Chemistry with an Artificial Primer of Polyhydroxybutyrate Synthase Suggests a Mechanism for Chain Termination

Polyhydroxybutyrate (PHB) synthases (PhaCs) catalyze the conversion of 3-(<i>R</i>)-hydroxybutyryl CoA (HBCoA) to PHB, which is deposited as granules in the cytoplasm of microorganisms. The class I PhaC from <i>Caulobacter crescentus</i> (PhaC_Cc) is a highly soluble protein with a turnover number of 75 s^–1 and no lag phase in coenzyme A (CoA) release. Studies with [1-¹⁴C]­HBCoA and PhaC_Cc monitored by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and autoradiography reveal that the rate of elongation is much faster than the rate of initiation. Priming with the artificial primer [³H]­sTCoA and monitoring for CoA release reveal a single CoA/PhaC, suggesting that the protein is uniformly loaded and that the elongation process could be studied. Reaction of sT-PhaC_Cc with [1-¹⁴C]­HBCoA revealed that priming with sTCoA increased the uniformity of elongation, allowing distinct polymerization species to be observed by SDS–PAGE and autoradiography. However, in the absence of HBCoA, [³H]­sT-PhaC unexpectedly generates [³H]­sDCoA with a rate constant of 0.017 s^–1. We propose that the [³H]­sDCoA forms via attack of CoA on the oxoester of the [³H]­sT-PhaC chain, leaving the synthase attached to a single HB unit. Comparison of the relative rate constants of thiolysis by CoA and elongation by PhaC_Cc, and the size of the PHB polymer generated in vivo, suggests a mechanism for chain termination and reinitiation

<i>Bacillus subtilis</i> Class Ib Ribonucleotide Reductase: High Activity and Dynamic Subunit Interactions

The class Ib ribonucleotide reductase (RNR) isolated from <i>Bacillus subtilis</i> was recently purified as a 1:1 ratio of NrdE (α) and NrdF (β) subunits and determined to have a dimanganic-tyrosyl radical (Mn^III₂-Y·) cofactor. The activity of this RNR and the one reconstituted from recombinantly expressed NrdE and reconstituted Mn^III₂-Y· NrdF using dithiothreitol as the reductant, however, was low (160 nmol min^–1 mg^–1). The apparent tight affinity between the two subunits, distinct from all class Ia RNRs, suggested that <i>B. subtilis</i> RNR might be the protein that yields to the elusive X-ray crystallographic characterization of an “active” RNR complex. We now report our efforts to optimize the activity of <i>B. subtilis</i> RNR by (1) isolation of NrdF with a homogeneous cofactor, and (2) identification and purification of the endogenous reductant(s). Goal one was achieved using anion exchange chromatography to separate apo-/mismetalated-NrdFs from Mn^III₂-Y· NrdF, yielding enzyme containing 4 Mn and 1 Y·/β₂. Goal two was achieved by cloning, expressing, and purifying TrxA (thioredoxin), YosR (a glutaredoxin-like thioredoxin), and TrxB (thioredoxin reductase). The success of both goals increased the specific activity to ∼1250 nmol min^–1 mg^–1 using a 1:1 mixture of NrdE:Mn^III₂-Y· NrdF and either TrxA or YosR and TrxB. The quaternary structures of NrdE, NrdF, and NrdE:NrdF (1:1) were characterized by size exclusion chromatography and analytical ultracentrifugation. At physiological concentrations (∼1 μM), NrdE is a monomer (α) and Mn^III₂-Y· NrdF is a dimer (β₂). A 1:1 mixture of NrdE:NrdF, however, is composed of a complex mixture of structures in contrast to expectations

Methodology To Probe Subunit Interactions in Ribonucleotide Reductases

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides, providing the monomeric precursors required for DNA replication and repair. <i>Escherichia coli</i> RNR is a 1:1 complex of two homodimeric subunits, α2 and β2. The interactions between α2 and β2 are thought to be largely associated with the C-terminal 20 amino acids (residues 356−375) of β2. To study subunit interactions, a single reactive cysteine has been introduced into each of 15 positions along the C-terminal tail of β2. Each cysteine has been modified with the photo-cross-linker benzophenone (BP) and the environmentally sensitive fluorophore dimethylaminonaphthalene (DAN). Each construct has been purified to homogeneity and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS−PAGE) and electrospray ionization mass spectrometry (ESI-MS). Each BP-β2 has been incubated with 1 equiv of α2 and photolyzed, and the results have been analyzed quantitatively by SDS−PAGE. Each DAN-β2 was incubated with a 50-fold excess of α2, and the emission maximum and intensity were measured. A comparison of the results from the two sets of probes reveals that sites with the most extensive cross-linking are also associated with the greatest changes in fluorescence. Titration of four different DAN-β2 variants (351, 356, 365, and 367) with α2 gave a <i>K</i>_d ≈ 0.4 μM for subunit interaction. Disruption of the interaction of the α2–DAN-β2 complex is accompanied by a decrease in fluorescence intensity and can serve as a high-throughput screen for inhibitors of subunit interactions

Formal Reduction Potential of 3,5-Difluorotyrosine in a Structured Protein: Insight into Multistep Radical Transfer

The reversible Y–O^•/Y–OH redox properties of the α₃Y model protein allow access to the electrochemical and thermodynamic properties of 3,5-difluorotyrosine. The unnatural amino acid has been incorporated at position 32, the dedicated radical site in α₃Y, by <i>in vivo</i> nonsense codon suppression. Incorporation of 3,5-difluorotyrosine gives rise to very minor structural changes in the protein scaffold at pH values below the apparent p<i>K</i> (8.0 ± 0.1) of the unnatural residue. Square-wave voltammetry on α₃(3,5)­F₂Y provides an <i>E</i>°′(Y–O^•/Y–OH) of 1026 ± 4 mV versus the normal hydrogen electrode (pH 5.70 ± 0.02) and shows that the fluoro substitutions lower the <i>E</i>°′ by −30 ± 3 mV. These results illustrate the utility of combining the optimized α₃Y tyrosine radical system with <i>in vivo</i> nonsense codon suppression to obtain the formal reduction potential of an unnatural aromatic residue residing within a well-structured protein. It is further observed that the protein <i>E°′</i> values differ significantly from peak potentials derived from irreversible voltammograms of the corresponding aqueous species. This is notable because solution potentials have been the main thermodynamic data available for amino acid radicals. The findings in this paper are discussed relative to recent mechanistic studies of the multistep radical-transfer process in <i>Escherichia coli</i> ribonucleotide reductase site-specifically labeled with unnatural tyrosine residues

Purification of Polyhydroxybutyrate Synthase from Its Native Organism, <i>Ralstonia eutropha</i>: Implications for the Initiation and Elongation of Polymer Formation in Vivo

Class I polyhydroxybutyrate (PHB) synthase (PhaC) from <i>Ralstonia eutropha</i> catalyzes the formation of PHB from (<i>R</i>)-3-hydroxybutyryl-CoA, ultimately resulting in the formation of insoluble granules. Previous mechanistic studies of <i>R. eutropha</i> PhaC, purified from <i>Escherichia coli</i> (PhaC_Ec), demonstrated that the polymer elongation rate is much faster than the initiation rate. In an effort to identify a factor(s) from the native organism that might prime the synthase and increase the rate of polymer initiation, an N-terminally Strep2-tagged <i>phaC</i> (Strep2-PhaC_Re) was constructed and integrated into the <i>R. eutropha</i> genome in place of wild-type <i>phaC</i>. Strep2-PhaC_Re was expressed and purified by affinity chromatography from <i>R. eutropha</i> grown in nutrient-rich TSB medium for 4 h (peak production PHB, 15% cell dry weight) and 24 h (PHB, 2% cell dry weight). Analysis of the purified PhaC by size exclusion chromatography, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and gel permeation chromatography revealed that it unexpectedly copurified with the phasin protein, PhaP1, and with soluble PHB (<i>M</i>_w = 350 kDa) in a “high-molecular weight” (HMW) complex and in monomeric/dimeric (M/D) forms with no associated PhaP1 or PHB. Assays for monitoring the formation of PHB in the HMW complex showed no lag phase in CoA release, in contrast to M/D forms of PhaC_Re (and PhaC_Ec), suggesting that PhaC in the HMW fraction has been isolated in a PHB-primed form. The presence of primed and nonprimed PhaC suggests that the elongation rate for PHB formation is also faster than the initiation rate in vivo. A modified micelle model for granule genesis is proposed to accommodate the reported observations

Formylglycinamide Ribonucleotide Amidotransferase from <i>Thermotoga maritima:</i> Structural Insights into Complex Formation

In the fourth step of the purine biosynthetic pathway, formyl glycinamide ribonucleotide (FGAR) amidotransferase, also known as PurL, catalyzes the conversion of FGAR, ATP, and glutamine to formyl glycinamidine ribonucleotide (FGAM), ADP, P_i, and glutamate. Two forms of PurL have been characterized, large and small. Large PurL, present in most Gram-negative bacteria and eukaryotes, consists of a single polypeptide chain and contains three major domains: the N-terminal domain, the FGAM synthetase domain, and the glutaminase domain, with a putative ammonia channel located between the active sites of the latter two. Small PurL, present in Gram-positive bacteria and archaea, is structurally homologous to the FGAM synthetase domain of large PurL, and forms a complex with two additional gene products, PurQ and PurS. The structure of the PurS dimer is homologous with the N-terminal domain of large PurL, while PurQ, whose structure has not been reported, contains the glutaminase activity. In <i>Bacillus subtilis</i>, the formation of the PurLQS complex is dependent on glutamine and ADP and has been demonstrated by size-exclusion chromatography. In this work, a structure of the PurLQS complex from <i>Thermotoga maritima</i> is described revealing a 2:1:1 stoichiometry of PurS:Q:L, respectively. The conformational changes observed in TmPurL upon complex formation elucidate the mechanism of metabolite-mediated recruitment of PurQ and PurS. The flexibility of the PurS dimer is proposed to play a role in the activation of the complex and the formation of the ammonia channel. A potential path for the ammonia channel is identified

Mechanism of Assembly of the Dimanganese-Tyrosyl Radical Cofactor of Class Ib Ribonucleotide Reductase: Enzymatic Generation of Superoxide Is Required for Tyrosine Oxidation via a Mn(III)Mn(IV) Intermediate

Ribonucleotide reductases (RNRs) utilize radical chemistry to reduce nucleotides to deoxynucleotides in all organisms. In the class Ia and Ib RNRs, this reaction requires a stable tyrosyl radical (Y^•) generated by oxidation of a reduced dinuclear metal cluster. The Fe^III₂-Y^• cofactor in the NrdB subunit of the class Ia RNRs can be generated by self-assembly from Fe^II₂-NrdB, O₂, and a reducing equivalent. By contrast, the structurally homologous class Ib enzymes require a Mn^III₂-Y^• cofactor in their NrdF subunit. Mn^II₂-NrdF does not react with O₂, but it binds the reduced form of a conserved flavodoxin-like protein, NrdI_hq, which, in the presence of O₂, reacts to form the Mn^III₂-Y^• cofactor. Here we investigate the mechanism of assembly of the Mn^III₂-Y^• cofactor in <i>Bacillus subtilis</i> NrdF. Cluster assembly from Mn^II₂-NrdF, NrdI_hq, and O₂ has been studied by stopped flow absorption and rapid freeze quench EPR spectroscopies. The results support a mechanism in which NrdI_hq reduces O₂ to O₂^•– (40–48 s^–1, 0.6 mM O₂), the O₂^•– channels to and reacts with Mn^II₂-NrdF to form a Mn^IIIMn^IV intermediate (2.2 ± 0.4 s^–1), and the Mn^IIIMn^IV species oxidizes tyrosine to Y^• (0.08–0.15 s^–1). Controlled production of O₂^•– by NrdI_hq during class Ib RNR cofactor assembly both circumvents the unreactivity of the Mn^II₂ cluster with O₂ and satisfies the requirement for an “extra” reducing equivalent in Y^• generation

Redox-Linked Changes to the Hydrogen-Bonding Network of Ribonucleotide Reductase β2

Ribonucleotide reductase (RNR) catalyzes conversion of nucleoside diphosphates (NDPs) to 2′-deoxynucleotides, a critical step in DNA replication and repair in all organisms. Class-Ia RNRs, found in aerobic bacteria and all eukaryotes, are a complex of two subunits: α2 and β2. The β2 subunit contains an essential diferric–tyrosyl radical (Y122O^•) cofactor that is needed to initiate reduction of NDPs in the α2 subunit. In this work, we investigated the Y122O^• reduction mechanism in Escherichia coli β2 by hydroxyurea (HU), a radical scavenger and cancer therapeutic agent. We tested the hypothesis that Y122OH redox reactions cause structural changes in the diferric cluster. Reduction of Y122O^• was studied using reaction-induced FT-IR spectroscopy and [¹³C]­aspartate-labeled β2. These Y122O^• minus Y122OH difference spectra provide evidence that the Y122OH redox reaction is associated with a frequency change to the asymmetric vibration of D84, a unidentate ligand to the diferric cluster. The results are consistent with a redox-induced shift in H-bonding between Y122OH and D84 that may regulate proton-transfer reactions on the HU-mediated inactivation pathway in isolated β2

Conformationally Dynamic Radical Transfer within Ribonucleotide Reductase

Ribonucleotide reductases (RNR) catalyze the reduction of nucleotides to deoxynucleotides through a mechanism involving an essential cysteine based thiyl radical. In the <i>E. coli</i> class 1a RNR the thiyl radical (C₄₃₉^•) is a transient species generated by radical transfer (RT) from a stable diferric-tyrosyl radical cofactor located >35 Å away across the α₂:β₂ subunit interface. RT is facilitated by sequential proton-coupled electron transfer (PCET) steps along a pathway of redox active amino acids (Y₁₂₂β ↔ [W₄₈β?] ↔ Y₃₅₆β ↔ Y₇₃₁α ↔ Y₇₃₀α ↔ C₄₃₉α). The mutant R₄₁₁A­(α) disrupts the H-bonding environment and conformation of Y₇₃₁, ostensibly breaking the RT pathway in α₂. However, the R₄₁₁A protein retains significant enzymatic activity, suggesting Y₇₃₁ is conformationally dynamic on the time scale of turnover. Installation of the radical trap 3-amino tyrosine (NH₂Y) by amber codon suppression at positions Y₇₃₁ or Y₇₃₀ and investigation of the NH₂Y^• trapped state in the active α₂:β₂ complex by HYSCORE spectroscopy validate that the perturbed conformation of Y₇₃₁ in R₄₁₁A-α₂ is dynamic, reforming the H-bond between Y₇₃₁ and Y₇₃₀ to allow RT to propagate to Y₇₃₀. Kinetic studies facilitated by photochemical radical generation reveal that Y₇₃₁ changes conformation on the ns−μs time scale, significantly faster than the enzymatic <i>k</i>_cat. Furthermore, the kinetics of RT across the subunit interface were directly assessed for the first time, demonstrating conformationally dependent RT rates that increase from 0.6 to 1.6 × 10⁴ s^–1 when comparing wild type to R₄₁₁A-α₂, respectively. These results illustrate the role of conformational flexibility in modulating RT kinetics by targeting the PCET pathway of radical transport

Importance of the Maintenance Pathway in the Regulation of the Activity of <i>Escherichia coli</i> Ribonucleotide Reductase

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The <i>Escherichia coli</i> class Ia RNR is composed of α and β subunits that form an α₂β₂ active complex. β contains the diferric tyrosyl radical (Y^•) cofactor that is essential for the reduction process that occurs on α. [Y^•] in vitro is proportional to RNR activity, and its regulation in vivo potentially represents a mechanism for controlling RNR activity. To examine this thesis, N- and C-terminal StrepII-tagged β under the control of an l-arabinose promoter were constructed. Using these constructs and with [l-arabinose] varying from 0 to 0.5 mM in the growth medium, [β] could be varied from 4 to 3300 µM. [Y^•] in vivo and on affinity-purified Strep-β in vitro was determined by EPR spectroscopy and Western analysis. In both cases, there was 0.1–0.3 Y^• radical per β. To determine if the substoichiometric Y^• level was associated with apo β or diferric β, titrations of crude cell extracts from these growths were carried out with reduced YfaE, a 2Fe2S ferredoxin involved in cofactor maintenance and assembly. Each titration, followed by addition of O₂ to assemble the cofactor and EPR analysis to quantitate Y^•, revealed that β is completely loaded with a diferric cluster even when its concentration in vivo is 244 µM. These titrations, furthermore, resulted in 1 Y^• radical per β, the highest levels reported. Whole cell Mössbauer analysis on cells induced with 0.5 mM arabinose supports high iron loading in β. These results suggest that modulation of the level of Y^• in vivo in <i>E. coli</i> is a mechanism of regulating RNR activity