37 research outputs found
Characterization of the Radical Intermediates of Dehaloperoxidase A and B from Amphitrite Ornata
High-throughput structures of protein–ligand complexes at room temperature using serial femtosecond crystallography
High-throughput X-ray crystal structures of protein–ligand complexes are critical to pharmaceutical drug development. However, cryocooling of crystals and X-ray radiation damage may distort the observed ligand binding. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) can produce radiation-damage-free room-temperature structures. Ligand-binding studies using SFX have received only modest attention, partly owing to limited beamtime availability and the large quantity of sample that is required per structure determination. Here, a high-throughput approach to determine room-temperature damage-free structures with excellent sample and time efficiency is demonstrated, allowing complexes to be characterized rapidly and without prohibitive sample requirements. This yields high-quality difference density maps allowing unambiguous ligand placement. Crucially, it is demonstrated that ligands similar in size or smaller than those used in fragment-based drug design may be clearly identified in data sets obtained from <1000 diffraction images. This efficiency in both sample and XFEL beamtime opens the door to true high-throughput screening of protein–ligand complexes using SFX
Characterization of the Radical Intermediates of Dehaloperoxidase A and B from Amphitrite Ornata
A Novel Approach for Rapid Preparation of Monophasic Microemulsions That Facilitates Penetration of Woody Biomass
Microemulsions are a straightforward,
efficient, and highly useful
complex media for flooding/wetting substrates as a result of their
low surface tension and viscosity. Among the four broad general classes
of microemulsions (Winsor-I, -II, -III, and -IV), Winsor-IV is by
far considered the ideal microemulsion type within the context of
woody biomass pretreatment because it is a single phase. In the present
study, a never-before reported titration method was developed with
the intent of providing a rapid online determination of Winsor-IV
type microemulsion formulations under fixed surfactant concentrations
for expressly treating woody biomass. A total of 108 surfactant-oil–water
formulations based on a sodium dodecylsulfate/pentanol/water/sodium
chloride/dodecane system were investigated for their phase behavior,
54 of which yielded Winsor-IV type microemulsions. The ability of
the selected microemulsions to affect the crystallinity of cellulose
was studied by X-ray diffraction as was the synergetic effect of microemulsion
surface tension and kinematic viscosity on wood penetration from liquid
uptake experiments. The general method described here enables rapid
preparation of Winsor-IV type microemulsions that exhibit rapid wood
penetration at room temperature and atmospheric pressure and potential
utility as a general means for screening surfactant-oil–water
formulations for effective wood biomass pretreatment or other materials
applications
Development of a Highly Efficient Pretreatment Sequence for the Enzymatic Saccharification of Loblolly Pine Wood
The
efficient pretreatment of lignocellulosic materials for bioenergy
production is a critical step upon which efficient saccharification
is highly dependent, particularly in softwoods due to both their high
lignin content and condensed lignin structures. In the present study,
preliminary pretreatment steps (e.g., Wiley milling, acetone extraction,
autohydrolysis, and disc refining) and economical subsequent/core-pretreatment
steps (e.g., reagents immersion, hydrothermolysis, dilute acid hydrolysis,
and ionic liquids treatment) were systematically investigated to identify
which combinations led to effective enzymatic saccharification of
loblolly pine wood, the dominant softwood resource in the US. The
results demonstrated that 85% phosphoric acid based immersions were
highly efficient for both cellulose crystallinity degradation and
enzymatic hydrolysis, and thus can be included as core pretreatment
steps. The highest glucan recovery yield obtained was 93.0% after
enzymatic hydrolysis when a pretreatment sequence consisting of autohydrolysis,
disc refining, acetone drying, and 85% phosphoric acid immersion (50
°C, 1 h) was employed
Compound ES of Dehaloperoxidase Decays via Two Alternative Pathways Depending on the Conformation of the Distal Histidine
Dehaloperoxidase (DHP) is a respiratory hemoglobin (Hb) that has been shown to catalyze the conversion of trihalophenols to dihaloquinones in the presence of hydrogen peroxide. Ferric heme states of the resting DHP and the free radical intermediates formed under H2O2 treatment were studied by low-temperature electron paramagnetic resonance spectroscopy in the range of reaction times from 50 ms to 2 min at three different pH values. Two high-spin ferric heme forms were identified in the resting enzyme and assigned to the open and closed conformations of the distal histidine, His55. Two free radicals were found in DHP activated by H2O2: the radical associated with Compound ES (the enzyme with the heme in the oxoferryl state and a radical on the polypeptide chain) has been assigned to Tyr34, and the other radical has been assigned to Tyr38. The Tyr34 radical is formed with a very high relative yield (almost 100% of heme), atypical of other globins. High-performance liquid chromatography analysis of the reaction products showed a pH-dependent formation of covalent heme-to-protein cross-links. The stable DHP Compound RH, formed under H2O2 in the absence of the trihalophenol substrates, is proposed to be a state with the ferric heme covalently cross-linked to Tyr34. A kinetic model of the experimental data suggests that formation of Compound RH and formation of the Tyr38 radical are two alternative routes of Compound ES decay. Which route is taken depends on the conformation of His55: in the less populated closed conformation, the Tyr38 radical is formed, but in the major open conformation, Compound ES decays, yielding Compound RH, a product of safe termination of the two oxidizing equivalents of H2O2 when no substrate is available. © 2010 American Chemical Society
Tyrosyl Radicals in Dehaloperoxidase
Dehaloperoxidase (DHP) from Amphitrite ornata, having been shown to catalyze the hydrogen peroxide-dependent oxidation of trihalophenols to dihaloquinones, is the first oxygen binding globin that possesses a biologically relevant peroxidase activity. The catalytically competent species in DHP appears to be Compound ES, a reactive intermediate that contains both a ferryl heme and a tyrosyl radical. By simulating the EPR spectra of DHP activated by H2O2, Thompson et al. (Thompson, M. K., Franzen, S., Ghiladi, R. A., Reeder, B. J., and Svistunenko, D. A. (2010) J. Am. Chem. Soc. 132, 17501-17510) proposed that two different radicals, depending on the pH, are formed, one located on either Tyr-34 or Tyr-28 and the other on Tyr-38. To provide additional support for these simulation-based assignments and to deduce the role(s) that tyrosyl radicals play in DHP, stoppedflow UV-visible and rapid-freeze-quench EPR spectroscopic methods were employed to study radical formation in DHP when three tyrosine residues, Tyr-28, Tyr-34, and Tyr-38, were replaced either individually or in combination with phenylalanines. The results indicate that radicals form on all three tyrosines in DHP. Evidence for the formation of DHP Compound I in several tyrosine mutants was obtained. Variants that formed Compound I showed an increase in the catalytic rate for substrate oxidation but also an increase in heme bleaching, suggesting that the tyrosines are necessary for protecting the enzyme from oxidizing itself. This protective role of tyrosines is likely an evolutionary adaptation allowing DHP to avoid selfinflicted damage in the oxidative environment. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc