Interaction of hydrolytic and phosphorolytic enzymes of starch metabolism in Kalanchoë daigremontiana

The degradation of starch by a protein fraction of Kalanchoë daigremontiana Hamet et Perrier, obtained by ammoniumsulfate precipitation (30–70%), was found to be catalyzed by -and -amylase (EC 3.2.1.1 and EC 3.2.1.2, respectively) and by starch phosphorylase (EC 2.4.1.1). The activity of these enzymes was determined by chromatographic analysis of the reaction products; separation and identification of -amylase was accomplished by heat-inactivation of -amylase and -glucosidase. When the interaction of amylolytic and phosphorolytic enzymes was comparatively studied, it was found that without inorganic phosphorus in the reaction mixture, ¹⁴C-starch was converted predominantly to maltose and glucose; supplementation with 1–10 mM orthophosphate (Pi) resulted in an increase in glucose-1-phosphate formation and a concomitant reduction of maltose production. Since the total volume of starch degradation remained approximately constant, Pi apparently inhibits -amylase (Ki about 3 mM Pi). Thus, free Pi in the cell participates in the regulation of starch catabolism, serving as a substrate for starch phosphorylase while simultaneously reducing the production of maltose. With respect to glucan synthesis, adenosinediphosphoglucose--1,4-glucosyltransferase (EC 2.4.1.22), maltose phosphorylase and maltoseglucosyltransferase were also found to be active. The last-named enzyme catalyzes an exchange between dextrins and is considered to provide primer carbohydrates for the synthesis of polyglucans.Abbreviations ADPG adenosinediphosphoglucose - G1P glucose-1-phosphate - PEG polyethylenglycol - PEP phosphoenolpyruvate - Pi orthophosphate     

N-Acetylglucosaminidases from CAZy Family GH3 Are Really Glycoside Phosphorylases,Thereby Explaining Their Use of Histidine as an Acid/Base Catalyst in Place of Glutamic Acid

CAZy glycoside hydrolase family GH3 consists primarily of stereochemistry-retaining β-glucosidases but also contains a subfamily of β-N-acetylglucosaminidases. Enzymes from this subfamily were recently shown to use a histidine residue within a His-Asp dyad contained in a signature sequence as their catalytic acid/base residue. Reasons for their use of His rather than the Glu or Asp found in other glycosidases were not apparent. Through studies on a representative member, the Nag3 β-N-acetylglucosaminidase from Cellulomonas fimi, we now show that these enzymes act preferentially as glycoside phosphorylases. Their need to accommodate an anionic nucleophile within the enzyme active site explains why histidine is used as an acid/base catalyst in place of the anionic glutamate seen in other GH3 family members. Kinetic and mechanistic studies reveal that these enzymes also employ a double-displacement mechanism involving a covalent glycosyl-enzyme intermediate, which was directly detected by mass spectrometry. Phosphate has no effect on the rates of formation of the glycosyl-enzyme intermediate, but it accelerates turnover of the N-acetylglucosaminyl-enzyme intermediate ∼3-fold, while accelerating turnover of the glucosyl-enzyme intermediate several hundredfold. These represent the first reported examples of retaining β-glycoside phosphorylases, and the first instance of free β-GlcNAc-1-phosphate in a biological context.     

Inorganic phosphate self-sufficient whole-cell biocatalysts containing two co-expressed phosphorylases facilitate cellobiose production

Cellobiose, a natural disaccharide, attracts extensive attention as a potential functional food/feed additive. In this study, we present an inorganic phosphate (Pi) self-sufficient biotransformation system to produce cellobiose by co-expressing sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP). The Bifidobacterium adolescentis SP (BASP) and Cellvibrio gilvus CBP (CGCBP) were co-expressed in Escherichia coli. Escherichia coli cells containing BASP and CGCBP were used as whole-cell catalysts to convert sucrose and glucose to cellobiose. The effects of reaction pH, temperature, Pi concentration, and substrate concentration were investigated. In the optimum biotransformation conditions, 800 mM cellobiose was produced from 1.0 M sucrose, 1.0 M glucose, and 50 mM Pi, within 12 hr. The by-product fructose and residual substrate (sucrose and glucose) were efficiently removed by treatment with yeast, to help purify the product cellobiose. The wider applicability of this Pi self-sufficiency strategy was demonstrated in the production of laminaribiose by co-expressing SP and laminaribiose phosphorylase. This study suggests that the Pi self-sufficiency strategy through co-expressing two phosphorylases has the advantage of great flexibility for enhanced production of cellobiose (or laminaribiose).     

Mg2+ Induces Conformational Changes in the Catalytic Subunit of Phosphorylase Kinase, Whether by Itself or as Part of the Holoenzyme Complex

Phosphorylase kinase (PhK) from skeletal muscle is a structurally complex, highly regulated, hexadecameric enzyme of subunit composition ()₄. Previous studies have revealed that the activity of its catalytic subunit is controlled by alterations in quaternary structure initiated at allosteric and covalent modification sites on PhK's three regulatory subunits; however, changes in the conformation of the holoenzyme initiated by the catalytic subunit have been more difficult to document. In this study a monoclonal antibody (mAb 79) has been generated against isolated subunit and used as a conformational probe of that subunit. The epitope recognized by this antibody is within the catalytic core of the subunit, between residues 100 and 240, and monovalent fragments of the antibody inhibit the catalytic activity of the holoenzyme, the -calmodulin binary complex, and the free subunit. Activation of PhK by a variety of mechanisms known or thought to act through its regulatory subunits (phosphorylation, ADP binding, or alkaline pH) increased the binding of the holoenzyme to immobilized mAb 79, indicating that activation by any of these distinct mechanisms involves repositioning of the portion of the catalytic domain of the subunit containing the epitope for mAb 79. The activating ligand Mg²⁺ also stimulated the binding of the PhK holoenzyme to immobilized mAb 79, as well as the binding of mAb 79 to immobilized subunit. Thus, Mg²⁺ increases the accessibility of the mAb 79 epitope in both the isolated subunit and in the holoenzyme. Our results suggest that previously reported influences of Mg²⁺ on the quaternary structure of the PhK holoenzyme are directly mediated by the subunit.     

Hypoglycemic action of the pectin present in the juice of the inflorescence stalk of plantain (Musa sapientum)—Mechanism of action

The pectin isolated from the juice of the inflorescence stalk of plantain (Musa sapientum) has been found to show significant hypoglycemic effect both in normoglycemic and alloxan diabetic rats. After its administration at a dose of 20mg/100g body weight, there was increase in the concentration of hepatic glycogen, increased glycogenesis as evident from the increased activity of glycogen synthetase and in normoglycemic rats increased incorporation of labelled glucose into hepatic glycogen. Glycogenolysis and glyconeogenesis were lower as was evident from the decreased activity of glycogen phosphorylase and gluconeogenic enzymes.     

Evolutionary and mechanistic relationships between glycosidases acting on alpha- and beta-bonds

Because of the fast accumulation of sequences derived from genome sequencing efforts, the sampling of the sequence space in glycosidase and related enzyme families is such that sensitive sequence similarity detection methods like PSI-BLAST are now able to reveal distant, but clear, structural and evolutionary relations between glycosidases acting on alpha- and beta-bonds. We have observed this trend within groups of glycosidases with completely different folds. We postulate that the evolutionary interconversion between alpha- and beta-acting glycosidases was greatly facilitated by the fact that both types share a similar axial orientation of the glycosidic bond in the reactive bound substrate. Glycosides in the beta anomeric configuration, require a sugar ring distortion, resulting in an axial orientation of the glycosidic bond, equivalent to that of an alpha glycosidic bond, prior to displacement by nucleophilic substitution.     

KINETIC PROPERTIES OF CELLULOMONAS SP. PURINE NUCLEOSIDE PHOSPHORYLASE WITH TYPICAL AND NON-TYPICAL SUBSTRATES: IMPLICATIONS FOR THE REACTION MECHANISM

Phosphorolysis catalyzed by Cellulomonas sp. PNP with typical nucleoside substrate, inosine (Ino), and non-typical 7-methylguanosine (m⁷Guo), with either nucleoside or phosphate (P_i) as the varied substrate, kinetics of the reverse synthetic reaction with guanine (Gua) and ribose-1-phosphate (R1P) as the varied substrates, and product inhibition patterns of synthetic and phosphorolytic reaction pathways were studied by steady-state kinetic methods. It is concluded that, like for mammalian trimeric PNP, complex kinetic characteristics observed for Cellulomonas enzyme results from simultaneous occurrence of three phenomena. These are sequential but random, not ordered binding of substrates, tight binding of one substrate purine bases, leading to the circumstances that for such substrates (products) rapid-equilibrium assumptions do not hold, and a dual role of P_i, a substrate, and also a reaction modifier that helps to release a tightly bound purine base.     

Oxyanion-Mediated Protein Stabilization: Differential Roles of Phosphate for Preventing Inactivation of Bacterial α-Glucan Phosphorylases

Maltodextrin phosphorylase (MalP) from Escherichia coli and starch phosphorylase (StP) from Corynebacterium callunae are significantly stabilized in the presence of phosphate against inactivation by elevated temperature or urea. The stabilizing effect of phosphate was observed at ion concentrations below 50 mM. Therefore, it is probably due to preferential binding of phosphate to the folded conformations of the phosphorylases. For StP, phosphate binding inhibited the dissociation of the active-site cofactor pyridoxal 5′-phosphate. Phosphate-liganded StP was at least 500-fold more stable at 60d`C than the free enzyme at the same temperature. It showed an apparent transition midpoint of 5.2 M for irreversible denaturation by urea, and this midpoint was increased by a denaturant concentration of 4M relative to the corresponding transition midpoint of free StP in urea. The mechanisms of inactivation and denaturation of MalP at 45d`C and by urea involve formation of a cofactor-containing, insoluble protein aggregate. Under denaturing conditions, phosphate was shown to inhibit aggregation of the reversibly inactivated MalP dimer.     

Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex

Phosphorylase kinase (PhK) is a hexadecameric (αβγδ)₄ complex that regulates glycogenolysis in skeletal muscle. Activity of the catalytic γ subunit is regulated by allosteric activators targeting the regulatory α, β, and δ subunits. Three-dimensional EM reconstructions of PhK show it to be two large (αβγδ)₂ lobes joined with D₂ symmetry through interconnecting bridges. The subunit composition of these bridges was unknown, although indirect evidence suggested the β subunits may be involved in their formation. We have used biochemical, biophysical, and computational approaches to not only address the quaternary structure of the β subunits within the PhK complex, i.e. whether they compose the bridges, but also their secondary and tertiary structures. The secondary structure of β was determined to be predominantly helical by comparing the CD spectrum of an αγδ subcomplex with that of the native (αβγδ)₄ complex. An atomic model displaying tertiary structure for the entire β subunit was constructed using chemical cross-linking, MS, threading, and ab initio approaches. Nearly all this model is covered by two templates corresponding to glycosyl hydrolase 15 family members and the A subunit of protein phosphatase 2A. Regarding the quaternary structure of the β subunits, they were directly determined to compose the four interconnecting bridges in the (αβγδ)₄ kinase core, because a β₄ subcomplex was observed through both chemical cross-linking and top-down MS of PhK. The predicted model of the β subunit was docked within the bridges of a cryoelectron microscopic density envelope of PhK utilizing known surface features of the subunit.     

Differential Activities of Protein Phosphatase Types 1 and 2A in Cytosolic and Participate Fractions from Rat Forebrain

Abstract: The activities and concentrations of protein phosphates type 1 (PP1) and type 2A (PP2A) were compared in cytosol and particulate fractions of rat forebrain. Although the activity of PP2A was highest in the cytosol, immunoblot analysis with a PP2A-specific antibody showed that there were significant levels of the enzyme in the particulate fraction. There was no significant difference between the concentration of PP2A in the cytosol and particulate fractions such that the low activity of PP2A in the particulate fraction represents an inactivation of this form of the enzyme. Similar analysis in skeletal muscle, heart, and liver showed this finding was unique to the brain. Similarly, the majority of PP1 activity was recovered in the cytosol, but most PP1 enzyme was associated with the particulate fraction. Comparison with other tissues showed that the activities of PP1 in the particulate fractions were similar but that the forebrain contained significantly more enzyme than the other tissues. Thus, like PP2A it appears that the specific activity of PP1 in the particulate fraction of rat forebrain is much lower than that of the cytosol and of the particulate fractions of other tissues. Elution of PP1 and PP2A from membranes with 0.5 M NaCl plus 0.3% Triton X-100 resulted in severalfold activation of both enzymes. That the majority of PP1 and PP2A in rat forebrain are associated with membrane structures but in a low activity state suggests that novel regulatory mechanisms exist that have considerable and unique potential for activation of protein dephosphorylation.