Growth of Rhodopseudomonas capsulata on l- and d-malic acid

d-malate replaced l-malate in supporting both photosynthetic (anaerobic, light) and heterotrophic (aerobic, dark) growth of Rhodopseudomonas capsulata. Growth rates and cell yields were nearly equivalent with both enantiomorphs. Addition of glucose to malate culture media increased the growth rate and doubled the cell yield of heterotrophic cultures, but had little effect on photosynthetic cultures. Aerobically-grown cells showed a higher level of substrate-dependent oxygen uptake with l-malate than with d-malate. This preference for l-malate occured even in cells grown on d-malate. No malic racemase activity was detected in extracts of heterotrophically- or photosynthetically-grown cells.     

Simultaneous determination of d- and l-thyroxine in human serum by liquid chromatography with electrochemical detection

A method for the determination of d- and l-thyroxine in human serum is described. The method involves extraction of thyroxine from serum and the separation of thyroxine enantiomers on a reversed-phase, high-performance liquid chromatographic column by use of a chiral eluent containing l-proline and cupric sulfate. Satisfactory resolution of the enantiomers of thyroxine, triiodothyronine, and reverse triiodothyronine can be achieved in 12 min and, employing amperometric detection to monitor the separation, the detection limit for serum thyroxine is in the range of 1–3 ng per injected sample.     

Phenylalanine ammonia-lyase: Mirror-image packing of d- and l-phenylalanine and d- and l-transition state analogs into the active site

The binding of substrate and product analogs to phenylalanine ammonia-lyase (EC 4.3.1.5) from maize has been studied by a protection method. The ligand dissociation constants, K_L, were estimated from the variation with [L] of the pseudo-first-order rate constants for enzyme inactivation by nitromethane. The phenylalanine analogs d- and l-2-aminooxy-3-phenylpropionic acid showed K_L, values over 20,000-fold lower than the K_m for l-phenylalanine. From these and other K_L values it is deduced that when the enzyme binds l-phenylalanine the structural free energy stored in the protein is higher than when it binds the superinhibitors. Models for binding d- and l-phenylalanine and the superinhibitors are described. The enantiomeric pairs are considered to have similar K_L values because they pack into the active site in a mirror-image relationship. If the elimination reaction approximates to the least-motion course deduced on stereoelectronic grounds, the mirror-image packing of the superinhibitors into the active site mimics the conformation inferred for a transition state in the elimination. It appears, therefore, that structural changes take place in the enzyme as the transition state conformation is approached causing stored free energy to be released. This lowers the activation free energy for the elimination reaction and accounts for the strong binding by the above analogs.     

l-Xylose and l-lyxose production from xylitol using Alcaligenes 701B strain and immobilized l-rhamnose isomerase enzyme

Xylitol was used as a raw material for production of l-xylose and l-lyxose using Alcaligenes 701B strain and immobilized l-rhamnose isomerase enzyme. Alcaligenes 701B converted xylitol to l-xylulose with a yield of 34% in the bioreactor. l-Xylulose was converted to l-xylose and l-lyxose using immobilized l-rhamnose isomerase enzyme. The final equilibrium between l-xylulose, l-xylose and l-lyxose was 53:26:21. The enzyme assays indicated that Alcaligenes 701B strain has an NAD-dependent xylitol dehydrogenase enzyme responsible for l-xylulose production. Furthermore, NAD(P)H-dependent l-xylulose reductase enzyme was active during conversion of xylitol to l-xylulose. The highest l-xylulose production rate corresponded with the highest growth rate. The Alcaligenes 701B strain used d-xylose for biomass growth, but xylitol was used only for l-xylulose production during conversion phase.     

Modification of optimal pH in l-arabinose isomerase from Geobacillus stearothermophilus for d-galactose isomerization

l-Arabinose isomerase from Geobacillus stearothermophilus (GSAI; EC 5.3.1.4) has been genetically evolved to increase the reaction rate toward d-galactose, which is not a natural substrate. To change the optimal pH of GSAI for d-galactose isomerization (pH optimum at 8.5), we investigated the single point mutations influencing the activity based on the sequences of the previously evolved enzymes. Among the seven point mutations found in the evolved enzymes, mutations at Val⁴⁰⁸ and Asn⁴⁷⁵ were determined to be highly influential mutation points for d-galactose isomerization activity. A random mutation was introduced into sites Val⁴⁰⁸ and Asn⁴⁷⁵ (X408V and X475N), and candidates were screened based on non-optimal pH conditions. Among the mutations of X408V and X475N, mutations of Q408V and R408V were selected. The optimal pH of the both mutations Q408V and R408V was shifted to pH 7.5. At the shifted optimal pH, the d-galactose isomerization activities of Q408V and R408V were 60 and 30% higher than that of the wild type at pH 8.5, respectively.     

Enzymatic epimerization of d-erythro-dihydroneopterin triphosphate to l-threo-dihydroneopterin triphosphate

An enzyme has been discovered in Escherichia coli that catalyzes the conversion of the triphosphate ester of 2-amino-4-hydroxy-6-(d-erythro-1′,2′,3′-trihydroxypropyl)-7,8-dihydropteridine, (i.e. d-erythro-dihydroneopterin triphosphate) to an epimer of this compound, l-threo-dihydroneopterin triphophate. The enzyme, which is here named “d-erythro-dihydroneopterin triphosphate 2′-epimerase,” needs a divalent cation (Mg²⁺ or Mn²⁺ is most effective) for maximal activity. Its molecular weight is estimated at 87 000–89 000. Little or no activity can be detected if either the monophosphate or the phosphate-free form of the substrate is incubated with the enzyme. Evidence is presented to establish that all three phosphate residues of the substrate are retained in the product and that the product is of the l-threo configuration.     

The effects of d- and l-glyceraldehyde on glucose oxidation, insulin secretion and insulin biosynthesis by pancreatic islets of the rat

d-glyceraldehyde stimulated insulin secretion from isolated rat pancreatic islets in static incubation and perifusion systems. At low concentrations (2–4 mM) d-glyceraldehyde was a more potent secretagogue than glucose. The insulinotropic action of 15 mM d-glyceraldehyde was not affected by d-mannoheptulose, was potentiated by cytochalasin B (5 μg/ml) and theophylline (4 mM), and was inhibited by both adrenalin (2 μM) and somatostatin (10 μg/ml). D-glyceraldehyde at a concentration of 1.5 mM produced a 10-fold increase of l-[4,5-³ H]leucine incorporation into proinsulin and insulin without a significant increase into other islet proteins. Glucose at 1.5 mM did not stimulate proinsulin biosynthesis. d-Glyceraldehyde at concentrations higher than 1.5 mM, in marked contrast to glucose, progressively inhibited incorporation of labelled leucine into proinsulin + insulin and other islet proteins. d-glyceraldehyde also inhibited the oxidation of glucose. l-Glyceraldehyde did not stimulate proinsulin biosynthesis and had less effect than the d-isomer on insulin release and glucose oxidation. The results strongly suggest that metabolites below d-glyceraldehyde-3-P are signals for insulin biosynthesisand release. Interaction of d-glyceraldehyde with a “membrane receptor” cannot, however, be excluded with certainty.     

d-Glucose- and d-mannose-based antimetabolites. Part 2. Facile synthesis of 2-deoxy-2-halo-d-glucoses and -d-mannoses

Modified d-glucose and d-mannose analogs are potentially clinically useful metabolic inhibitors. Biological evaluation of 2-deoxy-2-halo analogs has been impaired by limited availability and lack of efficient methods for their preparation. We have developed practical synthetic approaches to 2-deoxy-2-fluoro-, 2-chloro-2-deoxy-, 2-bromo-2-deoxy-, and 2-deoxy-2-iodo derivatives of d-glucose and d-mannose that exploit electrophilic addition reactions to a commercially available 3,4,6-tri-O-acetyl-d-glucal.     

Construction and co-expression of polycistronic plasmid encoding d-hydantoinase and d-carbamoylase for the production of d-amino acids

d-Hydantoinase and d-carbamoylase genes from Agrobacterium radiobacter TH572 were cloned by polymerase chain reaction (PCR). The plasmid pUCCH3 with a polycistronic structure that is controlled by the native hydantoinase promoter was constructed to co-express the two genes and transformed into Escherichia coli strain JM105. To obtain the highest level of expression of the d-carbamoylase and avoid intermediate accumulation, the d-carbamoylase gene was cloned closer to the promoter and the RBS region in the upstream of it was optimized. This resulted in high active expression of soluble d-hydantoinase and d-carbamoylase that is obtained without any inducer. Thus, by the constitutive recombinant JM105/pUCCH3, d-p-hydroxyphenylglycine (d-HPG) was obtained directly with 95.2% production yield and 96.3% conversion yield.     

A high-throughput assay for screening l- or d-amino acid specific aminotransferase mutant libraries

Aminotransferases are pyridoxal phosphate-dependent enzymes whose potential for the biocatalytic production of enantiopure amino acids is increasingly recognized. Because of this, there is a growing interest in engineering them to alter their substrate specificity and to increase their catalytic activity. Here, we report the development of a high-throughput assay for screening α-ketoglutarate-dependent aminotransferase mutant libraries. To achieve this, we exploited the l-glutamate dehydrogenase coupled assay that has previously been shown to allow for aminotransferase activity to be monitored in vitro. We adapted this assay to allow screening of mutant libraries of either l- or d-amino acid specific aminotransferases in a continuous fashion. This assay requiring clarified cell lysates is reproducible, rapid, and sensitive because it allowed for the identification of a catalytically active mutant of Bacillus sp. YM-1 d-amino acid aminotransferase displaying a decrease in k_cat/K_M of more than two orders of magnitude. In addition, this assay allowed us to discover a mutant of Escherichia coli branched-chain amino acid aminotransferase, F36W, which is approximately 60-fold more specific toward the natural substrate l-leucine than l-phenylalanine as compared with wild type. This result demonstrates the potential of our assay for the discovery of mutant aminotransferases displaying altered substrate specificity, an important goal of enzyme engineering.     

Efficient synthesis of d-xylo and d-ribo-phytosphingosines from methyl 2-amino-2-deoxy-β-d-hexopyranosides

A general and flexible synthetic approach to biologically important 5,6-unsaturated C₁₈-phytosphingosines was developed via olefin cross-metathesis employing truncated C₆-phytosphingosines as the key intermediates. These were efficiently prepared in high yields by zinc-mediated reductive opening of methyl 2-amino-2-deoxy-β-hexopyranosides.

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d-Amino acid production by E. coli co-expressed three genes encoding hydantoin racemase, d-hydantoinase and N-carbamoyl-d-amino acid amidohydrolase

Three genes respectively encoding d-specific hydantoinase (DHHase), N-carbamoyl-d-amino acid amidohydrolase (DCHase) and hydantoin racemase (HRase) were co-expressed in E. coli in a system designed for the efficient enzymatic production of d-amino acids via a combination of hydantoin hydrolysis and hydantoin racemization. With the use of whole cells, the d-forms of eight amino acids – d-phenylalanine, d-tyrosine, d-tryptophan, O-benzyl-d-serine, d-valine, d-norvaline, d-leucine and d-norleucine – were efficiently converted from the corresponding dl-5-monosubtituted hydantoin compounds.     

Overproduction of d-xylose isomerase in Escherichia coli by cloning the d-xylose isomerase gene

The Escherichia coli d-xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) gene, xylA, has been cloned on various E. coli plasmids. However, it has been found that high levels of overproduction of the d-xylose isomerase, the protein product of the xylA gene, cannot be accomplished by cloning the intact gene on high copy-number plasmids alone. This is believed to be due to the fact that the expression of the gene through its natural promoter is highly regulated in E. coli. In order to overcome this, the xylA structural gene has been fused with other strong promoters such as tac and lac, resulting in the construction of a number of fused genes. Analysis of the E. coli transformants containing the fused genes, cloned on high copy-number plasmids, indicated that a 20-fold overproduction of the enzyme can now be obtained. It is expected that overproduction of the enzyme in E. coli can still be substantially improved through additional manipulation with recombinant DNA techniques.     

Transport of l-ascorbic acid and dehydro-l-ascorbic acid across renal cortical basolateral membrane vesicles

The uptake of l-ascorbic acid and dehydro-l-ascorbic acid into renal cortical basolateral membrane vesicles has been characterized. The uptake systems for both solutes demonstrate saturation kinetics. The presence of structural analogs of l-ascorbic acid and dehydro-l-ascorbic acid results in cis-inhibition and trans-stimulation. Uptake of each substrate is Na⁺-independent, proceeding to an endpoint of substrate equilibrium across the vesicular membrane. The transport mechanism(s) for l-ascorbic acid and dehydro-l-ascorbic acid appears to be facilitated diffusion.     

Synthesis and antitumor activity of new d-seco and d-homo androstane derivatives

Starting from 3β-hydroxy-17-oxo-16,17-secoandrost-5-ene-16-nitrile (1), the new 16,17-secoandrostane derivatives 4–9 were synthesized. On the other hand, 3β-hydroxy-17-oxa-d-homoandrost-5-ene-16-one (10) yielded the new d-homo derivatives 12, 13 and 15. In vitro antiproliferative activity of selected compounds against three tumor cell lines (human breast adenocarcinoma ER+, MCF-7, human breast adenocarcinoma ER−, MDA-MB-231, prostate cancer AR−, PC-3, and normal fetal lung fibroblasts, MRC-5) was evaluated. Compounds 3 and 12 showed strong antiproliferative activity against PC-3 cells, the IC₅₀ values being 2 μM and 0.55 μM, respectively. Compounds 6 (10 μM) and 14 (9 μM) showed moderate activity against MDA-MB-231 cells. The synthesized compounds 1–3, 5–8, 10 and 12–15 were not toxic to normal fetal lung fibroblasts cells, MRC-5.     

Mechanism of ion transport through lipid bilayer-membranes mediated by peptide cyclo-(d-Val-l-Pro-l-Val-d-Pro)3

The cyclic dodecapeptide PV, cyclo-(d-Val-l-Pro-l-Val-d-Pro)₃, a structural analogue of the ion-carier valinomycin, increase the cation permeability of lipid bilayer membranes. This paper reports the results of two types of relaxation experiments, namely relaxation of the membrane current after a voltage jump and decay of the membrane voltage after a charge pulse in lipid bilayer membranes exposed to PV. From the relaxation data, the rate constant for the translocation of the ion carrier complex across the membrane, as well as the partition coefficient of the complex between water and membrane solution interface were computed and found to be about one order of magnitude less than the comparable values for valinomycin (Val). Furthermore, the dependence of the initial membrane conductivity on ion concentration was used to evaluate the equilibrium constant, K, of complexation between PV and some monovalent cations in water. The values of K yield the following selectivity sequence of PV: Na⁺ < NH₄⁺ < K⁺ < Cs⁺ < Rb⁺. These and earlier results are consistent with the idea that PV promotes cation movement across membranes by the solution complexation mechanism which involves complexation between ion and carrier in the aqueous phase and transport of the carrier across the membrane. In the particular form of the solution complexation mechanism operating here, the PV present in the PV-cation complex carrying charge across the membrane derives from the side from which the current is flowing (cis-mechanism). As shown previously, valinomycin, in contrast to PV, acts by an interfacial complexation mechanism in which the Val in the Val-cation complex derives from the side toward which current is flowing (trans-mechanims). The comparison of the kinetic properties of these two closely related compounds yields interesting insights into the relationship between chemical structure and function of ion carriers.     

Hydroxylation of l-proline to cis-3-hydroxy-l-proline by recombinant Escherichia coli expressing a synthetic l-proline-3-hydroxylase gene

A synthetic gene encoding a Streptomyces l-proline-3-hydroxylase was constructed and used to produce the hydroxylase protein in recombinant Escherichia coli. A fermentation process for growth of this recombinant E. coli for enzyme production was scaled-up to 250 L. A biotransformation process was developed using cell suspensions of the recombinant E. coli and subsequently scaled-up to 10 L for conversion of l-proline to cis-3-hydroxy-l-proline. A reaction yield of 85 M% and d.e. of 99.9% was obtained for cis-3-hydroxy-l-proline.     

An approach to stereoselective preparation of 3-C-glycosylated d- and l-glucals

An approach to stereoselective synthesis of α- or β-3-C-glycosylated l- or d-1,2-glucals starting from the corresponding α- or β-glycopyranosylethanals is described. The key step of the approach is the stereoselective cycloaddition of chiral vinyl ethers derived from both enantiomers of mandelic acid. The preparation of 1,5-anhydro-4,6-di-O-benzyl-2,3-dideoxy-3-C-[(2,3,4,6-tetra-O-benzyl-β-d-glucopyranosyl)methyl]-l-arabino-hex-1-enitol, 1,5-anhydro-4,6-di-O-benzyl-2,3-dideoxy-3-C-[(2,3,4,6-tetra-O-benzyl-β-d-glucopyranosyl)methyl]-d-arabino-hex-1-enitol, and 1,5-anhydro-4,6-di-O-benzyl-2,3-dideoxy-3-C-[(2,3,4-tri-O-benzyl-α-l-fucopyranosyl)methyl]-d-arabino-hex-1-enitol serves as an example of this approach.     

Guinea pig serum l-asparaginase: Purification, and immunological relationship to liver l-asparaginase and serum l-asparaginases in other mammals

l-asparaginase, an enzyme used in the treatment of acute lymphocytic leukemia, is found in the serum of only a few mammalian groups, including the guinea pig and its close relatives in the superfamily Cavioidea. This report describes the purification and characterization of l-asparaginase from guinea pig serum. Antiserum against the purified enzyme cross-reacted with sera from other Cavioidean species but not with mouse serum. Relatively weak cross-reaction with unpurified l-asparaginase in guinea pig liver indicates a significant degree of evolutionary divergence.