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Demand exists for a nicotine oxidase enzyme with high catalytic efficiency for a variety of applications including the in vivo detection of nicotine, therapeutic enzymatic blockade of nicotine from the CNS, and inactivation of toxic industrial wastes generated in the manufacture of tobacco products. Nicotine oxidase enzymes identified to date suffer from low efficiency, exhibiting either a high k cat or low K m , but not both. Here we present the crystal structure of the (S)-6-hydroxy-nicotine oxidase from Shinella sp HZN7 (NctB), an enzyme that oxidizes (S)-nicotine with a high k cat (>1 s−1), that possesses remarkable structural and sequence similarity to an enzyme with a nanomolar K m for (S)-nicotine, the (S)-nicotine oxidase from Pseudomonas putidia strain S16 (NicA2). Based on a comparison of our NctB structure and the previously published crystal structure of NicA2, we successfully employed a rational design approach to increase the rate of oxidative turnover of the NicA2 enzyme by ∼25% (0.011 s−1 to 0.014 s−1), and reduce the K m of the NctB protein by approximately 34% (940 μM–622 μM). While modest, these results are a step towards engineering a nicotine oxidase with kinetic parameters that fulfill the functional requirements of biosensing, waste remediation, and therapeutic applications. [ABSTRACT FROM AUTHOR]

The cyclooxygenase (COX) activity of prostaglandin endoperoxide H synthases (PGHSs) converts arachidonic acid and O2 to prostaglandin G2 (PGG2). PGHS peroxidase (POX) activity reduces PGG2 to PGH2. The first step in POX catalysis is formation of an oxyferryl heme radical cation (Compound I), which undergoes intramolecular electron transfer forming Intermediate II having an oxyferryl heme and a Tyr-385 radical required for COX catalysis. PGHS POX catalyzes heterolytic cleavage of primary and secondary hydroperoxides much more readily than H2O2, but the basis for this specificity has been unresolved. Several large amino acids form a hydrophobic "dome" over part of the heme, but when these residues were mutated to alanines there was little effect on Compound I formation from H2O2 or 15-hydroperoxyeicosatetraenoic acid, a surrogate substrate for PGG2. Ab initio calculations of heterolytic bond dissociation energies of the peroxyl groups of small peroxides indicated that they are almost the same. Molecular Dynamics simulations suggest that PGG2 binds the POX site through a peroxyl-iron bond, a hydrogen bond with His-207 and van der Waals interactions involving methylene groups adjoining the carbon bearing the peroxyl group and the protoporphyrin IX. We speculate that these latter interactions, which are not possible with H2O2, are major contributors to PGHS POX specificity. The distal GIn-203 four residues removed from His-207 have been thought to be essential for Compound I formation. However, Q203V PGHS-1 and PGHS-2 mutants catalyzed heterolytic cleavage of peroxides and exhibited native COX activity. PGHSs are homodimers with each monomer having a POX site and COX site. Cross-talk occurs between the COX sites of adjoining monomers. However, no cross-talk between the POX and COX sites of monomers was detected in a PGHS-2 heterodimer comprised of a Q203R monomer having an inactive POX site and a G533A monomer with an inactive COX site. [ABSTRACT FROM AUTHOR]

Cytochrome c oxidase (CcO) converts the energy from redox and oxygen chemistry to support proton translocation and create a transmembrane ΔμH+ used for ATP production. Molecular dynamics (MD) simulations were carried out to probe for the formation water chains capable of participating in proton translocation. Attention was focused on the region between and above the a and a3 hemes where well-defined water chains have not been identified in crystallographic studies. An arginine (R481) (Rhodobacter sphaeroides numbering), positioned between the D-propionates of the hemes, had been mutated in vivo to lysine and showed to have altered activity consistent with an altered proton conductance [Qian, J., Mills, D. A., Geren, L., Wang, K. F., Hoganson, C. W., Schmidt, B., Hiser, C., Babcock, G. 1., Durham, B., Millett, F., and Ferguson-Miller, S. (2004) Role of the conserved arginine pair in proton and electron transfer in cytochrome c oxidase, Biochemistry 43, 5748–5756; also see the accompanying paper by Mills et al.]. This mutant was created in silico, and the MD results for the mutant and wild type were compared to explore the effects on the formation of hydrogen-bonded water chains by this mutation. The simulations reveal the presence of hydrogen-bonded water chains that lead from E286 through the region above the hemes to the Mg2+, and from E286 to the heme a3 D-propionate and the binuclear center. The R481 K mutant does not form as many, or as extensive, water chains as wild-type CcO, due to a new conformation of residues in a large loop between helices Ill and IV in subunit I, indicating a reduction in the level of water chain formation in the mutant. This loop appears to play a role in controlling the formation of hydrogen-bonded water chains above the hemes. The results suggest a possible gating mechanism for proton movement that includes key residues W172 and Y175 on the loop and F282 on helix VI. [ABSTRACT FROM AUTHOR]

Prolactin (PRL) is the primary hormone that, in conjunction with local factors, leads to lobuloalveolar development during pregnancy. Recently, receptor activator of NF-κB ligand (RANKL) has been identified as one of the effector molecules essential for lobuloalveolar development. The molecular mechanisms by which PRL may induce RANKL expression have not been carefully examined. Here we report that RANKL expression in the mammary gland is developmentally regulated and dependent on PRL and progesterone, whereas its receptor RANK (receptor activator of NF-κB) and decoy receptor osteoprotegerin (OPG) are constitutively expressed at all stages in both normal (PRL[sup +/-]) and prolactin knockout (PRL[sup -/-]) mice. In vitro, PRL markedly increased RANKL expression in primary mammary epithelial cells and RANKL-luciferase reporter activity in CHOD6 cells, which constitutively express the PRL receptor. We identiffed a γ-interferon activation sequence (GAS) in the region between residues -965 to -725 of the RANKL promoter, which conferred a PRL response. Using dominant negative mutants of recombinant Jak2 and Stat5 in CHOD6 cells, and by reconstituting the Jak2/Stat5 pathway in COS7 cells, we determined that Jak2 and Stat5a are essential for the PRL-induced RANKL expression in mammary gland. [ABSTRACT FROM AUTHOR]

Examines the ovine prostaglandin H synthase (PGHS) with ethyl hydrogen peroxide. Use of electron paramagnetic resonance spectroscopy in determining the synthase; Formation of tyrosyl radical; Crystallographic structure of PGHS.

Examines the occurrence of peroxidase activity in prostaglandin endoperoxide H synthase (PGHS) -1 with a neutral histidine proximal heme ligand. Interaction of peroxides with PGHS heme sites; Evidence of neutral electrostatic state of proximal histidine ligand in oPGHS-1; Irrelevance of a strongly basic proximal ligand for peroxidase catalysis by oPGHS-1.

Klebsiella pneumoniae (Kp) is an infectious disease pathogen that poses a significant global health threat due to its potential to cause severe infections and its tendency to exhibit multidrug resistance. Understanding the enzymatic mechanisms of the oxygen‐insensitive nitroreductases (Kp‐NRs) from Kp is crucial for the development of effective nitrofuran drugs, such as nitrofurantoin, that can be activated as antibiotics. In this paper, three crystal structures of two Kp‐NRs (PDB entries 7tmf/7tmg and 8dor) are presented, and an analysis of their crystal structures and their flavin mononucleotide (FMN)‐binding mode is provided. The structures with PDB codes 7tmf (Kp‐NR1a), 7tmg (Kp‐NR1b) and 8dor (Kp‐NR2) were determined at resolutions of 1.97, 1.90 and 1.35 Å, respectively. The Kp‐NR1a and Kp‐NR1b structures adopt an αβ fold, in which four‐stranded antiparallel β‐sheets are surrounded by five helices. With domain swapping, the β‐sheet was expanded with a β‐strand from the other molecule of the dimer. The difference between the structures lies in the loop spanning Leu173–Ala185: in Kp‐NR1a the loop is disordered, whereas the loop adopts multiple conformations in Kp‐NR1b. The FMN interactions within Kp‐NR1/NR2 involve hydrogen‐bond and π‐stacking interactions. Kp‐NR2 contains four‐stranded antiparallel β‐sheets surrounded by eight helices with two short helices and one β‐sheet. Structural and sequence alignments show that Kp‐NR1a/b and Kp‐NR2 are homologs of the Escherichia coli oxygen‐insensitive NRs YdjA and NfnB and of Enterobacter cloacae NR, respectively. By homology inference from E. coli, Kp‐NR1a/b and Kp‐NR2 may detoxify polynitroaromatic compounds and Kp‐NR2 may activate nitrofuran drugs to cause bactericidal activity through a ping‐pong bi‐bi mechanism, respectively. [ABSTRACT FROM AUTHOR]

Reports that binding of ADP to the beta polypeptide isolated from the catalytic F(1) portion of the ATP synthase causes structural changes in the beta polypeptide of the enzyme. Details of materials and methods; Results and discussion.

Histamine dehydrogenase from the gram-negative bacterium Rhizobium sp. 4-9 (HaDHR) is a member of a small family of dehydrogenases containing a covalently attached FMN, and the only member so far identified to date that does not exhibit substrate inhibition. In this study, we present the 2.1 Å resolution crystal structure of HaDHR. This new structure allowed for the identification of the internal electron transfer pathway to abiological ferrocene-based mediators. Alanine 437 was identified as the exit point of electrons from the Fe 4 S 4 cluster. The enzyme was modified with a Ser436Cys mutation to facilitate covalent attachment of a ferrocene moiety. When modified with Fc-maleimide, this new construct demonstrated direct electron transfer from the enzyme to a gold electrode in a histamine concentration-dependent manner without the need for any additional electron mediators. [Display omitted] • The crystal structure for Histamine dehydrogenase from Rhizobium sp. 4-9 is described. • The electron transfer pathway from the active site to the surface of the protein is delineated. • Attachment of an abiological electron acceptor successfully captures electrons. • The modified protein undergoes direct electron transfer to an electrode surface. [ABSTRACT FROM AUTHOR]

We report the structural, biochemical, and functional characterization of the product of gene PA0962 from Pseudomonas aeruginosa PAO1. The protein, termed Pa Dps, adopts the Dps subunit fold and oligomerizes into a nearly spherical 12-mer quaternary structure at pH 6.0 or in the presence of divalent cations at neutral pH and above. The 12-Mer Pa Dps contains two di-iron centers at the interface of each subunit dimer, coordinated by conserved His, Glu, and Asp residues. In vitro, the di-iron centers catalyze the oxidation of Fe2+ utilizing H2O2 (not O2) as an oxidant, suggesting Pa Dps functions to aid P. aeruginosa to survive H2O2-mediated oxidative stress. In agreement, a P. aeruginosa Δdps mutant is significantly more susceptible to H2O2 than the parent strain. The Pa Dps structure harbors a novel network of Tyr residues at the interface of each subunit dimer between the two di-iron centers, which captures radicals generated during Fe2+ oxidation at the ferroxidase centers and forms di-tyrosine linkages, thus effectively trapping the radicals within the Dps shell. Surprisingly, incubating Pa Dps and DNA revealed unprecedented DNA cleaving activity that is independent of H2O2 or O2 but requires divalent cations and 12-mer Pa Dps. [ABSTRACT FROM AUTHOR]

Background: During elongation, multi-subunit RNA polymerases (RNAPs) cycle between phosphodiester bond formation and nucleic acid translocation. In the conformation associated with catalysis, the mobile "trigger loop" of the catalytic subunit closes on the nucleoside triphosphate (NTP) substrate. Closing of the trigger loop is expected to exclude water from the active site, and dehydration may contribute to catalysis and fidelity. In the absence of a NTP substrate in the active site, the trigger loop opens, which may enable translocation. Another notable structural element of the RNAP catalytic center is the "bridge helix" that separates the active site from downstream DNA. The bridge helix may participate in translocation by bending against the RNA/DNA hybrid to induce RNAP forward movement and to vacate the active site for the next NTP loading. The transition between catalytic and translocation conformations of RNAP is not evident from static crystallographic snapshots in which macromolecular motions may be restrained by crystal packing. Results: All atom molecular dynamics simulations of Thermus thermophilus (Tt) RNAP reveal flexible hinges, located within the two helices at the base of the trigger loop, and two glycine hinges clustered near the N-terminal end of the bridge helix. As simulation progresses, these hinges adopt distinct conformations in the closed and open trigger loop structures. A number of residues (described as "switch" residues) trade atomic contacts (ion pairs or hydrogen bonds) in response to changes in hinge orientation. In vivo phenotypes and in vitro activities rendered by mutations in the hinge and switch residues in Saccharomyces cerevisiae (Sc) RNAP II support the importance of conformational changes predicted from simulations in catalysis and translocation. During simulation, the elongation complex with an open trigger loop spontaneously translocates forward relative to the elongation complex with a closed trigger loop. Conclusions: Switching between catalytic and translocating RNAP forms involves closing and opening of the trigger loop and long-range conformational changes in the atomic contacts of amino acid side chains, some located at a considerable distance from the trigger loop and active site. Trigger loop closing appears to support chemistry and the fidelity of RNA synthesis. Trigger loop opening and limited bridge helix bending appears to promote forward nucleic acid translocation. [ABSTRACT FROM AUTHOR]

The enzyme 6-Hydroxy- l -Nicotine oxidase (HLNO) is a flavin-dependent enzyme that catalyzes the first step in the pyridine pathway of oxidation of nicotine as a source of energy and nitrogen in several bacteria. Recombinant Arthrobacter nicotinovorans HLNO also catalyzes oxidation of (s)-nicotine at a low but measurable rate (Fitzpatrick et al., 2016, Biochemistry 55, 697–703). Rational design and bioinformatics approaches, based on the known high-resolution structure of this enzyme (RCSB: 3NG7), were employed to further enhance the catalytic turnover and stability of the enzyme using (S)-nicotine as substrate. The active site residue Tyr311 forms a hydrogen bond with the hydroxyl group of (S)-6-OH-nicotine within the catalytic pocket. Its replacement by a tryptophan residue reduced the k cat for (S)-6-OH-nicotine by more than 6-fold and increased ~1.5-fold. Combining this mutation with two surface mutations that were predicted to enhance enzyme stability, further increased the k cat for nicotine resulting in a comparatively robust oxidation of (s)-nicotine (k cat >1 s−1) at 37 °C, at the same time reducing the specificity for (S)–OH-nicotine (k cat / K M) by more than 100-fold and increasing that for (S)-nicotine by more than 2-fold. Interestingly, adding a maltose-binding protein (MBP) tag onto the N-terminus of HLNO markedly increased the thermal stability of the enzyme, extending the half-life at 37 °C from ~2 h to ~22 h. This effect was due almost entirely to increased FAD retention, an observation that may prove useful to improve flavin retention in other flavin-dependent monoamine oxidases. [ABSTRACT FROM AUTHOR]