The evolution of RuBisCO stability at the thermal limit of photoautotrophy.

A long-standing question in evolutionary biology is how organisms adapt to novel environments. In North American hot springs, diversification of a clade of the cyanobacterium Synechococcus into hotter environments has resulted in the unique innovation of a light-driven ecosystem at temperatures up to 74°C, and temperature adaptation of photosynthetic carbon fixation with the Calvin cycle contributed to this process. Here, we investigated the evolution of thermostability of the Calvin cycle enzyme ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) during Synechococcus divergence. Circular dichroism thermal scans revealed that the RuBisCO of the most thermotolerant Synechococcus lineage is more stable than those of other lineages or of resurrected ancestral enzymes. Using site-directed mutagenesis, we next identified four amino acid substitutions that together increased stability and activity of this enzyme at higher temperatures. These are clustered near critical subunit interfaces distant from the active site. Each of the four amino acids is also observed in a less thermostable Synechococcus RuBisCO, and the impact on stability of three of these appears to be epistatic. Recombination analyses that allow for recurrent mutation as well as patterns of synonymous variation surrounding these sites suggest that the evolution of a more thermostable RuBisCO may have involved homologous recombination. Our results provide insights on the molecular evolutionary processes that shape niche differentiation and ecosystem function.

Dissociation of recombinant prion protein fibrils into short protofilaments: implications for the endocytic pathway and involvement of the N-terminal domain.

Fibril dissociation is necessary for efficient conversion of normal prion protein to its misfolded state and continued propagation into amyloid. Recent studies have revealed that conversion occurs along the endocytic pathway. To improve our understanding of the dissociation process, we have investigated the effect of low pH on the stability of recombinant prion fibrils. We show that under conditions that mimic the endocytic environment, amyloid fibrils made from full-length prion protein dissociate both laterally and axially to form protofilaments. Approximately 5% of the protofilaments are short enough to be considered soluble and contain ~100-300 monomers per structure; these also retain the biophysical characteristics of the filaments. We propose that protonation of His residues and charge repulsion in the N-terminal domain trigger fibril dissociation. Our data suggest that lysosomes and late endosomes are competent milieus for propagating the misfolded state not only by destabilizing the normal prion protein but also by accelerating the dissociation of fibrils into smaller structures that may act as seeds.

Use of microcalorimetry to determine the costs and benefits to Pseudomonas putida strain KT2440 of harboring cadmium efflux genes.

A novel microcalorimetric approach was used to analyze the responses of a metal-tolerant soil bacterium (Pseudomonas putida strain KT2440) to metal resistance gene deletions in cadmium-amended media. As hypothesized, under cadmium stress, the wild-type strain benefited from the resistance genes by entering the exponential growth phase earlier than two knockout strains. In the absence of cadmium, strain KT1, carrying a deletion in the main component (czcA1) of a Cd/Zn chemiosmotic efflux transporter (CzcCBA1), grew more efficiently than the wild type and released ∼700 kJ (per mole of biomass carbon) less heat than the wild-type strain, showing the energetic cost of maintaining CzcCBA1 in the absence of cadmium. A second mutant strain (KT4) carrying a different gene deletion, ΔcadA2, which encodes the main Cd/Pb efflux transporter (a P-type ATPase), did not survive beyond moderate cadmium concentrations and exhibited a decreased growth yield in the absence of cadmium. Therefore, CadA2 plays an essential role in cadmium resistance and perhaps serves an additional function. The results of this study provide direct evidence that heavy metal cation efflux mechanisms facilitate shorter lag phases in the presence of metals and that the maintenance and expression of tolerance genes carry quantifiable energetic costs and benefits.

Alternating pattern of stereochemistry in the nonactin macrocycle is required for antibacterial activity and efficient ion binding.

Nonactin is a polyketide antibiotic produced by Streptomyces griseus ETH A7796 and is an ionophore that is selective for K(+) ions. It is a cyclic tetraester generated from two monomers of (+)-nonactic acid and two of (-)-nonactic acid, arranged (+)-(-)-(+)-(-) so that nonactin has S4 symmetry and is achiral. To understand why achiral nonactin is the naturally generated diastereoisomer, we generated two alternate diastereoisomers of nonactin, one prepared solely from (+)-nonactic acid and one prepared solely from (-)-nonactic acid, referred to here as 'all-(+)-nonactin' and 'all-(-)-nonactin', respectively. Both non-natural diastereoisomers were 500-fold less active against gram positive organisms than nonactin confirming that the natural stereochemistry is necessary for biological activity. We used isothermal calorimetry to obtain the K(a), DeltaG, DeltaH, and DeltaS of formation for the K(+), Na(+), and NH(4)(+) complexes of nonactin and all-(-)-nonactin; the natural diastereoisomer bound K(+) 880-fold better than all-(-)-nonactin. A picrate partitioning assay confirmed that all-(-)-nonactin, unlike nonactin, could not partition K(+) ions into organic solvent. To complement the thermodynamic data we used a simple model system to show that K(+) transport was facilitated by nonactin but not by all-(-)-nonactin. Modeling of the K(+) complexes of nonactin and all-(-)-nonactin suggested that poor steric interactions in the latter complex precluded tight binding to K(+). Overall, the data show that both enantiomers of nonactic acid are needed for the formation of a nonactin diastereoisomer that can act as an ionophore and has antibacterial activity.

Hydroxylase activity of Met471Cys tyramine beta-monooxygenase.

A series of mutations was targeted at the methionine residue, Met471, coordinating the Cu(M) site of tyramine beta-monooxygenase (TbetaM). The methionine ligand at Cu(M) is believed to be key to dioxygen activation and the hydroxylation chemistry of the copper monooxygenases. The reactivity and copper binding properties of three TbetaM mutants, Met471Asp, Met471Cys, and Met471His, were examined. All three mutants show similar metal binding affinities to wild type TbetaM in the oxidized enzyme forms. EPR spectroscopy suggests that the Cu(II) coordination geometry is identical to that of the WT enzyme. However, substrate hydroxylation was observed for the reaction of tyramine solely with Met471Cys TbetaM. Met471Cys TbetaM provides the first example of an active mutant directed at the Cu(M) site of this class of hydroxylases. The reactivity and altered kinetics of the Met471Cys mutant further highlight the central role of the methionine residue in the enzyme mechanism. The sole ability of the cysteine residue to support activity among the series of alternate amino acids investigated is relevant to theoretical and biomimetic investigations of dioxygen activation at mononuclear copper centers.

Cloning, heterologous expression, and characterization of recombinant class II cytochromes c from Rhodopseudomonas palustris.

The cytochrome (cyt) c', cyt c(556), and cyt c(2) genes from Rhodopseudomonas palustris have been cloned; recombinant cyt c' and cyt c(556) have been expressed, purified, and characterized. Unlike mitochondrial cyt c, these two proteins are structurally similar to cyt b(562), in which the heme is embedded in a four-helix bundle. The hemes in both recombinant proteins form covalent thioether links to two Cys residues. UV/vis spectra of the Fe(II) and Fe(III) states of the recombinant cyts are identical with those of the corresponding native proteins. Equilibrium unfolding measurements in guanidine hydrochloride solutions confirm that native Fe(II)-cyt c(556) is more stable than the corresponding state of Fe(III)-cyt c(556) (DeltaDeltaG(f)(o) =22 kJ/mol).

Modulation of the electrochemical behavior of tyrosyl radicals by the electrode surface.

The ability to adsorb proteins and enzymes on electrode surfaces enhances opportunities for studying enzyme activity and redox-based catalysis. Proteins may be bound in a chosen orientation on surfaces so that specific sites within them may be preferentially studied, but to date no systematic study of a redox moiety from solvent to electrode surface to the protein milieu has been performed. We report the redox and ionization behavior of tyrosine-cysteine, using the cysteine residue to form covalent linkages with Au and self-assembled-monolayer (SAM)-modified Au surfaces and using the tyrosine for redox activity. In addition, the same redox fragment incorporated into a protein bound to a SAM is examined. We find that directly binding the dipeptide to Au causes the greatest change in properties, while binding it to the SAM causes a slight perturbation in redox potential and a significant perturbation in pK(a). When azurin with a surface-exposed tyrosine is bound to a SAM-modified electrode, the redox potential and pK(a) of the tyrosine are nearly unperturbed from the values found for the dipeptide free in solution. Finally, quantification of the voltammetric signal indicates that tyrosine oxidation in the protein triggers the additional oxidation of another nearby amino acid.

Probing structural differences in prion protein isoforms by tyrosine nitration.

Two conformational isomers of recombinant hamster prion protein (residues 90-232) have been probed by reaction with two tyrosine nitration reagents, peroxynitrite and tetranitromethane. Two conserved tyrosine residues (tyrosines 149 and 150) are not labeled by either reagent in the normal cellular form of the prion protein. These residues become reactive after the protein has been converted to the beta-oligomeric isoform, which is used as a model of the fibrillar form that causes disease. After conversion, a decrease in reactivity is noted for two other conserved residues, tyrosine 225 and tyrosine 226, whereas little to no effect was observed for other tyrosines. Thus, tyrosine nitration has identified two specific regions of the normal prion protein isoform that undergo a change in chemical environment upon conversion to a structure that is enriched in beta-sheet.

Expression and characterization of recombinant tyramine beta-monooxygenase from Drosophila: a monomeric copper-containing hydroxylase.

We report here the development of a robust recombinant expression system for Drosophila melanogaster tyramine beta-monooxygenase (TbetaM), the insect analog of mammalian dopamine beta-monooxygenase. Recombinant TbetaM is rapidly purified from the host cell media in three chromatographic steps. The expression system produces approximately 3-10 mg of highly purified, active protein per liter of culture. Recombinant TbetaM requires copper for activity and has a typical type 2 copper EPR spectrum. While TbetaM efficiently hydroxylates the aliphatic carbon of phenolic amines such as tyramine (the physiological substrate) and dopamine, phenethylamine is a poor substrate. TbetaM is most likely a monomer under physiological conditions, although under conditions of high pH and low ionic strength the dimeric form predominates. The lower oligomeric state of TbetaM may provide an advantage for structural studies over DbetaM, which exists as a mixture of dimer and tetramer.

Structural studies of Apo NosL, an accessory protein of the nitrous oxide reductase system: insights from structural homology with MerB, a mercury resistance protein.

The formation of the unique catalytic tetranuclear copper cluster (Cu(Z)) of nitrous oxide reductase, N(2)OR, requires the coexpression of a multiprotein assembly apparatus encoded by the nosDFYL operon. NosL, one of the proteins encoded by this transcript, is a 20 kDa lipoprotein of the periplasm that has been shown to bind copper(I), although its function has yet to be detemined. Cu(I) EXAFS data collected on the holo protein demonstrated that features of the copper binding site are consistent with a role for this protein as a metallochaperone, a class of metal ion transporters involved in metal resistance, homeostasis, and metallocluster biosynthesis. To test this hypothesis and to gain insight into other potential functional roles for this protein in the N(2)OR system, the three-dimensional solution structure of apo NosL has been solved by solution NMR methods. The structure of apo NosL consists of two relatively independent homologous domains that adopt an unusual betabetaalphabeta topology. The fold of apo NosL displays structural homology to only one other protein, MerB, an organomercury lyase involved in bacterial mercury resistance (Di Lello et al. (2004) Biochemistry 43, 8322-32). The structural similarity between apo NosL and MerB, together with the absolute conservation of Met109 in all NosL sequences, indicates that this residue may be involved in copper ligation, and that the metal binding site is likely to be solvent-accessible and contiguous with a large binding cleft. The structural observations suggest that NosL is exceptionally adapted for a role in copper and/or sulfur delivery and possibly for metallochaperone function.