Accumulation of d-Glucose from Pentoses by Metabolically Engineered Escherichia coli

Escherichia coli that is unable to metabolize d-glucose (with knockouts in ptsG, manZ, and glk) accumulates a small amount of d-glucose (yield of about 0.01 g/g) during growth on the pentoses d-xylose or l-arabinose as a sole carbon source. Additional knockouts in the zwf and pfkA genes, encoding, respectively, d-glucose-6-phosphate 1-dehydrogenase and 6-phosphofructokinase I (E. coli MEC143), increased accumulation to greater than 1 g/liter d-glucose and 100 mg/liter d-mannose from 5 g/liter d-xylose or l-arabinose. Knockouts of other genes associated with interconversions of d-glucose-phosphates demonstrate that d-glucose is formed primarily by the dephosphorylation of d-glucose-6-phosphate. Under controlled batch conditions with 20 g/liter d-xylose, MEC143 generated 4.4 g/liter d-glucose and 0.6 g/liter d-mannose. The results establish a direct link between pentoses and hexoses and provide a novel strategy to increase carbon backbone length from five to six carbons by directing flux through the pentose phosphate pathway.     

A Novel Type of Peptidoglycan-binding Domain Highly Specific for Amidated d-Asp Cross-bridge,Identified in Lactobacillus casei Bacteriophage Endolysins

Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-l-alanine amidase, whereas Lc-Lys-2 is a γ-d-glutamyl-l-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with d-Ala⁴→d-Asx-l-Lys³ in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting d-Ala⁴→l-Ala-(l-Ala/l-Ser)-l-Lys³; moreover, they do not lyse the L. lactis mutant containing only the nonamidated d-Asp cross-bridge, i.e. d-Ala⁴→d-Asp-l-Lys³. In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 l-Lys³-d-Asn-l-Lys³ bridges replacing the wild-type 4→3 d-Ala⁴-d-Asn-l-Lys³ bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly d-Asn but not PG with only the nonamidated d-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the d-Asn interpeptide bridge of PG.     

The enzymes forming isopentenyl pyrophosphate from 5-phosphomevalonate (mevalonate 5-phosphate) in the latex of Hevea brasiliensis

1. Phosphomevalonate kinase and 5-pyrophosphomevalonate decarboxylase have been purified from the freeze-dried latex serum of the commercial rubber tree Hevea brasiliensis. 2. The phosphomevalonate kinase was acid- and heat-labile and required the presence of a thiol to maintain activity. 3. The 5-pyrophosphomevalonate decarboxylase was relatively acid-stable and more heat-stable than the phosphokinase. 4. Maximum activity of the phosphokinase was achieved at pH 7.2 with 0.2mm-5-phosphomevalonate (K_m 0.042mm), 2.0mm-ATP (K_m 0.19mm) and 8mm-Mg²⁺ at 40°C. The apparent activation energy was 14.8kcal/mol. 5. Maximum activity of 5-pyrophosphomevalonate decarboxylase was achieved at pH5.5–6.5 with 0.1mm-5-pyrophosphomevalonate (K_m 0.004mm), 1.5mm-ATP (K_m 0.12mm) and 2mm-Mg²⁺. The apparent activation energy was 13.7kcal/mol. The enzyme was somewhat sensitive to inhibition by its products, isopentenyl pyrophosphate and ADP.     

d-Amino Acid Substitution of Peptide-Mediated NF-κB Suppression in mdx Mice Preserves Therapeutic Benefit in Skeletal Muscle,but Causes Kidney Toxicity

In Duchenne muscular dystrophy (DMD) patients and the mdx mouse model of DMD, chronic activation of the classical nuclear factor-κB (NF-κB) pathway contributes to the pathogenesis that causes degeneration of muscle fibers, inflammation and fibrosis. Prior studies demonstrate that inhibition of inhibitor of κB kinase (IKK)-mediated NF-κB activation using l-isomer NF-κB essential modulator (NEMO)-binding domain (NBD) peptide-based approaches reduce muscle pathology in the mdx mouse. For our studies, the NBD peptide is synthesized as a fusion peptide with an eight-lysine (8K) protein transduction domain to facilitate intracellular delivery. We hypothesized that the d-isoform peptide could have a greater effect than the naturally occurring l-isoform peptide due to the longer persistence of the d-isoform peptide in vivo. In this study, we compared systemic treatment with low (1 mg/kg) and high (10 mg/kg) doses of l- and d-isomer 8K-wild-type-NBD peptide in mdx mice. Treatment with both l- or d-isoform 8K-wild-type-NBD peptide resulted in decreased activation of NF-κB and improved histology in skeletal muscle of the mdx mouse. However, we observed kidney toxicity (characterized by proteinuria), increased serum creatinine, activation of NF-κB and pathological changes in kidney cortex that were most severe with treatment with the d-isoform of 8K-wild-type-NBD peptide. The observed toxicity was also seen in normal mice.     

High-Level Heterologous Production of d-Cycloserine by Escherichia coli

Previously, we successfully cloned a d-cycloserine (d-CS) biosynthetic gene cluster consisting of 10 open reading frames (designated dcsA to dcsJ) from d-CS-producing Streptomyces lavendulae ATCC 11924. In this study, we put four d-CS biosynthetic genes (dcsC, dcsD, dcsE, and dcsG) in tandem under the control of the T7 promoter in an Escherichia coli host. SDS-PAGE analysis demonstrated that the 4 gene products were simultaneously expressed in host cells. When l-serine and hydroxyurea (HU), the precursors of d-CS, were incubated together with the E. coli resting cell suspension, the cells produced significant amounts of d-CS (350 ± 20 μM). To increase the productivity of d-CS, the dcsJ gene, which might be responsible for the d-CS excretion, was connected downstream of the four genes. The E. coli resting cells harboring the five genes produced d-CS at 660 ± 31 μM. The dcsD gene product, DcsD, forms O-ureido-l-serine from O-acetyl-l-serine (OAS) and HU, which are intermediates in d-CS biosynthesis. DcsD also catalyzes the formation of l-cysteine from OAS and H₂S. To repress the side catalytic activity of DcsD, the E. coli chromosomal cysJ and cysK genes, encoding the sulfite reductase α subunit and OAS sulfhydrylase, respectively, were disrupted. When resting cells of the double-knockout mutant harboring the four d-CS biosynthetic genes, together with dcsJ, were incubated with l-serine and HU, the d-CS production was 980 ± 57 μM, which is comparable to that of d-CS-producing S. lavendulae ATCC 11924 (930 ± 36 μM).     

Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism

The first enzyme in the pathway for l-arabinose catabolism in eukaryotic microorganisms is a reductase, reducing l-arabinose to l-arabitol. The enzymes catalyzing this reduction are in general nonspecific and would also reduce d-xylose to xylitol, the first step in eukaryotic d-xylose catabolism. It is not clear whether microorganisms use different enzymes depending on the carbon source. Here we show that Aspergillus niger makes use of two different enzymes. We identified, cloned, and characterized an l-arabinose reductase, larA, that is different from the d-xylose reductase, xyrA. The larA is up-regulated on l-arabinose, while the xyrA is up-regulated on d-xylose. There is however an initial up-regulation of larA also on d-xylose but that fades away after about 4 h. The deletion of the larA gene in A. niger results in a slow growth phenotype on l-arabinose, whereas the growth on d-xylose is unaffected. The l-arabinose reductase can convert l-arabinose and d-xylose to their corresponding sugar alcohols but has a higher affinity for l-arabinose. The K_m for l-arabinose is 54 ± 6 mm and for d-xylose 155 ± 15 mm.     

Inositol Metabolism in Plants. V. Conversion of Myo-inositol to Uronic Acid and Pentose Units of Acidic Polysaccharides in Root-tips of Zea mays

The metabolism of myo-inositol-2-¹⁴C, d-glucuronate-1-¹⁴C, d-glucuronate-6-¹⁴C, and l-methionine-methyl-¹⁴C to cell wall polysaccharides was investigated in excised root-tips of 3 day old Zea mays seedlings. From myo-inositol, about one-half of incorporated label was recovered in ethanol insoluble residues. Of this label, about 90% was solubilized by treatment, first with a preparation of pectinase-EDTA, then with dilute hydrochloric acid. The only labeled constituents in these hydrolyzates were d-galacturonic acid, d-glucuronic acid, 4-O-methyl-d-glucuronic acid, d-xylose, and l-arabinose, or larger oligosaccharide fragments containing these units. Medium external to excised root-tips grown under sterile conditions in myo-inositol-2-¹⁴C contained labeled polysaccharide.     

Structural and Functional Characterization of VanG d-Ala:d-Ser Ligase Associated with Vancomycin Resistance in Enterococcus faecalis

d-Alanyl:d-lactate (d-Ala:d-Lac) and d-alanyl:d-serine ligases are key enzymes in vancomycin resistance of Gram-positive cocci. They catalyze a critical step in the synthesis of modified peptidoglycan precursors that are low binding affinity targets for vancomycin. The structure of the d-Ala:d-Lac ligase VanA led to the understanding of the molecular basis for its specificity, but that of d-Ala:d-Ser ligases had not been determined. We have investigated the enzymatic kinetics of the d-Ala:d-Ser ligase VanG from Enterococcus faecalis and solved its crystal structure in complex with ADP. The overall structure of VanG is similar to that of VanA but has significant differences mainly in the N-terminal and central domains. Based on reported mutagenesis data and comparison of the VanG and VanA structures, we show that residues Asp-243, Phe-252, and Arg-324 are molecular determinants for d-Ser selectivity. These residues are conserved in both enzymes and explain why VanA also displays d-Ala:d-Ser ligase activity, albeit with low catalytic efficiency in comparison with VanG. These observations suggest that d-Ala:d-Lac and d-Ala:d-Ser enzymes have evolved from a common ancestral d-Ala:d-X ligase. The crystal structure of VanG showed an unusual interaction between two dimers involving residues of the omega loop that are deeply anchored in the active site. We constructed an octapeptide mimicking the omega loop and found that it selectively inhibits VanG and VanA but not Staphylococcus aureus d-Ala:d-Ala ligase. This study provides additional insight into the molecular evolution of d-Ala:d-X ligases and could contribute to the development of new structure-based inhibitors of vancomycin resistance enzymes.     

Amino Acid Isomerization in the Production of l-Phenylalanine from d-Phenylalanine by Bacteria

To establish an advantageous method for the production of l-amino acids, microbial isomerization of d- and dl-amino acids to l-amino acids was studied. Screening experiments on a number of microorganisms showed that cell suspensions of Pseudomonas fluorescens and P. miyamizu were capable of isomerizing d- and dl-phenylalanines to l-phenylalanine. Various conditions suitable for isomerization by these organisms were investigated. Cells grown in a medium containing d-phenylalanine showed highest isomerization activity, and almost completely converted d- or dl-phenylalanine into l-phenylalanine within 24 to 48 hr of incubation. Enzymatic studies on this isomerizing system suggested that the isomerization of d- or dl-phenylalanine is not catalyzed by a single enzyme, “amino acid isomerase,” but the conversion proceeds by a two step system as follows: d-pheylalanine is oxidized to phenylpyruvic acid by d-amino acid oxidase, and the acid is converted to l-phenylalanine by transamination or reductive amination.     

d-Amino Acids Indirectly Inhibit Biofilm Formation in Bacillus subtilis by Interfering with Protein Synthesis

The soil bacterium Bacillus subtilis forms biofilms on surfaces and at air-liquid interfaces. It was previously reported that these biofilms disassemble late in their life cycle and that conditioned medium from late-stage biofilms inhibits biofilm formation. Such medium contained a mixture of d-leucine, d-methionine, d-tryptophan, and d-tyrosine and was reported to inhibit biofilm formation via the incorporation of these d-amino acids into the cell wall. Here, we show that l-amino acids were able to specifically reverse the inhibitory effects of their cognate d-amino acids. We also show that d-amino acids inhibited growth and the expression of biofilm matrix genes at concentrations that inhibit biofilm formation. Finally, we report that the strain routinely used to study biofilm formation has a mutation in the gene (dtd) encoding d-tyrosyl-tRNA deacylase, an enzyme that prevents the misincorporation of d-amino acids into protein in B. subtilis. When we repaired the dtd gene, B. subtilis became resistant to the biofilm-inhibitory effects of d-amino acids without losing the ability to incorporate at least one noncanonical d-amino acid, d-tryptophan, into the peptidoglycan peptide side chain. We conclude that the susceptibility of B. subtilis to the biofilm-inhibitory effects of d-amino acids is largely, if not entirely, due to their toxic effects on protein synthesis.     

FBXO22 Protein Is Required for Optimal Synthesis of the N-Methyl-d-Aspartate (NMDA) Receptor Coagonist d-Serine

d-Serine is a physiological activator of NMDA receptors (NMDARs) in the nervous system that mediates several NMDAR-mediated processes ranging from normal neurotransmission to neurodegeneration. d-Serine is synthesized from l-serine by serine racemase (SR), a brain-enriched enzyme. However, little is known about the regulation of d-serine synthesis. We now demonstrate that the F-box only protein 22 (FBXO22) interacts with SR and is required for optimal d-serine synthesis in cells. Although FBXO22 is classically associated with the ubiquitin system and is recruited to the Skip1-Cul1-F-box E3 complex, SR interacts preferentially with free FBXO22 species. In vivo ubiquitination and SR half-life determination indicate that FBXO22 does not target SR to the proteasome system. FBXO22 primarily affects SR subcellular localization and seems to increase d-serine synthesis by preventing the association of SR to intracellular membranes. Our data highlight an atypical role of FBXO22 in enhancing d-serine synthesis that is unrelated to its classical effects as a component of the ubiquitin-proteasome degradation pathway.     

In Vitro Analysis of the H-Hexose Symporter on the Plasma Membrane of Sugarbeets (Beta vulgaris L.)

The mechanism of hexose transport into plasma membrane vesicles isolated from mature sugarbeet leaves (Beta vulgaris L.) was investigated. The initial rate of glucose uptake into the vesicles was stimulated approximately fivefold by imposing a transmembrane pH gradient (ΔpH), alkaline inside, and approximately fourfold by a negative membrane potential (ΔΨ), generated as a K⁺-diffusion potential, negative inside. The -fold stimulation was directly related to the relative ΔpH or ΔΨ gradient imposed, which were determined by the uptake of acetate or tetraphenylphosphonium, respectively. ΔΨ- and ΔpH-dependent glucose uptake showed saturation kinetics with a K_m of 286 micromolar for glucose. Other hexose molecules (e.g. 2-deoxy-d-glucose, 3-O-methyl-d-glucose, and d-mannose) were also accumulated into plasma membrane vesicles in a ΔpH-dependent manner. Inhibition constants of a number of compounds for glucose uptake were determined. Effective inhibitors of glucose uptake included: 3-O-methyl-d-glucose, 5-thio-d-glucose, d-fructose, d-galactose, and d-mannose, but not 1-O-methyl-d-glucose, d- and l-xylose, l-glucose, d-ribose, and l-sorbose. Under all conditions of proton motive force magnitude and glucose and sucrose concentration tested, there was no effect of sucrose on glucose uptake. Thus, hexose transport on the sugarbeet leaf plasma membrane was by a H⁺-hexose symporter, and the carrier and possibly the energy source were not shared by the plasma membrane H⁺-sucrose symporter.     

DdlN from Vancomycin-Producing Amycolatopsis orientalis C329.2 Is a VanA Homologue with d-Alanyl-d-Lactate Ligase Activity

Vancomycin-resistant enterococci acquire high-level resistance to glycopeptide antibiotics through the synthesis of peptidoglycan terminating in d-alanyl-d-lactate. A key enzyme in this process is a d-alanyl-d-alanine ligase homologue, VanA or VanB, which preferentially catalyzes the synthesis of the depsipeptide d-alanyl-d-lactate. We report the overexpression, purification, and enzymatic characterization of DdlN, a VanA and VanB homologue encoded by a gene of the vancomycin-producing organism Amycolatopsis orientalis C329.2. Evaluation of kinetic parameters for the synthesis of peptides and depsipeptides revealed a close relationship between VanA and DdlN in that depsipeptide formation was kinetically preferred at physiologic pH; however, the DdlN enzyme demonstrated a narrower substrate specificity and commensurately increased affinity for d-lactate in the C-terminal position over VanA. The results of these functional experiments also reinforce the results of previous studies that demonstrated that glycopeptide resistance enzymes from glycopeptide-producing bacteria are potential sources of resistance enzymes in clinically relevant bacteria.The origin of antibiotic resistance determinants is of significant interest for several reasons, including the prediction of the emergence and spread of resistance patterns, the design of new antimicrobial agents, and the identification of potential reservoirs for resistance elements. Antibiotic resistance can occur either through spontaneous mutation in the target or by the acquisition of external genetic elements such as plasmids or transposons which carry resistance genes (7). The origins of these acquired genes are varied, but it has long been recognized that potential reservoirs are antibiotic-producing organisms which naturally harbor antibiotic resistance genes to protect themselves from the actions of toxic compounds (6).High-level resistance to glycopeptide antibiotics such as vancomycin and teicoplanin in vancomycin-resistant enterococci (VRE) is conferred by the presence of three genes, vanH, vanA (or vanB), and vanX, which, along with auxiliary genes necessary for inducible gene expression, are found on transposons integrated into plasmids or the bacterial genome (1, 20). These three genes are essential to resistance and serve to change the C-terminal peptide portion of the peptidoglycan layer from d-alanyl-d-alanine (d-Ala-d-Ala) to d-alanyl-d-lactate (d-Ala-d-Lac). This change results in the loss of a critical hydrogen bond between vancomycin and the d-Ala-d-Ala terminus and in a 1,000-fold decrease in binding affinity between the antibiotic and the peptidoglycan layer, which is the basis for the bactericidal action of this class of compounds (5). The vanH gene encodes a d-lactate dehydrogenase which provides the requisite d-Lac (3, 5), while the vanX gene encodes a highly specific dd-peptidase which cleaves only d-Ala-d-Ala produced endogenously while leaving d-Ala-d-Lac intact (19, 21). The final gene, vanA or vanB, encodes an ATP-dependent d-Ala-d-Lac ligase (4, 8, 10). This enzyme has sequence homology with the chromosomal d-Ala-d-Ala ligases, which are essential for peptidoglycan synthesis but which generally lack the ability to synthesize d-Ala-d-Lac (9).We have recently cloned vanH, vanA, and vanX homologues from two glycopeptide antibiotic-synthesizing organisms: Amycolatopsis orientalis C329.2, which produces vancomycin, and Streptomyces toyocaensis NRRL 15009, which produces {"type":"entrez-nucleotide","attrs":{"text":"A47934","term_id":"2301796","term_text":"A47934"}}A47934 (14). In addition, the vanH-vanA-vanX gene cluster was identified in several other glycopeptide producers. We have also demonstrated that the VanA homologue from S. toyocaensis NRRL 15009 can synthesize d-Ala-d-Lac in vitro and in the glycopeptide-sensitive host Streptomyces lividans (15, 16). We now report the expression of the A. orientalis C329.2 VanA homologue DdlN in Escherichia coli, its purification, and its enzymatic characterization. These data reinforce the striking similarity between vancomycin resistance elements in VRE and glycopeptide-producing organisms and support the possibility of a common origin for these enzymes.

Expression, purification, and specificity of DdlN.

DdlN was overexpressed in E. coli under the control of the bacteriophage T7 promoter. The construct gave good yields of highly purified enzyme following a four-step purification procedure (Table ​(Table1;1; Fig. ​Fig.1).1). Like other dd-ligases, DdlN behaved like a dimer in solution (not shown).

TABLE 1

Purification of DdlN from E. coli BL21 (DE3)/pETDdlN

alpha-l-Arabinofuranosidase from Radish (Raphanus sativus L.) Seeds

An α-l-arabinofuranosidase has been purified 1043-fold from radish (Raphanus sativus L.) seeds. The purified enzyme was a homogeneous glycoprotein consisting of a single polypeptide with an apparent molecular weight of 64,000 and an isoelectric point value of 4.7, as evidenced by denaturing gel electrophoresis and reversed-phase or size-exclusion high-performance liquid chromatography and isoelectric focusing. The enzyme characteristically catalyzes the hydrolysis of p-nitrophenyl α-l-arabinofuranoside and p-nitrophenyl β-d-xylopyranoside in a constant ratio (3:1) of the initial velocities at pH 4.5, whereas the corresponding α-l-arabinopyranoside and β-d-xylofuranoside are unsusceptible. The following evidence was provided to support that a single enzyme with one catalytic site was responsible for the specificity: (a) high purity of the enzyme preparation, (b) an invariable ratio of the activities toward the two substrates throughout the purification steps, (c) a parallelism of the activities in activation with bovine serum albumin and in heat inactivation of the enzyme as well as in the inhibition with heavy metal ions and sugars such as Hg²⁺, Ag⁺, l-arabino-(1→4)-lactone, and d-xylose, and (d) results of the mixed substrate kinetic analysis using the two substrates. The enzyme was shown to split off α-l-arabinofuranosyl residues in sugar beet arabinan, soybean arabinan-4-galactan, and radish seed and leaf arabinogalactan proteins. Arabinose and xylose were released by the action of the enzyme on oat-spelt xylan. Synergistic action of α-l-arabinofuranosidase and β-d-galactosidase on radish seed arabinogalactan protein resulted in the extensive degradation of the carbohydrate moiety.     

Utilization of d-Ribitol by Lactobacillus casei BL23 Requires a Mannose-Type Phosphotransferase System and Three Catabolic Enzymes

Lactobacillus casei strains 64H and BL23, but not ATCC 334, are able to ferment d-ribitol (also called d-adonitol). However, a BL23-derived ptsI mutant lacking enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was not able to utilize this pentitol, suggesting that strain BL23 transports and phosphorylates d-ribitol via a PTS. We identified an 11-kb region in the genome sequence of L. casei strain BL23 (LCABL_29160 to LCABL_29270) which is absent from strain ATCC 334 and which contains the genes for a GlpR/IolR-like repressor, the four components of a mannose-type PTS, and six metabolic enzymes potentially involved in d-ribitol metabolism. Deletion of the gene encoding the EIIB component of the presumed ribitol PTS indeed prevented d-ribitol fermentation. In addition, we overexpressed the six catabolic genes, purified the encoded enzymes, and determined the activities of four of them. They encode a d-ribitol-5-phosphate (d-ribitol-5-P) 2-dehydrogenase, a d-ribulose-5-P 3-epimerase, a d-ribose-5-P isomerase, and a d-xylulose-5-P phosphoketolase. In the first catabolic step, the protein d-ribitol-5-P 2-dehydrogenase uses NAD⁺ to oxidize d-ribitol-5-P formed during PTS-catalyzed transport to d-ribulose-5-P, which, in turn, is converted to d-xylulose-5-P by the enzyme d-ribulose-5-P 3-epimerase. Finally, the resulting d-xylulose-5-P is split by d-xylulose-5-P phosphoketolase in an inorganic phosphate-requiring reaction into acetylphosphate and the glycolytic intermediate d-glyceraldehyde-3-P. The three remaining enzymes, one of which was identified as d-ribose-5-P-isomerase, probably catalyze an alternative ribitol degradation pathway, which might be functional in L. casei strain 64H but not in BL23, because one of the BL23 genes carries a frameshift mutation.     

Substrate Specificity of the H-Sucrose Symporter on the Plasma Membrane of Sugar Beets (Beta vulgaris L.) : Transport of Phenylglucopyranosides

Previous results (TJ Buckhout, Planta [1989] 178: 393-399) indicated that the structural specificity of the H⁺-sucrose symporter on the plasma membrane from sugar beet leaves (Beta vulgaris L.) was specific for the sucrose molecule. To better understand the structural features of the sucrose molecule involved in its recognition by the symport carrier, the inhibitory activity of a variety of phenylhexopyranosides on sucrose uptake was tested. Three competitive inhibitors of sucrose uptake were found, phenyl-α-d-glucopyranoside, phenyl-α-d-thioglucopyranoside, and phenyl-α-d-4-deoxythioglucopyranoside (PDTGP; K_i = 67, 180, and 327 micromolar, respectively). The K_m for sucrose uptake was approximately 500 micromolar. Like sucrose, phenyl-α-d-thioglucopyranoside and to a lesser extent, PDTGP induced alkalization of the external medium, which indicated that these derivatives bound to and were transported by the sucrose symporter. Phenyl-α-d-3-deoxy-3-fluorothioglucopyranoside, phenyl-α-d-4-deoxy-4-fluorothioglucopyranoside, and phenyl-α-d-thioallopyranoside only weakly but competively inhibited sucrose uptake with K_i values ranging from 600 to 800 micromolar, and phenyl-α-d-thiomannopyranoside, phenyl-β-d-glucopyranoside, and phenylethyl-β-d-thiogalactopyranoside did not inhibit sucrose uptake. Thus, the hydroxyl groups of the fructose portion of sucrose were not involved in a specific interaction with the carrier protein because phenyl and thiophenyl derivatives of glucose inhibited sucrose uptake and, in the case of phenyl-α-d-thioglucopyranoside and PDTGP, were transported.     

Partial Purification and Properties of l-Glutamine d-Fructose 6-Phosphate Amidotransferase from Phaseolus aureus

l-Glutamine d-fructose 6-phosphate amidotransferase (EC 2.6.1.16) was extracted and purified 600-fold by acetone fractionation and diethylaminoethyl cellulose column chromatography from mung bean seeds (Phaseolus aureus). The partially purified enzyme was highly specific for l-glutamine as an amide nitrogen donor, and l-asparagine could not replace it. The enzyme showed a pH optimum in the range of 6.2 to 6.7 in phosphate buffer. Km values of 3.8 mm and 0.5 mm were obtained for d-fructose 6-phosphate and l-glutamine, respectively. The enzyme was competitively inhibited with respect to d-fructose 6-phosphate by uridine diphosphate-N-acetyl-d-glucosamine which had a Ki value of 13 μm. Upon removal of l-glutamine and its replacement by d-fructose 6-phosphate and storage over liquid nitrogen, the enzyme was completely desensitized to inhibition by uridine diphosphate-N-acetyl-d-glucosamine. This indicates that the inhibitor site is distinct from the catalytic site and that uridine diphosphate-N-acetyl-d-glucosamine acts as a feedback inhibitor of the enzyme.