The Biochemical Impact of Extracting an Embedded Adenylate Kinase Domain Using Circular Permutation.

Adenylate kinases (AKs) have evolved AMP-binding and lid domains that are encoded as continuous polypeptides embedded at different locations within the discontinuous polypeptide encoding the core domain. A prior study showed that AK homologues of different stabilities consistently retain cellular activity following circular permutation that splits a region with high energetic frustration within the AMP-binding domain into discontinuous fragments. Herein, we show that mesophilic and thermophilic AKs having this topological restructuring retain activity and substrate-binding characteristics of the parental AK. While permutation decreased the activity of both AK homologues at physiological temperatures, the catalytic activity of the thermophilic AK increased upon permutation when assayed >30 °C below the melting temperature of the native AK. The thermostabilities of the permuted AKs were uniformly lower than those of native AKs, and they exhibited multiphasic unfolding transitions, unlike the native AKs, which presented cooperative thermal unfolding. In addition, proteolytic digestion revealed that permutation destabilized each AK in differing manners, and mass spectrometry suggested that the new termini within the AMP-binding domain were responsible for the increased proteolysis sensitivity. These findings illustrate how changes in contact order can be used to tune enzyme activity and alter folding dynamics in multidomain enzymes.

Structural diversity and substrate preferences of three tannase enzymes encoded by the anaerobic bacterium Clostridium butyricum.

Tannins are secondary metabolites that are enriched in the bark, roots, and knots in trees and are known to hinder microbial attack. The biological degradation of water-soluble gallotannins, such as tannic acid, is initiated by tannase enzymes (EC 3.1.1.20), which are esterases able to liberate gallic acid from aromatic-sugar complexes. However, only few tannases have previously been studied in detail. Here, for the first time, we biochemically and structurally characterize three tannases from a single organism, the anaerobic bacterium Clostridium butyricum, which inhabits both soil and gut environments. The enzymes were named CbTan1-3, and we show that each one exhibits a unique substrate preference on a range of galloyl ester model substrates; CbTan1 and 3 demonstrated preference toward galloyl esters linked to glucose, while CbTan2 was more promiscuous. All enzymes were also active on oak bark extractives. Furthermore, we solved the crystal structure of CbTan2 and produced homology models for CbTan1 and 3. In each structure, the catalytic triad and gallate-binding regions in the core domain were found in very similar positions in the active site compared with other bacterial tannases, suggesting a similar mechanism of action among these enzymes, though large inserts in each enzyme showcase overall structural diversity. In conclusion, the varied structural features and substrate specificities of the C. butyricum tannases indicate that they have different biological roles and could further be used in development of new valorization strategies for renewable plant biomass.

The Oxidation of Oxygen and Sulfur-Containing Heterocycles by Cytochrome P450 Enzymes.

The cytochrome P450 (CYP) superfamily of monooxygenase enzymes play important roles in the metabolism of molecules which contain heterocyclic, aromatic functional groups. Here we study how oxygen- and sulfur-containing heterocyclic groups interact with and are oxidized using the bacterial enzyme CYP199A4. This enzyme oxidized both 4-(thiophen-2-yl)benzoic acid and 4-(thiophen-3-yl)benzoic acid almost exclusively via sulfoxidation. The thiophene oxides produced were activated towards Diels-Alder dimerization after sulfoxidation, forming dimeric metabolites. Despite X-ray crystal structures demonstrating that the aromatic carbon atoms of the thiophene ring were located closer to the heme than the sulfur, sulfoxidation was still favoured with 4-(thiophen-3-yl)benzoic acid. These results highlight a preference of this cytochrome P450 enzyme for sulfoxidation over aromatic hydroxylation. Calculations predict a strong preference for homodimerization of the enantiomers of the thiophene oxides and the formation of a single major product, in broad agreement with the experimental data. 4-(Furan-2-yl)benzoic acid was oxidized to 4-(4'-hydroxybutanoyl)benzoic acid using a whole-cell system. This reaction proceeded via a γ-keto-α,ß-unsaturated aldehyde species which could be trapped in vitro using semicarbazide to generate a pyridazine species. The combination of the enzyme structures, the biochemical data and theoretical calculations provides detailed insight into the formation of the metabolites formed from these heterocyclic compounds.

Investigating the Active Oxidants Involved in Cytochrome P450 Catalyzed Sulfoxidation Reactions.

Cytochrome P450 (CYP) heme-thiolate monooxygenases catalyze the hydroxylation of the C-H bonds of organic molecules. This reaction is initiated by a ferryl-oxo heme radical cation (Cpd I). These enzymes can also catalyze sulfoxidation reactions and the ferric-hydroperoxy complex (Cpd 0) and the Fe(III)-H2 O2 complex have been proposed as alternative oxidants for this transformation. To investigate this, the oxidation of 4-alkylthiobenzoic acids and 4-methoxybenzoic acid by the CYP199A4 enzyme from Rhodopseudomonas palustris HaA2 was compared using both monooxygenase and peroxygenase pathways. By examining mutants at the mechanistically important, conserved acid alcohol-pair (D251N, T252A and T252E) the relative amounts of the reactive intermediates that would form in these reactions were disturbed. Substrate binding and X-ray crystal structures helped to understand changes in the activity and enabled an attempt to evaluate whether multiple oxidants can participate in these reactions. In peroxygenase reactions the T252E mutant had higher activity towards sulfoxidation than O-demethylation but in the monooxygenase reactions with the WT enzyme the activity of both reactions was similar. The peroxygenase activity of the T252A mutant was greater for sulfoxidation reactions than the WT enzyme, which is the reverse of the activity changes observed for O-demethylation. The monooxygenase activity and coupling efficiency of sulfoxidation and oxidative demethylation were reduced by similar degrees with the T252A mutant. These observations infer that while Cpd I is required for O-dealkylation, another oxidant may contribute to sulfoxidation. Based on the activity of the CYP199A4 mutants it is proposed that this is the Fe(III)-H2 O2 complex which would be more abundant in the peroxide-driven reactions.

Enabling Aromatic Hydroxylation in a Cytochrome P450 Monooxygenase Enzyme through Protein Engineering.

The cytochrome P450 (CYP) family of heme monooxygenases catalyse the selective oxidation of C-H bonds under ambient conditions. The CYP199A4 enzyme from Rhodopseudomonas palustris catalyses aliphatic oxidation of 4-cyclohexylbenzoic acid but not the aromatic oxidation of 4-phenylbenzoic acid, due to the distinct mechanisms of aliphatic and aromatic oxidation. The aromatic substrates 4-benzyl-, 4-phenoxy- and 4-benzoyl-benzoic acid and methoxy-substituted phenylbenzoic acids were assessed to see if they could achieve an orientation more amenable to aromatic oxidation. CYP199A4 could catalyse the efficient benzylic oxidation of 4-benzylbenzoic acid. The methoxy-substituted phenylbenzoic acids were oxidatively demethylated with low activity. However, no aromatic oxidation was observed with any of these substrates. Crystal structures of CYP199A4 with 4-(3'-methoxyphenyl)benzoic acid demonstrated that the substrate binding mode was like that of 4-phenylbenzoic acid. 4-Phenoxy- and 4-benzoyl-benzoic acid bound with the ether or ketone oxygen atom hydrogen-bonded to the heme aqua ligand. We also investigated whether the substitution of phenylalanine residues in the active site could permit aromatic hydroxylation. Mutagenesis of the F298 residue to a valine did not significantly alter the substrate binding position or enable the aromatic oxidation of 4-phenylbenzoic acid; however the F182L mutant was able to catalyse 4-phenylbenzoic acid oxidation generating 2'-hydroxy-, 3'-hydroxy- and 4'-hydroxy metabolites in a 83 : 9 : 8 ratio, respectively. Molecular dynamics simulations, in which the distance and angle of attack were considered, demonstrated that in the F182L variant, in contrast to the wild-type enzyme, the phenyl ring of 4-phenylbenzoic acid attained a productive geometry for aromatic oxidation to occur.

To Be, or Not to Be, an Inhibitor: A Comparison of Azole Interactions with and Oxidation by a Cytochrome P450 Enzyme.

The cytochrome P450 (CYP) superfamily of heme monooxygenases is involved in a range of important chemical biotransformations across nature. Azole-containing molecules have been developed as drugs that bind to the heme center of these enzymes, inhibiting their function. The optical spectrum of CYP enzymes after the addition of these inhibitors is used to assess how the molecules bind. Here we use the bacterial CYP199A4 enzyme, from Rhodopseudomonas palustris HaA2, to compare how imidazolyl and triazolyl inhibitors bind to ferric and ferrous heme. 4-(Imidazol-1-yl)benzoic acid induced a red shift in the Soret wavelength (424 nm) in the ferric enzyme along with an increase and a decrease in the intensities of the δ and α bands, respectively. 4-(1H-1,2,4-Triazol-1-yl)benzoic acid binds to CYP199A4 with a 10-fold lower affinity and induces a smaller red shift in the Soret band. The crystal structures of CYP199A4 with these two inhibitors confirmed that these differences in the optical spectra were due to coordination of the imidazolyl ligand to the ferric Fe, but the triazolyl inhibitor interacts with, rather than displaces, the ferric aqua ligand. Additional water molecules were present in the active site of 4-(1H-1,2,4-triazol-1-yl)benzoic acid-bound CYP199A4. The space required to accommodate these additional water molecules in the active site necessitates changes in the position of the hydrophobic phenylalanine 298 residue. Upon reduction of the heme, the imidazole-based inhibitor Fe-N ligation was not retained. A 5-coordinate heme was also the predominant species in 4-(1H-1,2,4-triazol-1-yl)benzoic acid-bound ferrous CYP199A4, but there was an obvious shoulder at 447 nm indicative of some degree of Fe-N coordination. Rather than inhibit CYP199A4, 4-(imidazol-1-yl)benzoic acid was a substrate and was oxidized to generate a metabolite derived from ring opening of the imidazolyl ring: 4-[[2-(formylamino)acetyl]amino]benzoic acid.

The Stereoselective Oxidation of para-Substituted Benzenes by a Cytochrome P450 Biocatalyst.

The serine 244 to aspartate (S244D) variant of the cytochrome P450 enzyme CYP199A4 was used to expand its substrate range beyond benzoic acids. Substrates, in which the carboxylate group of the benzoic acid moiety is replaced were oxidised with high activity by the S244D mutant (product formation rates >60 nmol.(nmol-CYP)-1 .min-1 ) and with total turnover numbers of up to 20,000. Ethyl α-hydroxylation was more rapid than methyl oxidation, styrene epoxidation and S-oxidation. The S244D mutant catalysed the ethyl hydroxylation, epoxidation and sulfoxidation reactions with an excess of one stereoisomer (in some instances up to >98 %). The crystal structure of 4-methoxybenzoic acid-bound CYP199A4 S244D showed that the active site architecture and the substrate orientation were similar to that of the WT enzyme. Overall, this work demonstrates that CYP199A4 can catalyse the stereoselective hydroxylation, epoxidation or sulfoxidation of substituted benzene substrates under mild conditions resulting in more sustainable transformations using this heme monooxygenase enzyme.

Evidence for Semiochemical Divergence Between Sibling Bark Beetle Species: Dendroctonus brevicomis and Dendroctonus barberi.

We investigated geographic variation in the semiochemistry of major disturbance agents of western North American pine forests, Dendroctonus brevicomis Le Conte and Dendroctonus barberi Hopkins (Coleoptera: Curculionidae: Scolytinae), species separated by the Great Basin in the USA that until recently were synonymous. At 15 sites in the western USA and northern Mexico, beetle populations were examined to determine (1) pheromone production by solitary, mining females, (2) male electroantennogram amplitudes in response to known semiochemicals for the genus, or (3) relative attractiveness of two female-produced pheromone components (endo- and exo-brevicomin) and two host odors (alpha-pinene and myrcene) to beetles in the field. Compared to female beetles collected east of the Great Basin (D. barberi), western females (D. brevicomis) produced a consistently higher proportion of, and male antenna were correspondingly more sensitive to, the exo- than the endo-isomer of brevicomin. With the exception of one sampling location (where no preference was observed), beetles west of the Great Basin were more attracted to exo- than endo- brevicomin trap lures, whereas eastern beetles displayed the reverse preference. In contrast, there was not a consistent difference between these populations regarding relative attraction or olfactory response to myrcene or alpha-pinene, although some geographic variability was evident. These data show that the semiochemical systems of D. brevicomis and D. barberi have diverged and corroborate genetic and morphological evidence that they are distinct, allopatric species.

Biophysical Techniques for Distinguishing Ligand Binding Modes in Cytochrome P450 Monooxygenases.

The cytochrome P450 superfamily of heme monooxygenases catalyzes important chemical reactions across nature. The changes in the optical spectra of these enzymes, induced by the addition of substrates or inhibitors, are critical for assessing how these molecules bind to the P450, enhancing or inhibiting the catalytic cycle. Here we use the bacterial CYP199A4 enzyme (Uniprot entry Q2IUO2), from Rhodopseudomonas palustris HaA2, and a range of substituted benzoic acids to investigate different binding modes. 4-Methoxybenzoic acid elicits an archetypal type I spectral response due to a ≥95% switch from the low- to high-spin state with concomitant dissociation of the sixth aqua ligand. 4-(Pyridin-3-yl)- and 4-(pyridin-2-yl)benzoic acid induced different type II ultraviolet-visible (UV-vis) spectral responses in CYP199A4. The former induced a greater red shift in the Soret wavelength (424 nm vs 422 nm) along with a larger overall absorbance change and other differences in the α-, ß-, and δ-bands. There were also variations in the ferrous UV-vis spectra of these two substrate-bound forms with a spectrum indicative of Fe-N bond formation with 4-(pyridin-3-yl)benzoic acid. The crystal structures of CYP199A4, with the pyridinyl compounds bound, revealed that while the nitrogen of 4-(pyridin-3-yl)benzoic acid is coordinated to the heme, with 4-(pyridin-2-yl)benzoic acid an aqua ligand remains. Continuous wave and pulse electron paramagnetic resonance data in frozen solution revealed that the substrates are bound in the active site in a form consistent with the crystal structures. The redox potential of each CYP199A4-substrate combination was measured, allowing correlation among binding modes, spectroscopic properties, and the observed biochemical activity.

Structural insights into the role of the acid-alcohol pair of residues required for dioxygen activation in cytochrome P450 enzymes.

The cytochrome P450 heme monooxygenases commonly use an acid-alcohol pair of residues, within the I-helix, to activate iron-bound dioxygen. This work aims to clarify conflicting reports on the importance of the alcohol functionality in this process. Mutants of the P450, CYP199A4 (CYP199A4D251N and CYP199A4T252A), were prepared, characterised and their crystal structures were solved. The acid residue of CYP199A4 is not part of a salt bridge network, a key feature of paradigmatic model system P450cam. Instead, there is a direct proton delivery network, via a chain of water molecules, extending to the surface. Nevertheless, CYP199A4D251N dramatically reduced the activity of the enzyme consistent with a role in proton delivery. CYP199A4T252A decreased the coupling efficiency of the enzyme with a concomitant increase in the hydrogen peroxide uncoupling pathway. However, the effect of this mutation was much less pronounced than reported with P450cam. Its crystal structures revealed fewer changes at the I-helix, compared to the P450cam system. The structural changes observed within the I-helix of P450cam during oxygen activation do not seem to be required in this P450. These differences are due to the presence of a second threonine residue at position 253, which is absent in P450cam. This threonine forms part of the hydrogen bonding network, resulting in subtle structural changes and is also present across the majority of the P450 superfamily. Overall, the results suggest that while the acid-alcohol pair is important for dioxygen activation this process and the method of proton delivery can differ across P450s.Graphic abstract.

Exploring the biological functional mechanism of the HMGB1/TLR4/MD-2 complex by surface plasmon resonance.

BACKGROUND: High Mobility Group Box 1 (HMGB1) was first identified as a nonhistone chromatin-binding protein that functions as a pro-inflammatory cytokine and a Damage-Associated Molecular Pattern molecule when released from necrotic cells or activated leukocytes. HMGB1 consists of two structurally similar HMG boxes that comprise the pro-inflammatory (B-box) and the anti-inflammatory (A-box) domains. Paradoxically, the A-box also contains the epitope for the well-characterized anti-HMGB1 monoclonal antibody "2G7", which also potently inhibits HMGB1-mediated inflammation in a wide variety of in vivo models. The molecular mechanisms through which the A-box domain inhibits the inflammatory activity of HMGB1 and 2G7 exerts anti-inflammatory activity after binding the A-box domain have been a mystery. Recently, we demonstrated that: 1) the TLR4/MD-2 receptor is required for HMGB1-mediated cytokine production and 2) the HMGB1-TLR4/MD-2 interaction is controlled by the redox state of HMGB1 isoforms. METHODS: We investigated the interactions of HMGB1 isoforms (redox state) or HMGB1 fragments (A- and B-box) with TLR4/MD-2 complex using Surface Plasmon Resonance (SPR) studies. RESULTS: Our results demonstrate that: 1) intact HMGB1 binds to TLR4 via the A-box domain with high affinity but an appreciable dissociation rate; 2) intact HMGB1 binds to MD-2 via the B-box domain with low affinity but a very slow dissociation rate; and 3) HMGB1 A-box domain alone binds to TLR4 more stably than the intact protein and thereby antagonizes HMGB1 by blocking HMGB1 from interacting with the TLR4/MD-2 complex. CONCLUSIONS: These findings not only suggest a model whereby HMGB1 interacts with TLR4/MD-2 in a two-stage process but also explain how the A-box domain and 2G7 inhibit HMGB1.

Correction to: Exploring the biological functional mechanism of the HMGB1/TLR4/MD-2 complex by surface plasmon resonance.

After publication of this article (He et al., 2018), the corresponding authors recognised an error in Scheme 1, in particular to section "A. HMGB1/TLR4/MD-2 complex formation". Above "Step 2: B box binding to MD-2", the text incorrectly read: "Low affinity / extremely slow off". In addition, some text was omitted below "TLR4/MD-2". The correct version of Scheme 1 is included in this Correction article. The original article (He et al., 2018) has been corrected.

The importance of the benzoic acid carboxylate moiety for substrate recognition by CYP199A4 from Rhodopseudomonas palustris HaA2.

BACKGROUND: The cytochrome P450 enzyme CYP199A4 can efficiently demethylate 4-methoxybenzoic acid. The substrate is positioned in the enzyme active site with the methoxy group ideally positioned for demethylation. This occurs through interactions of hydrophobic benzene ring with aromatic phenylalanine residues and the charged carboxylate group with polar and basic amino acids. METHODS: In vitro substrate binding and kinetic turnover assays coupled with HPLC and GC-MS analysis and whole-cell oxidation turnovers. RESULTS: Modification of the carboxylate group to an amide or aldehyde resulted in substrate binding, as judged by the almost total shift of the spin state to the high-spin form, but binding was three orders of magnitude weaker. Changing the carboxylate to phenol alcohol, ketone, ester and nitro groups and boronic, sulfinic and sulfonic acids resulted in a dramatic reduction in the binding affinity. Even phenylacetic acids were mediocre substrates for CYP199A4, despite maintaining a carboxylate group. The weaker binding of all of these substrates results in lower levels of turnover activity and product formation compared to 4-methoxybenzoic acid. CONCLUSION: Substrate binding to CYP199A4 is tightly regulated by interactions between the 4-methoxybenzoic acid and the amino acids in the active site. The benzoic acid carboxylate moiety is critical for optimal substrate binding and turnover activity with CYP199A4. GENERAL SIGNIFICANCE: An understanding of how the CYP199A4 enzyme has evolved to be highly selective for para-substituted benzoic acids. This provides valuable insight into how other, as yet structurally uncharacterised, monooxygenase enzymes may bind benzoic acid substrates.

Pedestrian traffic deaths among residents, visitors, and homeless persons--Clark County, Nevada, 2008-2011.

Motor vehicle collisions and crashes are a leading cause of death among Nevada residents aged 5-34 years, representing 14% of all injury deaths in that age group in 2010. During 2008-2011, a total of 173 pedestrian deaths from motor vehicle collisions occurred in Nevada, accounting for 16% of motor vehicle deaths in the state. Approximately 75% (2 million persons) of Nevada residents live in Clark County, which includes the city of Las Vegas. To analyze pedestrian traffic deaths in Clark County among residents, visitors, and homeless persons, the Southern Nevada Health District used coroner's office data and death certificate data for the period 2008-2011. The results indicated that the average annual pedestrian traffic death rates from motor vehicle collisions during this period were 1.4 per 100,000 population for residents, 1.1 for visitors, and 30.7 for homeless persons. Among the three groups, time of day, location of motor vehicle collisions, and pedestrian blood alcohol concentration (BAC) differed. Effective interventions to increase roadway safety, such as lowering speed limits in areas with greater pedestrian traffic, targeting interventions during hours when alcohol-impaired walking is more likely, and modifying roadway designs to increase protection of pedestrians, might decrease pedestrian deaths among all three groups.

Sanitation options for managing oak wood infested with the invasive goldspotted oak borer (Coleoptera: Buprestidae) in southern California.

Movement of invasive wood-boring insects in wood products presents a threat to forest health and a management challenge for public and private land managers. The goldspotted oak borer, Agrilus auroguttatus Schaeffer (Coleoptera: Buprestidae), is a new pest in San Diego and Riverside Cos., CA, believed to have been introduced on firewood. This beetle has caused elevated levels of oak mortality since 2002. From 2009-2011, we tested several sanitation methods, applicable to large and small land parcels, to reduce or prevent goldspotted oak borer emergence from infested oak wood. In most experiments, emergence of goldspotted oak borer adults from the positive controls demonstrated that the beetle could complete development in firewood-sized pieces of cut oak wood. In 2009, adult emergence from sun-exposed oak wood began and peaked 2- to 4-wks earlier at a low elevation site than at a high elevation site (late May to late June). However, there were no significant effects of elevation or host species on the emergence response of goldspotted oak borer by solarization treatment in this study. Solarization of infested wood with thick (6 mil) and thin (1 mil) plastic tarpaulins (tarps) did not significantly reduce emergence of adults despite recordings of greater mean and maximum daily temperatures in both tarped treatments and greater relative humidity in the thick-tarped treatment (all compared with nontarped controls). Grinding wood with a 3"-minus screen (< or = 7.6 cm) significantly reduced goldspotted oak borer emergence compared with control treatments, and this was the best method for reducing adult emergence among those tested. In a separate grinding study, no adults emerged when wood was ground to 9"-minus (22.9 cm), 2"-minus (5.1 cm), or 1"-minus (2.5 cm) screen sizes, but a low level of adult emergence from the positive controls limited any inferences from this experiment. Debarking cut wood pieces eliminated goldspotted oak borer emergence from the wood fraction, but adults emerged from the shaved bark and phloem.

Yeasts Have Evolved Divergent Enzyme Strategies To Deconstruct and Metabolize Xylan.

Together with bacteria and filamentous fungi, yeasts actively take part in the global carbon cycle. Over 100 yeast species have been shown to grow on the major plant polysaccharide xylan, which requires an arsenal of carbohydrate active enzymes. However, which enzymatic strategies yeasts use to deconstruct xylan and what specific biological roles they play in its conversion remain unclear. In fact, genome analyses reveal that many xylan-metabolizing yeasts lack expected xylanolytic enzymes. Guided by bioinformatics, we have here selected three xylan-metabolizing ascomycetous yeasts for in-depth characterization of growth behavior and xylanolytic enzymes. The savanna soil yeast Blastobotrys mokoenaii displays superior growth on xylan thanks to an efficient secreted glycoside hydrolase family 11 (GH11) xylanase; solving its crystal structure revealed a high similarity to xylanases from filamentous fungi. The termite gut-associated Scheffersomyces lignosus, in contrast grows more slowly, and its xylanase activity was found to be mainly cell surface-associated. The wood-isolated Wickerhamomyces canadensis, surprisingly, could not utilize xylan as the sole carbon source without the addition of xylooligosaccharides or exogenous xylanases or even co-culturing with B. mokoenaii, suggesting that W. canadensis relies on initial xylan hydrolysis by neighboring cells. Furthermore, our characterization of a novel W. canadensis GH5 subfamily 49 (GH5_49) xylanase represents the first demonstrated activity in this subfamily. Our collective results provide new information on the variable xylanolytic systems evolved by yeasts and their potential roles in natural carbohydrate conversion. IMPORTANCE Microbes that take part in the degradation of the polysaccharide xylan, the major hemicellulose component in plant biomass, are equipped with specialized enzyme machineries to hydrolyze the polymer into monosaccharides for further metabolism. However, despite being found in virtually every habitat, little is known of how yeasts break down and metabolize xylan and what biological role they may play in its turnover in nature. Here, we have explored the enzymatic xylan deconstruction strategies of three underexplored yeasts from diverse environments, Blastobotrys mokoenaii from soil, Scheffersomyces lignosus from insect guts, and Wickerhamomyces canadensis from trees, and we show that each species has a distinct behavior regarding xylan conversion. These findings may be of high relevance for future design and development of microbial cell factories and biorefineries utilizing renewable plant biomass.

Cytochrome P450-catalyzed oxidation of halogen-containing substrates.

Cytochrome P450 (CYP) enzymes are heme-thiolate monooxygenases which catalyze the oxidation of aliphatic and aromatic C-H bonds and other reactions. The oxidation of halogens by cytochrome P450 enzymes has also been reported. Here we use CYP199A4, from the bacterium Rhodopseudomonas palustris strain HaA2, with a range of para-substituted benzoic acid ligands, which contain halogens, to assess if this enzyme can oxidize these species or if the presence of these electronegative atoms can alter the outcome of P450-catalyzed reactions. Despite binding to the enzyme, there was no detectable oxidation of any of the 4-halobenzoic acids. CYP199A4 was, however, able to efficiently catalyze the oxidation of both 4-chloromethyl- and 4-bromomethyl-benzoic acid to 4-formylbenzoic acid via hydroxylation of the α­carbon. The 4-chloromethyl substrate bound in the enzyme active site in a similar manner to 4-ethylbenzoic acid. This places the benzylic α­carbon hydrogens in an unfavorable position for abstraction indicating a degree of substrate mobility must be possible within the active site. CYP199A4 catalyzed oxidations of 4-(2'-haloethyl)benzoic acids yielding α-hydroxylation and desaturation metabolites. The α-hydroxylation product was the major metabolite. The desaturation pathway is significantly disfavored compared to 4-ethylbenzoic acid. This may be due to the electron-withdrawing halogen atom or a different positioning of the substrate within the active site. The latter was demonstrated by the X-ray crystal structures of CYP199A4 with these substrates. Overall, the presence of a halogen atom positioned close to the heme iron can alter the binding orientation and outcomes of enzyme-catalyzed oxidation.

Monkeypox virus-infected individuals mount comparable humoral immune responses as Smallpox-vaccinated individuals.

In early 2022, a cluster of monkeypox virus (MPXV) infection (mpox) cases were identified within the UK with no prior travel history to MPXV-endemic regions. Subsequently, case numbers exceeding 80,000 were reported worldwide, primarily affecting gay, bisexual, and other men who have sex with men (GBMSM). Public health agencies worldwide have offered the IMVANEX Smallpox vaccination to these individuals at high-risk to provide protection and limit the spread of MPXV. We have developed a comprehensive array of ELISAs to study poxvirus-induced antibodies, utilising 24 MPXV and 3 Vaccinia virus (VACV) recombinant antigens. Panels of serum samples from individuals with differing Smallpox-vaccine doses and those with prior MPXV infection were tested on these assays, where we observed that one dose of Smallpox vaccination induces a low number of antibodies to a limited number of MPXV antigens but increasing with further vaccination doses. MPXV infection induced similar antibody responses to diverse poxvirus antigens observed in Smallpox-vaccinated individuals. We identify MPXV A27 as a serological marker of MPXV-infection, whilst MPXV M1 (VACV L1) is likely IMVANEX-specific. Here, we demonstrate analogous humoral antigen recognition between both MPXV-infected or Smallpox-vaccinated individuals, with binding to diverse yet core set of poxvirus antigens, providing opportunities for future vaccine (e.g., mRNA) and therapeutic (e.g., mAbs) design.

Screening of wild deer populations for exposure to SARS-CoV-2 in the United Kingdom, 2020-2021.

Following findings in Northern America of SARS-CoV-2 infections in white-tailed deer, there is concern of similar infections in European deer and their potential as reservoirs of SARS-CoV-2 including opportunities for the emergence of new variants. UK deer sera were collected in 2020-2021 from 6 species and a hybrid with 1748 tested using anti-spike and anti-nucleocapsid serology assays. No samples were positive on both assays nor by surrogate neutralization testing. There is no evidence that spill-over infections of SARS-CoV-2 occurred from the human population to UK deer or that SARS-CoV-2 has been circulating in UK deer (over the study period). Although it cannot be ruled out, study results indicate that spill-over infections followed by circulation of SARS-CoV-2 to the most common European deer species is small.

Exploring the Factors which Result in Cytochrome P450 Catalyzed Desaturation Versus Hydroxylation.

The cytochrome P450 family of monooxygenase enzymes have essential biological roles involving the selective oxidation of carbon-hydrogen bonds. They can also catalyze other important metabolic reactions including desaturation to form alkenes. Currently the factors that control the partition between P450 hydroxylation and desaturation pathways are poorly defined. The CYP199A4 enzyme from the bacterium Rhodopseudomonas palustris HaA2 catalyzes the oxidation of 4-ethyl- and 4-isopropyl- benzoic acids with hydroxylation and desaturation occurring in significant quantities. Here we demonstrate that 4-cyclopropylbenzoic acid is regioselectively hydroxylated by CYP199A4 at the benzylic carbon. In contrast, the oxidation of 4-n-propylbenzoic acid by CYP199A4 results in three major metabolites: an alkene from desaturation and two hydroxylation products at the benzylic (Cα) and Cß carbons in similar quantities. Extending the length of the alkyl substituent resulted in 4-n-butylbenzoic acid being oxidized at the benzylic position (45%) and desaturated (55%). In contrast, 4-isobutylbenzoic generated very little alkene (5%) but was hydroxylated at the benzylic position (54%) and at the tertiary Cß position (41%). The oxidation of 4-n-propylbenzoic acid by the F298 V mutant of CYP199A4 occurred with no hydroxylation at Cß and a significant increase in metabolites arising from desaturation (73%). The X-ray crystal structures of CYP199A4 with each substrate revealed that they bind in the active site with the alkyl substituent positioned over the heme. However, the longer alkylbenzoic acids were bound in a different conformation as was 4-n-propylbenzoic acid in the F298 V mutant. Overall, the changes in metabolite distribution could be ascribed to bond strength differences and the position of the alkyl group relative to the heme.