Richard B. Herbert

Purification and properties of the Escherichia coli nucleoside transporter NupG, a paradigm for a major facilitator transporter sub-family

NupG from Escherichia coli is the archetype of a family of nucleoside transporters found in several eubacterial groups and has distant homologues in eukaryotes, including man. To facilitate investigation of its molecular mechanism, we developed methods for expressing an oligohistidine-tagged form of NupG both at high levels (>20% of the inner membrane protein) in E. coli and in Xenopus laevis oocytes. In E. coli recombinant NupG transported purine (adenosine) and pyrimidine (uridine) nucleosides with apparent Km values of ∼20–30 μM and transport was energized primarily by the membrane potential component of the proton motive force. Competition experiments in E. coli and measurements of uptake in oocytes confirmed that NupG was a broad-specificity transporter of purine and pyrimidine nucleosides. Importantly, using high-level expression in E. coli and magic-angle spinning cross-polarization solid-state nuclear magnetic resonance, we have for the first time been able directly to measure the binding of the permeant ([1′-13C]uridine) to the protein and to assess its relative mobility within the binding site, under non-energized conditions. Purification of over-expressed NupG to near homogeneity by metal chelate affinity chromatography, with retention of transport function in reconstitution assays, was also achieved. Fourier transform infrared and circular dichroism spectroscopy provided further evidence that the purified protein retained its 3D conformation and was predominantly α-helical in nature, consistent with a proposed structure containing 12 transmembrane helices. These findings open the way to elucidating the molecular mechanism of transport in this key family of membrane transporters.

Solid-state NMR spectroscopy detects interactions between tryptophan residues of the E. coli sugar transporter GalP and the alpha-anomer of the D-glucose substrate.

An experimental approach is described in which high resolution 13C solid-state NMR (SSNMR) spectroscopy has been used to detect interactions between specific residues of membrane-embedded transport proteins and weakly binding noncovalent ligands. This procedure has provided insight into the binding site for the substrate D-glucose in the Escherichia coli sugar transport protein GalP. Cross-polarization magic-angle spinning (CP-MAS) SSNMR spectra of GalP in its natural membrane at 4 degrees C indicated that the alpha- and beta-anomers of D-[1-(13)C]glucose were bound by GalP with equal affinity and underwent fast exchange between the free and bound environments. Further experiments confirmed that by lowering the measurement temperature to -10 degrees C, peaks could be detected selectively from the substrate when restrained within the binding site. Dipolar-assisted rotational resonance (DARR) SSNMR experiments at -10 degrees C showed a selective interaction between the alpha-anomer of D-[1-(13)C]glucose and 13C-labels within [13C]tryptophan-labeled GalP, which places the carbon atom at C-1 in the alpha-anomer of D-glucose to within 6 A of the carbonyl carbon of one or more tryptophan residues in the protein. No interaction was detected for the beta-isomer. The role of tryptophan residues in substrate binding was investigated further in CP-MAS experiments to detect D-[1-(13)C]glucose binding to the GalP mutants W371F and W395F before and after the addition of the inhibitor forskolin. The results suggest that both mutants bind D-glucose with similar affinities, but have different affinities for forskolin. This work highlights a useful general experimental strategy for probing the binding sites of membrane proteins, using methodology which overcomes the problems associated with the unfavorable dynamics of weak ligands.

The improved synthesis of β-D-glucuronides using TEMPO and t-butyl hypochlorite

Abstract TEMPO/t-BuOCl is used to oxidise β-D-glucosides to β-D-glucuronides in high yield as a pivotal step in the preparation of labelled glucuronides from labelled glucose samples.

The Biosynthesis of the phenethylisoquinoline alkaloid colchicine. Early and intermediate stages

Abstract Experiments with 14 C- and 3 H-labelled compounds in Colchicum byzantinum and C. autumnale show that in the early stages of biosynthesis the major pathway to colchicine (7) and demecolcine (6) is (13)→(15)→(16)→(19). Condensation of the aldehyde (19) with dopamine affords a set of phenethylisoquinolines which are precursors for the alkaloids (6) and (7) and (21) is identified as the first of these followed by (22) and then (1); (39) is by contrast also an exellent alkaloid precursor; it is deduced that neither of two phenethylisoquinolines [as (37) and (38)] which contain a side-chain double bond are involved in alkaloid biosynthesis

The biosynthesis of the β-carboline alkaloids, harman and eleagnine

1-Methyl-1,2,3,4-tetrahydro-β-carboline-1-carboxylic acid (3) is shown to be an intact precursor for harman (5) in Passiflora edulis and for eleagnine (6) in Eleagnus angustifolia; it is a natural constituent of both these plants; (3) is at least an 8-fold better precursor for (5) than is N-acetyltryptamine (11).

Use of iron (III) chloride in the efficient preparation of the alkaloid kreysigine by oxidative coupling

Abstract Reaction of the isoquinoline (1) with iron (III) chloride in dichloromethane followed by reductive treatment with methanol gives kreysigine (4) in 71% yield; (2) gives (4) in lower yield as does treatment with water instead of methanol; (8) is the product of a thallium (III) oxidation.

Stereospecific lysis of a range of β-hydroxy-α-amino acids catalysed by a novel aldolase from Streptomyces amakusaensis

Cell-free preparations of Streptomyces amakusaensis contain a novel aldolase which catalyses the conversion of β-hydroxy-α-amino acids (as 2) into the corresponding aldehyde plus glycine; the aldolase is stable, shows broad substrate tolerance, is highly selective for threo stereochemistry and, where examined, is Stereospecific for the (2S,3R) configuration.

The biosynthesis of the antibiotic obafluorin from p-aminophenylalanine in Pseudomonas fluorescens

Abstract The separate units which are used to construct the unique β-lactone antibiotic obafluorin (1) in Pseudomonas fluorescens are defined by the results of [U-13C]glucose incorporation. A key intermediate in the biosynthesis of (1) is established to be L-p-aminophenylalanine (7); L-p-nitrophenylalanine (8) is a relatively insignificant precursor. Similar results were obtained for p-nitrophenylacetic acid (9) which is also a metabolite of Ps. fluorescens. L-phenylalanine is an insignificant precursor for obafluorin (1).

Do mammals make their own morphine

We are such stuff As dreams are made on, and our little life Is rounded with a sleep. Shakespeare, The Tempest, act 4, scene 1

The biosynthesis of nikkomycin X from histidine in Streptomyces tendae

Abstract L-Histidine (4) is the source in Streptomyces tendae for the imidazolone moiety (1) of nikkomycin X (2). It is deduced to be incorporated with stereospecific retention of the β - pro - S proton. Histamine (5), 4-formylimidazole (7) and 4-hydroxymethylimidazole (8) are not significant precursors. Urocanic acid (6) is a less efficient precursor than (4). Aldolase activity is identified in S.tendae which catalyses cleavage of threo -(4-hydroxyphenyl)serine (9) but not significantly of threo - or erythro - β -hydroxyhistidine.