein; MalF and MalG, maltodextrin ABC transport permease proteins; MalL, oligo-1,6-glucosidase; MalN, neopullulanase; MalP, Lactobacillus acidophilus NCFM maltose phosphorylase; MalR, transcriptional regulator of the LacI-GalR family; MsmK, maltodextrin import ATP-binding protein; b-Glc 1-P, b-glucose 1-phosphate.
Reaction Engineering Aspects of α-l,4-D-Glucan Phosphorylase Catalysis
Applied Biochemistry and Biotechnology, 1997
Some important process properties of (x-l,4-D-glucan phosphorylases isolated from the bacterium Corynebacterium callunae and potato tubers (Solanum tuberosum) were compared. Apart from minor differences in their stability and specificity (represented by the maximum degree of maltodextrin conversion) and a 10-fold higher affinity of the plant phosphorylase for maltodextrin (KM of 1.3 g/L at 300 mM of orthophosphate), the performances of both enzymes in a continuous ultra filtration membrane reactor were almost identical. Product synthesis was carried out over a time course of 300-400 h in the presence or absence of auxiliary pullulanase (increasing the accessibility of the glucan substrate for phosphorolytic attack up to 15-20%). The effect of varied dilution rate and reaction temperature on the resulting productivities was quantitated, and a maximum operational temperature of 40~ was identified.