Search

Galactosyltransferase (GalT) is an important enzyme in synthesizing exopolysaccharide (EPS), the major polymer of biofilms protecting cells from severe conditions. However, the contribution to, and regulatory mechanism of GalT, in stressor resistance are still unclear. Herein, we successfully overexpressed GalT in Lactobacillus acidophilus NCFM by genetic engineering. The GalT activity and freeze-drying survival rate of the recombinant strain were significantly enhanced. The EPS yield also increased by 17.8%, indicating a positive relationship between freeze-drying resistance and EPS. RNA-Seq revealed that GalT could regulate the flux of the membrane transport system, pivotal sugar-related metabolic pathways, and promote quorum sensing to facilitate EPS biosynthesis, which enhanced freeze-drying resistance. The findings concretely prove that the mechanism of GalT regulating EPS biosynthesis plays an important role in protecting lactic acid bacteria from freeze-drying stress., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Introduction: The rapid emergence of antibiotic resistance among various bacterial pathogens has been one of the major concerns of health organizations across the world. In this context, for the development of novel inhibitors against antibiotic-resistant bacterial pathogens, UDP-N-Acetylmuramoyl-L-Alanine-D-Glutamate Ligase (MurD) enzyme represents one of the most apposite targets., Body: The present review focuses on updated advancements on MurD-targeted inhibitors in recent years along with genetic regulation, structural and functional characteristics of the MurD enzyme from various bacterial pathogens. A concise account of various crystal structures of MurD enzyme, submitted into Protein Data Bank is also discussed., Discussion: MurD, an ATP dependent cytoplasmic enzyme is an important target for drug discovery. The genetic organization of MurD enzyme is well elucidated and many crystal structures of MurD enzyme are submitted into Protein Data bank. Various inhibitors against MurD enzyme have been developed so far with an increase in the use of in-silico methods in the recent past. But cell permeability barriers and conformational changes of MurD enzyme during catalytic reaction need to be addressed for effective drug development. So, a combination of in-silico methods along with experimental work is proposed to counter the catalytic machinery of MurD enzyme., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Abstract: Since the 1970s, with Heinrich as a pioneer in the field, numerous kinetic models of erythrocyte glycolysis have been constructed. A functional comparison of eight of these models indicates that the production of ATP and GSH in the red blood cell is largely controlled by the demand reactions. The rate characteristics for the supply and demand blocks indicate a good homeostatic control of ATP and GSH concentrations at different work loads for the pathway, while the production rates of ATP and GSH can be adjusted as needed by the demand reactions. [Copyright &y& Elsevier]

The solution form of IIANtr from Escherichia coli and its interaction with its partner protein, NPr, were characterized by nuclear magnetic resonance (NMR) spectroscopy. The diffusion coefficient of the protein (1.13 × 10−6 cm/sec) falls between that of HPr (′9 kDa) and the N-terminal domain of E. coli enzyme I (∼30 kDa), indicating that the functional form of IIANtr is a monomer (∼18 kDa) in solution. Thus, the dimeric structure of the protein found in the crystal is an artifact of crystal packing. The residual dipolar coupling data of IIANtr (covering residues 11-155) measured in the absence and presence of a 4% polyethyleneglycol-hexanol liquid crystal alignment medium fit well to the coordinates of both molecule A and molecule B of the dimeric crystal structure, indicating that the 3D structures in solution and in the crystal are indeed similar for that protein region. However, only molecule A possesses an N-terminal helix identical to that derived from chemical shifts of IIANtr in solution. Further, the 15N heteronuclear nuclear Overhauser effect (NOE) data also support molecule A as the representative structure in solution, with the terminal residues 1-8 and 158-163 more mobile. Chemical shift mapping identified the surface on IIANtr for NPr binding. Residues Gly61, Asp115, Ser125, Thr156, and nearby regions of IIANtr are more perturbed and participate in interaction with NPr. The active-site His73 of IIANtr for phosphoryl transfer was found in the Nδ1-H tautomeric state. This work lays the foundation for future structure and function studies of the signal transducing proteins from this nitrogen pathway. [ABSTRACT FROM AUTHOR]

Using a recently developed program (SCOPmap) designed to automatically assign new protein structures to existing evolutionary-based classification schemes, we identify a evolutionarily conserved domain (EDD) common to three different folds: mannose transporter EIIA domain (EIIA-man), dihydroxyacetone kinase (Dak), and DegV. Several lines of evidence support unification of these three folds into a single superfamily: statistically significant sequence similarity detected by PSI-BLAST; 'closed structural grouping' using DALI Z-scores (each protein inside a group finds all other group members with scores higher than those to proteins outside the group) that includes only these proteins sharing a unique α-helical hairpin at the C-terminus and excludes all other proteins with similar topology; similar domain fusions connect Dak and DegV, and genomic neighborhood organizations connect Dak and EIIA-man. Finally, both Dak and EIIA-man perform similar phosphotransfer reactions, suggesting a phosphotransferase activity for the DegV-like family of proteins, whose function other than lipid binding revealed in the crystal structure remains unknown. [ABSTRACT FROM AUTHOR]

1,5-Anhydroglucitol (1,5-AG) is a glucose analogue, which is decreased in hyperglycemic individuals. We report the technical performance of an assay (GlycoMark™) on a chemistry analyzer, evaluation of analyte stability and determination of reference intervals for 1,5-AG in a non-diabetic US population.NCCLS protocols were followed to evaluate the reagent on a Hitachi 917 chemistry analyzer.Intra- and interassay imprecision ranged from 1.3% to 3.8% and 0.79% to 3.7%, respectively. The assay was linear to 110 μg/ml. Interference from triglyceride, hemoglobin and bilirubin was <10% to concentrations of 12.6 mmol/l, 12.1 and 911.4 μmol/l, respectively. Correlation coefficients between lot numbers on the Hitachi 917 and between analyses on the Hitachi 917 and the Hitachi 7170 analyzers were >0.99. The lowest limit of detection was 0.49 μg/ml (mean±2 S.D.). 1,5-AG was stable at 4 °C for 7 days, at 22 °C for 5 days, at −80 °C for 14 days and for three freeze–thaw cycles at −80 °C. The US reference intervals (nonparametric 2.5th–97.5th percentiles) were 10.2–33.8 μg/ml (males) and 5.9–31.8 μg/ml (females).The performance of the GlycoMark™ assay for the measurement of 1,5-AG was acceptable on the Hitachi 917 analyzer. [Copyright &y& Elsevier]

Infrequent structural fluctuations of a globular protein is seldom detected and studied in detail. One tyrosine ring of HPr from Staphylococcus carnosus, an 88-residue phosphocarrier protein with no disulfide bonds, undergoes a very slow ring flip, the pressure and temperature dependence of which is studied in detail using the on-line cell high-pressure nuclear magnetic resonance technique in the pressure range from 3 MPa to 200 MPa and in the temperature range from 257 K to 313 K. The ring of Tyr6 is buried sandwiched between a β-sheet and α-helices (the water-accessible area is less than 0.26 nm2), its hydroxyl proton being involved in an internal hydrogen bond. The ring flip rates101∼105 s−1 were determined from the line shape analysis of Hδ1, δ2 and Hε1,ε2 of Tyr6, giving an activation volume Δ V‡ of 0.044 ± 0.008 nm3 (27 mL mol−1), an activation enthalpy Δ H‡ of 89 ± 10 kJ mol−1, and an activation entropy Δ S‡ of 16 ± 2 JK−1 mol−1. The Δ V‡ and Δ H‡ values for HPr found previously for Tyr and Phe ring flips of BPTI and cytochrome c fall within the range of Δ V‡ of 28 to 51 mL mol−1 and Δ H‡ of 71 to 155 kJ mol−1. The fairly common Δ V‡ and Δ H‡ values are considered to represent the extra space or cavity required for the ring flip and the extra energy required to create a cavity, respectively, in the core part of a globular protein. Nearly complete cold denaturation was found to take place at 200 MPa and 257 K independently from the ring reorientation process. [ABSTRACT FROM AUTHOR]

Computational analysis of ADP-glucose pyrophosphorylases predicts a fold with two domains. Co-expression of two polypeptides comprising residues 1–323 and 328–431 from the Escherichia coli ADP-glucose pyrophosphorylase yielded an enzyme form as active as the wild type. The only difference from the wild type was a slightly modified affinity for allosteric effectors. The two polypeptides could not be separated by chromatographic procedures. Separate expression of these polypeptides produced inactive unstable forms. All these results indicated that the ADP-glucose pyrophosphorylase comprises two domains with a strong interaction between them. That interaction is important for allosteric properties and structural stability. [Copyright &y& Elsevier]

Human neuron-specific enolase (NSE) or isozyme γ has been expressed with a C-terminal His-tag in Escherichia coli. The enzyme has been purified, crystallized and its crystal structure determined. In the crystals the enzyme forms the asymmetric complex NSE·Mg2·SO4/NSE·Mg·Cl, where “/” separates the dimer subunits. The subunit that contains the sulfate (or phosphate) ion and two magnesium ions is in the closed conformation observed in enolase complexes with the substrate or its analogues; the other subunit is in the open conformation observed in enolase subunits without bound substrate or analogues. This indicates negative cooperativity for ligand binding between subunits. Electrostatic charge differences between isozymes α and γ, -19 at physiological pH, are concentrated in the regions of the molecular surface that are negatively charged in α, i.e. surface areas negatively charged in α are more negatively charged in γ, while areas that are neutral or positively charged tend to be charge-conserved. [Copyright &y& Elsevier]

3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) catalyzes the first reaction of the aromatic biosynthetic pathway in bacteria, fungi, and plants, the condensation of phosphoenolpyruvate (PEP) and d-erythrose-4-phosphate (E4P) with the formation of DAHP. Crystals of DAHPS from Thermotoga maritima (DAHPSTm) were grown in the presence of PEP and metal cofactor, Cd2+, and then soaked with E4P at 4 °C where the catalytic activity of the enzyme is negligible. The crystal structure of the “frozen” reaction complex was determined at 2.2 Å resolution. The subunit of the DAHPSTm homotetramer consists of an N-terminal ferredoxin-like (FL) domain and a (β/α)8-barrel domain. The active site located at the C-end of the barrel contains Cd2+, PEP, and E4P, the latter bound in a non-productive conformation. The productive conformation of E4P is suggested and a catalytic mechanism of DAHPS is proposed. The active site of DAHPSTm is nearly identical to the active sites of the other two known DAHPS structures from Escherichia coli (DAHPSEc) and Saccharomyces cerevisiae (DAHPSSc). However, the secondary, tertiary, and quaternary structures of DAHPSTm are more similar to the functionally related enzyme, 3-deoxy-d-manno-octulosonate-8-phosphate synthase (KDOPS) from E. coli and Aquiflex aeolicus, than to DAHPSEc and DAHPSSc. Although DAHPSTm is feedback-regulated by tyrosine and phenylalanine, it lacks the extra barrel segments that are required for feedback inhibition in DAHPSEc and DAHPSSc. A sequence similarity search revealed that DAHPSs of phylogenetic family Iβ possess a FL domain like DAHPSTm while those of family Iα have extra barrel segments similar to those of DAHPSEc and DAHPSSc. This indicates that the mechanism of feedback regulation in DAHPSTm and other family Iβ enzymes is different from that of family Iα enzymes, most likely being mediated by the FL domain. [Copyright &y& Elsevier]

Biocatalysis has become an established technology for the industrial manufacture of fine chemicals. In recent years, a multitude of chemical companies have embraced biocatalysis for the manufacture of desired stereoisomers, and new or improved methods for the synthesis of enantiomerically pure α- and β-amino acids, amines, amides, peptides, nitriles, alcohols, organic acids and epoxides have emerged. Furthermore, the selectivity and mild operational conditions of biocatalysts are increasingly applied in industry to modify complex target molecules. These recent innovations in the manufacture of industrial fine chemicals using biocatalysis are discussed from an industrial perspective. [Copyright &y& Elsevier]

Krebs cycle enzyme activities and levels of five metabolites were determined from livers of old mice (30 months) maintained either on control or on long-term caloric restriction (CR) diets (28 months). In CR mice, the cycle was divided into two major blocks, the first containing citrate synthase, aconitase and NAD-dependent isocitrate dehydrogenase which showed decreased activities, while the second block, containing the remaining enzymes, displayed increased activity (except for fumarase, which was unchanged). CR also resulted in decreased levels of citrate, glutamate and α-ketoglutarate, increased levels of malate, and unchanged levels of aspartate. The α-ketoglutarate/glutamate and malate/α-ketoglutarate ratios were higher in CR, in parallel with previously reported increases with CR in pyruvate carboxylase activity and glucagon levels, respectively. The results indicate that long-term CR induces a differential regulation of Krebs cycle in old mice and this regulation may be the result of changes in gene expression levels, as well as a complex interplay between enzymes, hormones and other effectors. Truncation of Krebs cycle by CR may be an important adaptation to utilize available substrates for the gluconeogenesis necessary to sustain glycolytic tissues, such as brain. [Copyright &y& Elsevier]

Glycolysis in Trypanosoma brucei was modeled using a reaction transport simulator and tested for possible complex dynamics. The glycolytic model is multi-compartmentalized and accounts for the exchange of metabolites between the glycosomes, cytosol, mitochondrion and the host medium. The model is used to examine the effects of a range of culture medium concentrations of oxygen on the glycolysis of T. brucei. Our results are in good agreement with steady-state experiments. We also find that under aerobic conditions, increasing the activity of glycerol-3-phosphate dehydrogenase induces complex dynamics in the system. We report the presence of three distinct types of these dynamics. Varying the oxygen concentration in the medium can induce the transition between these dynamics. [Copyright &y& Elsevier]

We observed that in vivo and in vitro a small fraction of the glycolytic enzyme enolase became covalently modified by its substrate 2-phosphoglycerate (2-PG). In modified Escherichia coli enolase, 2-PG was bound to Lys341, which is located in the active site. An identical reversible modification was observed with other bacterial enolases, but also with enolase from Saccharomyces cerevisiae and rabbit muscle. An equivalent of Lys341, which plays an important role in catalysis, is present in enolase of all organisms. Covalent binding of 2-PG to this amino acid rendered the enzyme inactive. Replacement of Lys341 of E. coli enolase with other amino acids prevented the automodification and in most cases strongly reduced the activity. As reported for other bacteria, a significant fraction of E. coli enolase was found to be exported into the medium. Interestingly, all Lys341 substitutions prevented not only the automodification, but also the export of enolase. The K341E mutant enolase was almost as active as the wild-type enzyme and therefore allowed us to establish that the loss of enolase export correlates with the loss of modification and not the loss of glycolytic activity. [Copyright &y& Elsevier]

Molar calorimetric enthalpy changes ΔrHm(cal) have been measured for the biochemical reactions {cAMP(aq) + H2O(l)=AMP(aq)} and {PEP(aq) + H2O(l)=pyruvate(aq) + phosphate(aq)}. The reactions were catalyzed, respectively, by phosphodiesterase 3′,5′-cyclic nucleotide and by alkaline phosphatase. The results were analyzed by using a chemical equilibrium model to obtain values of standard molar enthalpies of reaction ΔrHm∘ for the respective reference reactions {cAMP−(aq) + H2O(l)=HAMP−(aq)} and {PEP3−(aq) + H2O(l)=pyruvate−(aq) + HPO2−4(aq)}. Literature values of the apparent equilibrium constants K′ for the reactions {ATP(aq)=cAMP(aq) + pyrophosphate(aq)}, {ATP(aq) + pyruvate(aq)=ADP(aq) + PEP(aq)}, and {ATP(aq) + pyruvate(aq) + phosphate(aq)=AMP(aq) + PEP(aq) + pyrophosphate(aq)} were also analyzed by using the chemical equilibrium model. These calculations yielded values of the equilibrium constants K and standard molar Gibbs free energy changes ΔrGm∘ for ionic reference reactions that correspond to the overall biochemical reactions. Combination of the standard molar reaction property values (K, ΔrHm∘, and ΔrGm∘) with the standard molar formation properties of the AMP, ADP, ATP, pyrophosphate, and pyruvate species led to values of the standard molar enthalpy ΔfHm∘ and Gibbs free energy of formation ΔfGm∘ and the standard partial molar entropy Sm∘ of the cAMP and PEP species. The thermochemical network appears to be reasonably well reinforced and thus lends some confidence to the accuracy of the calculated property values of the variety of species involved in the several reactions considered herein. [Copyright &y& Elsevier]

A first generation genetically modified strain of Corynebacterium glutamicum has been assessed for its potential to synthesise and accumulate the vitamin pantothenic acid in the medium using fed-batch cultivation technology, with biomass concentration controlled by isoleucine limitation. Kinetic analysis of specific rates throughout the process has been used to model carbon flux through both central metabolism and the specific pathways involved in product formation. Flux towards pantothenic acid is potentially high but much of this flux is dissipated as by-products within associated pathways, notably linked to amino acid synthesis. The major limitation of vitamin production in this strain is linked to the tenfold higher flux of keto-isovalerate towards valine rather than pantothenic acid. Attempts to modify this ratio by imposing nitrogen limitation provoked carbon overflow as unidentified non-nitrogenous compounds. The observed accumulation of glycine suggests that the flux towards pantothenate production may by limited by the rate of the pathway intermediate (5,10-methylene-tetrahydrofolate) regeneration. [Copyright &y& Elsevier]

The activity of enzyme I (EI), the first protein in the bacterial PEP:sugar phosphotransferase system, is regulated by a monomer-dimer equilibrium where a Mg2+-dependent autophosphorylation by PEP requires the homodimer. Using inactive EI(H189A), in which alanine is substituted for the active-site His189, substrate-binding effects can be separated from those of phosphorylation. Whereas 1 mM PEP (with 2 mM Mg2+) strongly promotes dimerization of EI(H189A) at pH 7.5 and 20°C, 5 mM pyruvate (with 2 mM Mg2+) has the opposite effect. A correlation between the coupling of N- and C-terminal domain unfolding, measured by differential scanning calorimetry, and the dimerization constant for EI, determined by sedimentation equilibrium, is observed. That is, when the coupling between N- and C-terminal domain unfolding produced by 0.2 or 1.0 mM PEP and 2 mM Mg2+ is inhibited by 5 mM pyruvate, the dimerization constant for EI(H189A) decreases from > 108 to < 5 × 105 or 3 × 107 M−1, respectively. Incubation of the wild-type, dephospho-enzyme I with the transition-state analog phosphonopyruvate and 2 mM Mg2+ also increases domain coupling and the dimerization constant ∼42-fold. With 2 mM Mg2+ at 15-25°C and pH 7.5, PEP has been found to bind to one site/monomer of EI(H189A) with KA′ ≈ 106 M−1 (Δ G′ = −8.05 ± 0.05 kcal/mole and Δ H = +3.9 kcal/mole at 20°C); Δ Cp = −0.33 kcal K−1 mole−1. The binding of PEP to EI(H189A) is synergistic with that of Mg2+. Thus, physiological concentrations of PEP and Mg2+ increase, whereas pyruvate and Mg2+ decrease the amount of dimeric, active, dephospho-enzyme I. [ABSTRACT FROM AUTHOR]

The glycolytic enzymes of the trypanosomatids, that cause a variety of medically and agriculturally important diseases, are validated targets for drug design. Design of species-specific inhibitors is facilitated by the availability of structural data. Irreversible inhibitors, that bound covalently to the parasite enzyme alone, would be potentially particularly effective. Here we determine the crystal structure of enolase from Trypanosoma brucei and show that two cysteine residues, located in a water-filled cavity near the active-site, are modified by iodoacetamide leading to loss of catalytic activity. Since these residues are specific to the Trypanosomatidae lineage, this finding opens the way for the development of parasite-specific, irreversibly binding enolase inhibitors. In the present structure, the catalytic site is partially occupied by sulphate and two zinc ions. Surprisingly, one of these zinc ions illustrates the existence of a novel enolase-binding site for divalent metals. Evidence suggests that this is the first direct visualization of the elusive inhibitory metal site, whose existence has hitherto only been inferred from kinetic data. [Copyright &y& Elsevier]

Chromoplasts were extracted from the perianth of the wild Meadow Buttercup (Ranunculus acris L.) and purified by sucrose density gradient centrifugation. Analysis of marker enzymes indicated that chromoplast preparations were essentially free of contamination by cytosol, mitochondria and microbodies. The distribution and recovery of glycolytic enzymes indicated an incomplete sequence of enzymes for this pathway in chromoplasts. Fructose 1, 6-bisphosphate aldolase (aldolase) was the only glycolytic enzyme for which unequivocal evidence of localisation within chromoplast preparations could not be obtained. Sub-cellular fractionation and immunological studies using antibodies raised against spinach cytosolic and chloroplastic aldolase, revealed that over 40% of its activity was associated with a washed particulate/membrane fraction in chromoplasts, but this could be removed with 1.2 M NaCl. It is concluded that Ranunculus chromoplasts do not possess a plastidial form of aldolase, and that the aldolase measured in the plastid fraction is a cytosolic form associating with plastid membranes. Isolated buttercup chromoplasts possess a functional oxidative pentose-phosphate pathway (OPPP) which could be monitored as 14CO2 release following incubation with [U-14C]-glucose 6-phosphate (Glc6P). 14CO2 was not evolved when [U-14C]-glucose 1-phosphate (Glc1P) was supplied to chromoplasts suggesting this compound is not transported into the intact organelle and that no interconversion of hexose-phosphate by phosphoglucomutase occurred outside the plastid. Transport experiments using native chromoplast vesicles preloaded with inorganic orthophosphate indicated that these organelles transported Glc6P and dihydroxyacetone phosphate (DHAP), but not Glc1P. The chromoplasts isolated from this wild plant have similar enzymic components and transport properties to other chromoplasts studied to date. [Copyright &y& Elsevier]

A cDNA clone encoding pyruvate kinase (PK) was isolated from a skeletal muscle cDNA library of globefish (Fugu rubripes), which is a kind of lower vertebrate. The full-length cDNA of globefish skeletal muscle pyruvate kinase (FM-PK) is approximately 2 kb and encodes a protein comprising 530 amino acids. The FM-PK gene is spanning approximately 4.8 kb and consists of 11 exons. FM-PK mRNA was detected in muscle and heart using Northern blots. The recombinant FM-PK (rFM-PK) was expressed in a baculovirus-insect cell system and purified using ion-exchange chromatography. The purified rFM-PK was shown to exist a 230 kDa homotetramer composed of 57 kDa subunits. Gel filtration showed 230 000 as the tetramer of the subunit. The apparent Km (or S0.5) and the Hill coefficient for phosphoenolpyruvate (PEP) and ADP are 0.14 mM, 1.3 and 0.30 mM 0.98 at pH 7.4, respectively, when the enzyme is saturated with the second substrate. The rFM-PK is strongly activated by fructose-1,6-bisphosphate, the apparent Km for PEP changes to 0.059 mM and the Hill coefficient to 1.1. ATP, which is the product of the enzyme reaction, inhibits activity. This is the first report to show the full-length cDNA and amino acid sequence of PK for a species of fish. [Copyright &y& Elsevier]

Following acetate, propionate is the second most abundant low molecular mass carbon compound found in soil. Many microorganisms, including most, if not all fungi, as well as several aerobic bacteria, such as Escherichia coli and Salmonella enterica oxidize propionate via the methylcitrate cycle. The enzyme 2-methylisocitrate lyase (PrpB) from Escherichia coli catalysing the last step of this cycle, the cleavage of 2-methylisocitrate to pyruvate and succinate, was crystallised and its structure determined to a resolution of 1.9 A˚. The enzyme, which strictly depends on Mg2+ for catalysis, belongs to the isocitrate lyase protein family. A common feature of members of this enzyme family is the movement of a so-called “active site loop” from an open into a closed conformation upon substrate binding thus shielding the reactants from the surrounding solvent. Since in the presented structure, PrpB contains, apart from a Mg2+, no ligand, the active site loop is found in an open conformation. This conformation, however, differs significantly from the open conformation present in the so far known structures of ligand-free isocitrate lyases. A possible impact of this observation with respect to the different responses of isocitrate lyases and PrpB upon treatment with the common inhibitor 3-bromopyruvate is discussed. Based on the structure of ligand-bound isocitrate lyase from Mycobacterium tuberculosis a model of the substrate-bound PrpB enzyme in its closed conformation was created which provides hints towards the substrate specificity of this enzyme. [Copyright &y& Elsevier]

The phosphoenolpyruvate transferase system (PTS) is the major pathway by which bacteria import hexose sugars across the plasma membrane. The PTS transfers a phosphoryl group sequentially via several components from the glycolytic intermediate phosphoenolpyruvate (PEP) to the translocated sugar. It is comprised of the two general proteins enzyme I and HPr, and a sugar-specific enzyme II complex. Sugar translocation is through the membrane domain of the enzyme II complex. The enzyme II complex can belong to one of six families based upon sequence similarity, with the sorbose transporter from Klebsiella pneumoniae a member of the mannose family.The structure of the IIBSor domain was solved to 1.75 A˚ resolution by molecular replacement. It has a central core of seven parallel β-strands surrounded by a total of six α-helices. Three helices cover the front face, one the back face with the remaining two capping the central β-sheet at the top and bottom. The catalytic His15 residue is situated on the surface-exposed loop between strand 1 and helix 1. In addition to the features previously observed in the homologous IIBLev domain from Bacillus subtilis we see new features in the IIBSor structure. First, the catalytic His15 side-chain is fixed in a specific conformation by forming a short hydrogen bond with Asp10, which in turn makes a salt-bridge with Arg8. Second, as observed in other phosphoproteins, an arginine residue (Arg12) is well poised to stabilize a phosphoryl group on His15. Third, we see an Asp/His pair reminiscent of that observed in the IIAMan domain from Escherichia coli. Finally, docking of IIAMan to IIBSor shows that Arg12 in its current conformation is well positioned to assist the subsequent transfer of the phosphoryl group onto the sugar in line with previous mutagenesis studies. [Copyright &y& Elsevier]

The kinetic properties of a microsomal gill (Na+,K+)-ATPase from the blue crab Callinectes danae were analyzed using the substrate p-nitrophenylphosphate. The (Na+,K+)-ATPase hydrolyzed PNPP obeying cooperative kinetics (n=1.5) at a rate of V=125.4±7.5 U mg−1 with K0.5=1.2±0.1 mmol l−1; stimulation by potassium (V=121.0±6.1 U mg−1; K0.5=2.1±0.1 mmol l−1) and magnesium ions (V=125.3±6.3 U mg−1; K0.5=1.0±0.1 mmol l−1) was cooperative. Ammonium ions also stimulated the enzyme through site–site interactions (nH=2.7) to a rate of V=126.1±4.8 U mg−1 with K0.5=13.7±0.5 mmol l−1. However, K+-phosphatase activity was not stimulated further by K+ plus NH4+ ions. Sodium ions (KI=36.7±1.7 mmol l−1), ouabain (KI=830.3±42.5 μmol l−1) and orthovanadate (KI=34.0±1.4 nmol l−1) completely inhibited K+-phosphatase activity. The competitive inhibition by ATP (KI=57.2±2.6 μmol l−1) of PNPPase activity suggests that both substrates are hydrolyzed at the same site on the enzyme. These data reveal that the K+-phosphatase activity corresponds strictly to a (Na+,K+)-ATPase in C. danae gill tissue. This is the first known kinetic characterization of K+-phosphatase activity in the portunid crab C. danae and should provide a useful tool for comparative studies. [Copyright &y& Elsevier]

Long-term caloric restriction (CR) has been shown to extend maximum life span in laboratory rodents. We investigated the activities of gluconeogenic and transaminase enzymes in the livers of old and young mice fed either control or calorie-restricted diets. Livers were sampled 48 h after the last scheduled feeding time. Old mice on CR showed significant increases in the activities of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase and glucose-6-phosphatase when compared with controls, indicating increased gluconeogenesis. Increased activities of tyrosine, tryptophan, histidine, phenylalanine, alanine and aspartate transaminases, as well as of malate and glutamate dehydrogenases were also observed, while branched-chain amino acid transaminase was unchanged. Young mice on CR showed a significant increase only in the phosphoenolpyruvate carboxykinase activity in the gluconeogenic pathway, while transaminases were increased significantly, except for tryptophan and branched-chain amino acid transaminases. Glutamate dehydrogenase also showed increased activity but malate dehydrogenase was unchanged. Increases in the level of acetyl-CoA and [Acetyl-CoA]/[CoA] ratio were observed only in the old CR mice. Our results demonstrate increased gluconeogenic activity in CR mice and are consistent with a state of increased hepatic gluconeogenesis and protein turnover during CR. [Copyright &y& Elsevier]

The influence of caloric restriction (CR) from 2 months of age on the activities of liver glycolytic enzymes and metabolite levels was studied in young and old mice. Livers were sampled 48 h after the last scheduled feeding time. Old mice on CR showed significant decreases in the activities of all the enzymes studied, except for aldolase, triosephosphate isomerase and phosphoglycerate mutase, which were unchanged. The metabolites glucose, glucose-6-phosphate, fructose-6-phosphate, pyruvate and lactate were lower while fructose-1,6-bisphosphate, glyceraldehyde-3-phosphate, dihydroxyacetone phosphate, 3-phosphoglycerate and phosphoenolpyruvate were increased in old CR. Young mice on CR also showed reduced enzyme activities, except for aldolase, triosephosphate isomerase and enolase which were unchanged when compared with young controls. The metabolites glucose, glucose-6-phosphate, fructose-6-phosphate and pyruvate were decreased when compared with young controls, while phosphoenolpyruvate was increased. Ketone bodies increased (65%) in old, but not young, CR mice while fructose-2,6-bisphosphate decreased in both young (22%) and old CR (28%) mice. The results indicate that decreased hepatic glucose levels in CR mice are associated with decreased enzyme activities but not a uniform decrease in metabolite levels. Increased ketone body levels indicate increased utilization of non-carbohydrate fuels while decreased fructose-2,6-bisphosphate level suggests its importance in the control of glycolysis in CR. [Copyright &y& Elsevier]

An NAD-malate dehydrogenase (NAD-MDH, EC 1.1.1.37) was purified and characterized from leaves of Aptenia cordifolia L. f. (Schwant). This plant performs crassulacean acid metabolism (CAM), as indicated by: (a) elevated levels of phosphoenolpyruvate carboxylase (PEPC) and NAD(P) malic enzyme; (b) regulation of PEPC compatible with its function during the night; (c) characteristic day/night changes in titratable acidity; and (d) gas exchange profile consistent with that shown by CAM plants. These features remained unchanged by water availability or salt stress, suggesting constitutive CAM. The purified MDH showed a subunit molecular mass of 39.4 kDa, a native mass of 83 kDa (dimer) and a pI of 5.8. It cross-reacted with antibodies against cytosolic malate dehydrogenase (cMDH) from pineapple. Maximum activities for oxaloacetate (OAA) reduction or malate oxidation were observed at pH 7.0 and between pH 7.2 and 8.4, respectively. The enzyme was inhibited by excess OAA, in a pH-dependent manner. A discontinuity was observed in Arrhenius plots at 33 °C, with an activation energy twice as high below this temperature. Although immunologically related, some physical and kinetic dissimilarities between the A. cordifolia and pineapple enzymes suggest that diverse CAM metabolic subtypes may require different MDH isozymes to carry out OAA reduction. [Copyright &y& Elsevier]

The activity of rabbit muscle pyruvate kinase (PK) is regulated by metabolites. Besides requiring the presence of its substrates, PEP and ADP, the enzyme requires Mg++ and K+ for activity. PK is allosterically inhibited by Phe for activity. The presence of PEP or Phe has opposing effects on the hydrodynamic properties of the enzyme without an apparent change in secondary structure. In this study, the structural perturbation induced by ligand binding was investigated by Fourier transform infrared (FT-IR) spectroscopy. Furthermore, the structural dynamics of PK was probed by H/D exchange monitored by FT-IR. Substrates and activating metal ions induce PK to assume a more dynamic structure while Phe exerts an opposite effect. In all cases there is no significant interconversion of secondary structures. PEP is the most efficient ligand in inducing a change in the microenvironments of both helices and sheets so much so that they can be detected spectroscopically as separate bands. These results provide the first evidence for a differential effect of ligand binding on the dynamics of structural elements in PK. Furthermore, the data support the model that allosteric regulation of PK is the consequence of perturbation of the distribution of an ensemble of states in which the observed change in hydrodynamic properties represent the two extreme end states. [Copyright &y& Elsevier]

Two isoforms of NADP-dependent malic enzyme (NADP-ME) with the same molecular mass 72 kDa and different isoelectric points, 6.1 and 6.4, were found in crude extracts from the leaves of Aptenia cordifolia, a constitutive CAM plant. In the roots, only one isoform of 72 kDa was found, with a pI of 6.1. The isoform of pI 6.4 was partially purified from leaves to a final specific activity of 30.14 U mg−1, a value similar to the photosynthetic isozymes. This enzyme showed a native mass of 264 kDa, suggesting a homotetramer. An optimal pH of 7.3 and Km values for NADP and l-malate 13 μM and 1.1 mM, respectively, were determined. The enzymatic activities and the level of immunoreactive protein did not vary with the day/night cycle. The enzyme was strongly and competitively inhibited by oxaloacetate (OAA), l-aspartate and phosphoenolpyruvate (PEP) and to a lesser degree by citrate, suggesting that NADP-ME activity might be subject to metabolite control. At night, high levels of OAA, l-aspartate and citrate might inhibit NADP-ME, avoiding a futile cycle of carboxylation/decarboxylation mediated by PEP carboxylase, malate dehydrogenase and NADP-ME. During the day, the low levels of these metabolites would allow the decarboxylation of l-malate. [Copyright &y& Elsevier]

Aquifex aeolicus 3-deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) by favoring the activation of a water molecule coordinated to the active-site metal ion. Cys11, His185, Glu222 and Asp233 are the other metal ligands. Wild-type KDO8PS is purified with Zn2+ or Fe2+ in the active site, but maximal activity in vitro is achieved when the endogenous metal is replaced with Cd2+. The H185G enzyme retains 8% of the wild-type activity. ICP mass spectrometry analysis indicates that loss of His185 decreases the enzyme affinity for Fe2+, but not for Zn2+. However, maximal activity is again achieved by substitution of the endogenous metal with Cd2+. We have determined the X-ray structures of the Cd2+ H185G enzyme in its substrate-free form, and in complex with PEP, and PEP plus A5P. These structures show a normal amount of Cd2+ bound, suggesting that coordination by His185 is not essential to retain Cd2+ in the active site. Nonetheless, there are significant changes in the coordination sphere of Cd2+ with respect to the wild-type enzyme, as the carboxylate moiety of PEP binds directly to the metal ion and replaces water and His185 as ligands. These observations indicate that the primary function of His185 in A. aeolicus KDO8PS is to orient PEP in the active site of the enzyme in such a way that a water molecule on the sinister (si) side of PEP can be activated by direct coordination to the metal ion. [Copyright &y& Elsevier]

The extension of 13C-nuclear magnetic resonance (NMR) techniques to study cellular metabolism over recent years has provided valuable data supporting the occurrence, diversity and extent of carbon cycling in the carbohydrate metabolism of micro-organisms. The occurrence of such cycles, resulting from the simultaneous operation of different and sometimes opposite individual steps, is inherently related to the network organisation of cellular metabolism. These cycles are tentatively classified here as ‘reversibility’, ‘metabolic’ and ‘substrate’ cycles on the basis of their balance in carbon and cofactors. Current hypotheses concerning the physiological relevance of carbohydrate cycles are discussed in light of the 13C-NMR data. They most likely represent system-level mechanisms for coherent and timely partitioning of carbon resources to fit with the various biosynthetic, energetic or redox needs of cells and/or additional strategies in the adaptive capacity of micro-organisms to face variation in environmental conditions. [Copyright &y& Elsevier]

The modulation by Na+, K+, NH4+ and ATP of the (Na+,K+)-ATPase in a microsomal fraction from Callinectes danae gills was analyzed. ATP was hydrolyzed at high-affinity binding sites at a maximal rate of V=35.4±2.1 U mg−1 and K0.5=54.0±3.6 nM, obeying cooperative kinetics (nH=3.6). At low-affinity sites, the enzyme hydrolyzed ATP obeying Michaelis–Menten kinetics with KM=55.0±3.0 μM and V=271.5±17.2 U mg−1. This is the first demonstration of a crustacean (Na+,K+)-ATPase with two ATP hydrolyzing sites. Stimulation by sodium (K0.5=5.80±0.30 mM), magnesium (K0.5=0.48±0.02 mM) and potassium ions (K0.5=1.61±0.06 mM) exhibited site–site interactions, while that by ammonium ions obeyed Michaelis–Menten kinetics (KM=4.61±0.27 mM). Ouabain (KI=147.2±7.2 μM) and orthovanadate (KI=11.2±0.6 μM) completely inhibited ATPase activity, indicating the absence of contaminating ATPase and/or neutral phosphatase activities. Ammonium and potassium ions synergistically stimulated the enzyme, increasing specific activities up to 90%, suggesting that these ions bind to different sites on the molecule. The presence of each ion modulates enzyme stimulation by the other. The modulation of (Na+,K+)-ATPase activity by ammonium ions, and the excretion of NH4+ in benthic crabs are discussed. [Copyright &y& Elsevier]

3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS), the first enzyme of the aromatic biosynthetic pathway in microorganisms and plants, catalyzes the aldol-like condensation of phosphoenolpyruvate and d-erythrose-4-phosphate with the formation of 3-deoxy-d-arabino-heptulosonate-7-phosphate. In Escherichia coli, there are three isoforms of DAHPS, each specifically feedback-regulated by one of the three aromatic amino acid end products. The crystal structure of the phenylalanine-regulated DAHPS from E. coli in complex with its inhibitor, l-phenylalanine, phosphoenolpyruvate, and metal cofactor, Mn2+, has been determined to 2.8 A˚ resolution. Phe binds in a cavity formed by residues of two adjacent subunits and is located about 20 A˚ from the closest active site. A model for the mechanism of allosteric inhibition has been derived from conformational differences between the Phe-bound and previously determined Phe-free structures. Two interrelated paths of conformational changes transmit the inhibitory signal from the Phe-binding site to the active site of DAHPS. The first path involves transmission within a single subunit due to the movement of adjacent segments of the protein. The second involves alterations in the contacts between subunits. The combination of these two paths changes the conformation of one of the active site loops significantly and shifts the other slightly. This alters the interaction of DAHPS with both of its substrates. Upon binding of Phe, the enzyme loses the ability to bind d-erythrose-4-phosphate and binds phosphoenolpyruvate in a flipped orientation. [Copyright &y& Elsevier]

In order to investigate the influence of cytoskeletal organization and dynamics on cellular biochemistry, a mathematical model was formulated based on our own experimental evidence. The model couples microtubular protein (MTP) dynamics to the glycolytic pathway and its branches: the Krebs cycle, ethanolic fermentation, and the pentose phosphate (PP) pathway. Results show that the flux through glycolysis coherently and coordinately increases or decreases with increased or decreased levels of polymerized MTP, respectively. The rates of individual enzymatic steps and metabolite concentrations change with the polymeric status of MTP throughout the metabolic network. Negative control is exerted by the PP pathway on the glycolytic flux, and the extent of inhibition depends inversely on the polymerization state of MTP, i.e. a high degree of polymerization relieves the negative control. The stability of the model''s steady state dynamics for a wide range of variation of metabolic parameters increased with the degree of polymerized MTP. The findings indicate that the organization of the cytoskeleton bestows coherence and robustness to the coordination of cellular metabolism. [Copyright &y& Elsevier]

The crystal structure of the glycosomal enzyme pyruvate phosphate dikinase from the African protozoan parasite Trypanosoma brucei has been solved to 3.0 A˚ resolution by molecular replacement. The search model was the 2.3 A˚ resolution structure of the Clostridium symbiosum enzyme. Due to different relative orientations of the domains and sub-domains in the two structures, molecular replacement could be achieved only by positioning these elements (four bodies altogether) sequentially in the asymmetric unit of the P21212 crystal, which contains one pyruvate phosphate dikinase (PPDK) subunit. The refined model, comprising 898 residues and 188 solvent molecules per subunit, has a crystallographic residual index Rf=0.245 (cross-validation residual index Rfree=0.291) and displays satisfactory stereochemistry. Eight regions, comprising a total of 69 amino acid residues at the surface of the molecule, are disordered in this crystal form. The PPDK subunits are arranged around the crystallographic 2-fold axis as a dimer, analogous to that observed in the C. symbiosum enzyme. Comparison of the two structures was carried out by superposition of the models. Although the fold of each domain or sub-domain is similar, the relative orientations of these constitutive elements are different in the two structures. The trypanosome enzyme is more “bent” than the bacterial enzyme, with bending increasing from the center of the molecule (close to the molecular 2-fold axis) towards the periphery where the N-terminal domain is located. As a consequence of this increased bending and of the differences in relative positions of sub-domains, the nucleotide-binding cleft in the amino-terminal domain is wider in T. brucei PPDK: the N-terminal fragment of the amino-terminal domain is distant from the catalytic, phospho-transfer competent histidine 482 (ca 10 A˚ away). Our observations suggest that the requirements of domain motion during enzyme catalysis might include widening of the nucleotide-binding cleft to allow access and departure of the AMP or ATP ligand. [Copyright &y& Elsevier]

DNA supercoiling by DNA gyrase involves the cleavage of a DNA helix, the passage of another helix through the break, and the religation of the first helix. The cleavage–religation reaction involves the formation of a 5′-phosphotyrosine intermediate with the GyrA subunit of the gyrase (A2B2) complex. We report the characterization of mutations near the active-site tyrosine residue in GyrA predicted to affect the cleavage–religation reaction of gyrase. We find that mutations at Arg32, Arg47, His78 and His80 inhibit DNA supercoiling and other reactions of gyrase. These effects are caused by the involvement of these residues in the DNA cleavage reaction; religation is largely unaffected by these mutations. We show that these residues cooperate with the active-site tyrosine residue on the opposite subunit of the GyrA dimer during the cleavage–religation reaction. [Copyright &y& Elsevier]

Myocardial hibernation represents an adaptation to sustained ischemia to maintain tissue vitality during severe supply–demand imbalance which is characterized by an increased glucose uptake. To elucidate this adaptive protective mechanism, the regulation of anaerobic glycolysis was investigated using human biopsies. In hibernating myocardium showing an increase in anaerobic glycolytic flux metabolizing exogenous glucose, the adjustment of flux through this pathway was analyzed by flux:metabolite co-responses. By this means, a previously unknown pattern of regulation using multisite modulation was found which largely differs from traditional concepts of metabolic control of the Embden–Meyerhof pathway in normal and diseased myocardium. [Copyright &y& Elsevier]

The activity of pyruvate kinase of Leishmania mexicana is allosterically regulated by fructose 2,6-bisphosphate (F-2,6-P2), contrary to the pyruvate kinases from other eukaryotes that are usually stimulated by fructose 1,6-bisphosphate (F-1,6-P2). Based on the comparison of the three-dimensional structure of Saccharomyces cerevisiae pyruvate kinase crystallized with F-1,6-P2 present at the effector site (R-state) and the L. mexicana enzyme crystallized in the T-state, two residues (Lys453 and His480) were proposed to bind the 2-phospho group of the effector. This hypothesis was tested by site-directed mutagenesis. The allosteric activation by F-2,6-P2 appeared to be entirely abrogated in the mutated enzymes confirming our predictions. [Copyright &y& Elsevier]

Yarrowia lipolytica is a widely-used chassis cell in biotechnological applications. It has recently gained extensive research interest owing to its extraordinary ability of producing industrially valuable biochemicals from a variety of carbon sources. Genome-scale metabolic models (GSMMs) enable analyses of cellular metabolism for engineering various industrial hosts. In the present study, we developed a high-quality GSMM i Yli21 for Y. lipolytica type strain W29 by extensive manual curation with Biolog experimental data. The model showed a high accuracy of 85.7% in predicting nutrient utilization. Transcriptomics data were integrated to delineate cellular metabolism of utilizing six individual metabolites as sole carbon sources. Comparisons showed that 302 reactions were commonly used, including those from TCA cycle, oxidative phosphorylation, and purine metabolism for energy and material supply. Whereas glycolytic reactions were employed only when glucose and glycerol used as sole carbon sources, gluconeogenesis and fatty acid oxidation reactions were specifically employed when fatty acid, alkane and glycerolipid were the sole carbon sources. Further test of 46 substrates for generating 5 products showed that hexanoate outcompeted other compounds in terms of maximum theoretical yield owing to the lowest carbon loss for energy supply. This newly generated model i Yli21 will be a valuable tool in dissecting metabolic mechanism and guiding metabolic engineering of this important industrial cell factory., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Cell-free protein synthesis (CFPS) reactions have grown in popularity with particular interest in applications such as gene construct prototyping, biosensor technologies and the production of proteins with novel chemistry. Work has frequently focussed on optimising CFPS protocols for improving protein yield, reducing cost, or developing streamlined production protocols. Here we describe a statistical Design of Experiments analysis of 20 components of a popular CFPS reaction buffer. We simultaneously identify factors and factor interactions that impact on protein yield, rate of reaction, lag time and reaction longevity. This systematic experimental approach enables the creation of a statistical model capturing multiple behaviours of CFPS reactions in response to components and their interactions. We show that a novel reaction buffer outperforms the reference reaction by 400% and importantly reduces failures in CFPS across batches of cell lysates, strains of E. coli , and in the synthesis of different proteins. Detailed and quantitative understanding of how reaction components affect kinetic responses and robustness is imperative for future deployment of cell-free technologies., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)