Jennifer L. Beck

Properties of a purple phosphatase from red kidney bean: a zinc-iron metalloenzyme

A purple phosphatase from the red kidney bean Phaseolus vulgaris has been purified to homogeneity and characterized. It resembles sweet potato purple acid phosphatase in being a dimer of approx. 130 kDa and in its amino acid composition and visible absorption spectrum. The red kidney bean enzyme contains one atom of iron and one atom of zinc per subunit, whereas the sweet potato enzyme is reported to contain manganese. The visible absorption spectrum shows a λmax at 560 nm with e{lunate}560 (per iron) = 3360 M- · cm-1, and is destroyed by dithionite treatment. Red kidney bean phosphatase shows a marked preference for ATP as substrate over p-nitrophenyl phosphate and ADP. Stable esters such as AMP and β-glycerophosphate are very poor substrates. The enzyme is compared with other purple phosphatases from plant and animal sources.

Antibacterial activity of berberine-NorA pump inhibitor hybrids with a methylene ether linking group

Conjugation of the NorA substrate berberine and the NorA inhibitor 5-nitro-2-phenyl-1H-indole via a methylene ether linking group gave the 13-substituted berberine-NorA inhibitor hybrid, 3. A series of simpler arylmethyl ether hybrid structures were also synthesized. The hybrid 3 showed excellent antibacterial activity (MIC Staphylococcus aureus, 1.7 microM), which was over 382-fold more active than the parent antibacterial berberine, against this bacterium. This compound was also shown to block the NorA efflux pump in S. aureus.

Use of electrospray ionization mass spectrometry to study binding interactions between a replication terminator protein and DNA

Tus protein binds tightly to specific DNA sequences (Ter) on the Escherichia coli chromosome halting replication. We report here conditions for detecting the 1 : 1 Tus–Ter complex by electrospray ionization mass spectrometry (ESI‐MS). ESI mass spectra of a mixture of Tus and nonspecific DNA showed ions predominantly from uncomplexed Tus protein, indicating that the Tus–Ter complex observed in the gas phase was the result of a specific interaction rather than nonspecific associations in the ionization source. The Tus–Ter complex was very stable using a spray solvent of 10 mM ammonium acetate at pH 8.0, and initial attempts to distinguish binding affinities of Tus and mutant Tus proteins for Ter DNA were unsuccessful. Increasing the ammonium acetate concentration in the electrospray solvent (800 mM at pH 8.0) increased the dissociation constants sufficiently such that relative orders of binding affinity for Tus and various mutant Tus proteins for various DNA sequences could be determined. These were in agreement with the dissociation constants determined in solution studies. A dissociation constant of 700 × 10−9 M for the binding of the mutant Tus protein A173T (where residue 173 is changed from alanine to threonine) to Ter DNA was estimated, compared with a value of ≤2 × 10−9 M for Tus where A173 was unchanged. This is the first example in which ESI‐MS has been used to compare binding affinities of a DNA‐binding protein with mutant proteins for specific DNA recognition sequences. It was also possible to estimate the strength of the interaction between Tus and a DNA sequence (TerH) that had been identified by database searching.

Translational incorporation of L-3,4-dihydroxyphenylalanine into proteins

An Escherichia coli cell‐free transcription/translation system was used to explore the high‐level incorporation of l‐3,4‐dihydroxyphenylalanine (DOPA) into proteins by replacing tyrosine with DOPA in the reaction mixtures. ESI‐MS showed specific incorporation of DOPA in place of tyrosine. More than 90% DOPA incorporation at each tyrosine site was achieved, allowing the recording of clean 15N‐HSQC NMR spectra. A redox‐staining method specific for DOPA was shown to provide a sensitive and generally applicable method for assessing the cell‐free production of proteins. Of four proteins produced in soluble form in the presence of tyrosine, two resulted in insoluble aggregates in the presence of high levels of DOPA. DOPA has been found in human proteins, often in association with various disease states that implicate protein aggregation and/or misfolding. Our results suggest that misfolded and aggregated proteins may result, in principle, from ribosome‐mediated misincorporation of intracellular DOPA accumulated due to oxidative stress. High‐yield cell‐free protein expression systems are uniquely suited to obtain rapid information on solubility and aggregation of nascent polypeptide chains.

Telomeric G-quadruplexes are a substrate and site of localization for human telomerase

It has been hypothesized that G-quadruplexes can sequester the 3′ end of the telomere and prevent it from being extended by telomerase. Here we purify and characterize stable, conformationally homogenous human telomeric G-quadruplexes, and demonstrate that human telomerase is able to extend parallel, intermolecular conformations in vitro. These G-quadruplexes align correctly with the RNA template of telomerase, demonstrating that at least partial G-quadruplex resolution is required. A highly purified preparation of human telomerase retains this extension ability, establishing that the core telomerase enzyme complex is sufficient for partial G-quadruplex resolution and extension. The parallel-specific G-quadruplex ligand N-methyl mesoporphyrin IX (NMM) causes an increase in telomeric G-quadruplexes, and we show that telomerase colocalizes with a subset of telomeric G-quadruplexes in vivo. The ability of telomerase to partially unwind, extend and localize to these structures implies that parallel telomeric G-quadruplexes may play an important biological role.

Enzymatically active zinc, copper and mercury derivatives of the one-iron form of pig allantoic fluid acid phosphatase

Derivatives of the violet, iron-containing acid phosphatase of pig allantoic fluid have been prepared in which one of the two iron atoms present in the native enzyme has been replaced by zinc, copper or mercury. The derivatives so formed are enzymatically active: the Zn-Fe, Cu-Fe and Hg-Fe enzymes have specific activities of about 80%, 25% and 17% respectively, of the maximum specific activity of the Fe-Fe enzyme in the standard assay at pH 4.9 with p-nitrophenyl phosphate as substrate. In contrast to the Fe-Fe enzyme, the mixed metal derivatives are not rapidly inactivated by H2O2. Visible absorption spectra of the derivatives confirm that all of the visible absorption of the Fe-Fe enzyme is due to one of the iron atoms. Attempts to prepare an active Cu-Cu enzyme were unsuccessful.

A direct proofreader-clamp interaction stabilizes the Pol III replicase in the polymerization mode

Processive DNA synthesis by the αεθ core of the Escherichia coli Pol III replicase requires it to be bound to the β2 clamp via a site in the α polymerase subunit. How the ε proofreading exonuclease subunit influences DNA synthesis by α was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non‐proofreading activity of ε. Combination of mutagenesis with biophysical studies and single‐molecule leading‐strand replication assays traced this activity to a novel β‐binding site in ε that, in conjunction with the site in α, maintains a closed state of the αεθ–β2 replicase in the polymerization mode of DNA synthesis. The ε–β interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein–protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states.

Derivatives of the purple phosphatase from red kidney bean: Replacement of zinc with other divalent metal ions

Abstract Apoenzyme, containing ⩽0.1 zinc atoms and ⩽0.2 Fe atoms per subunit and with ⩽3% of the phosphatase activity, has been prepared from native red kidney bean purple phosphatase. Treatment of this apoenzyme with Fe 3+ or Zn 2+ separately gave very little recovery of activity, whereas treatment with both Fe 3+ and Zn 2+ resulted in complete restoration of activity, indicating that both metal ions are essential. ZnFe enzyme with close to one iron and one zinc atom per subunit has been reconstituted by this procedure. Essentially full reactivation was also achieved by addition of Fe 3+ together with Fe 2+ or Co 2+ to the apoenzyme; Fe 3+ and Cd 2+ gave 27% restoration of activity, whereas Fe 3+ with Mn 2+ , Cu 2+ , Ni 2+ or Hg 2+ gave little or no increase in activity. Kinetic parameters for the hydrolysis of p -nitrophenyl phosphate and ATP by the FeFe derivative are reported.

Improving recombinant Rubisco biogenesis, plant photosynthesis and growth by coexpressing its ancillary RAF1 chaperone

Significance Using a translational photosynthesis approach, we successfully increased CO2-assimilation in leaf chloroplasts of the model plant tobacco. Phylogenetic analysis revealed parallel evolutionary linkages between the large (L-) subunit of the CO2-fixing enzyme Rubisco and its molecular chaperone Rubisco accumulation factor 1 (RAF1). We experimentally tested and exploited this correlation using plastome transformation, producing plants that demonstrated the role of RAF1 in L-subunit assembly and resolve the RAF1 quaternary structure as a dimer. We show the increase in Rubisco biogenesis translated to improvements in leaf photosynthesis and growth of the plants. The outcomes have application to the growing interest into identifying and implementing strategies to supercharge photosynthesis to improve crop productivity and stem global food-security concerns. Enabling improvements to crop yield and resource use by enhancing the catalysis of the photosynthetic CO2-fixing enzyme Rubisco has been a longstanding challenge. Efforts toward realization of this goal have been greatly assisted by advances in understanding the complexities of Rubisco’s biogenesis in plastids and the development of tailored chloroplast transformation tools. Here we generate transplastomic tobacco genotypes expressing Arabidopsis Rubisco large subunits (AtL), both on their own (producing tobAtL plants) and with a cognate Rubisco accumulation factor 1 (AtRAF1) chaperone (producing tobAtL-R1 plants) that has undergone parallel functional coevolution with AtL. We show AtRAF1 assembles as a dimer and is produced in tobAtL-R1 and Arabidopsis leaves at 10–15 nmol AtRAF1 monomers per square meter. Consistent with a postchaperonin large (L)-subunit assembly role, the AtRAF1 facilitated two to threefold improvements in the amount and biogenesis rate of hybrid L8AS8t Rubisco [comprising AtL and tobacco small (S) subunits] in tobAtL-R1 leaves compared with tobAtL, despite >threefold lower steady-state Rubisco mRNA levels in tobAtL-R1. Accompanying twofold increases in photosynthetic CO2-assimilation rate and plant growth were measured for tobAtL-R1 lines. These findings highlight the importance of ancillary protein complementarity during Rubisco biogenesis in plastids, the possible constraints this has imposed on Rubisco adaptive evolution, and the likely need for such interaction specificity to be considered when optimizing recombinant Rubisco bioengineering in plants.

A mass spectrometric investigation of the binding of gold antiarthritic agents and the metabolite [Au(CN)2]− to human serum albumin

Electrospray ionisation (ESI) mass spectrometry was used to examine the reactions of the clinically used antiarthritic agent [Au(S2O3)2]3−, and AuPEt3Cl, a derivative of another clinically used agent auranofin, with human serum albumin (HSA) obtained from a human volunteer. Both compounds reacted readily with HSA to form complexes containing one or more covalently attached gold fragments. In the case of AuPEt3Cl, binding was accompanied by the loss of the chloride ligand, while for [Au(S2O3)2]3− the mass spectral data indicated binding of Au(S2O3) groups. Experiments performed using HSA with Cys34 blocked by reaction with iodoacetamide were consistent with reaction of both gold compounds with this amino acid. Separate blocking experiments using diethylpyrocarbonate and AuPEt3Cl also provided evidence for histidine residues acting as lower-affinity binding sites for this gold compound. ESI mass spectra of solutions containing [Au(S2O3)2]3− or [Au(CN)2]−, and HSA, provided evidence for the formation of protein complexes in which intact gold molecules were non-covalently bound. In the case of [Au(S2O3)2]3−, these non-covalent complexes proved to be transitory in nature. However, for [Au(CN)2]− a non-covalent complex containing a single gold molecule bound to HSA was found to be stable, and constituted the main adduct formed in solutions containing low-to-medium Au-to-HSA ratios. Evidence was also obtained for the formation of a covalent adduct in which a single Au(CN) moiety was bonded to Cys34 of the protein. AuPEt3Cl reacted to a much lower extent with HSA that had Cys34 modified by formation of a disulfide bond to added cysteine, than with unmodified HSA. This suggests that the extent of modification of the protein in vivo may have an important influence on the transport and bioavailability of gold antiarthritic drugs.