Search

This study aimed to adapt and validate the Knee Osteoarthritis Fears and Beliefs Questionnaire (KOFBeQ) for Chinese patients, thereby advancing the understanding of fear-avoidance behaviors. Adopting a cross-sectional design, data were collected for 241 subjects (78.8% women; mean age 68.0 ± 7.8 years) across various healthcare settings in Hong Kong. Exploratory factor analysis resulted in an 11-item questionnaire with three distinct subscales, covering fears and beliefs related to physicians and disease (six items), daily living activities (three items), and sports and leisure activities (two items). The overall Cronbach's α coefficient was 0.86, indicating strong internal consistency. The questionnaire exhibited favorable convergent validity. Confirmatory factor analyses confirmed a good model fit. Test-retest reliability analysis indicated a high intraclass correlation coefficient of 0.93 (95% confidence interval: 0.88, 0.96), and a Bland-Altman plot revealed a slight bias in two measurements (0.97 [0.19]) without a systematic trend. The adapted Chinese version of the KOFBeQ demonstrated robust psychometric properties in terms of validity and reliability, providing an effective tool for surveying Chinese patients with knee osteoarthritis. These findings offer valuable insights for clinicians and patients, aiding in informed decision-making and improved rehabilitation strategies.

Competing Interests: The authors have no conflicts of interest to declare.

Background: Physical and social distancing recommendations aimed at limiting exposure during the COVID-19 pandemic have forced residency programs to increasingly rely on videoconferencing and web-based resources., Objective: In this pilot study, we aimed to explore the effects of the COVID-19 pandemic on residency training experience, and to delineate the perceived barriers to the successful implementation of web-based medical education., Methods: A 19-item survey was compiled and distributed electronically using Qualtrics. This anonymous survey included information on the training level of each resident, their participation in formal didactics before and during the pandemic, and their perception of the ease and limitations of virtual didactics. The resident's opinions on specific educational resources were assessed, and the effectiveness of new delivery methods on resident engagement and learning was examined., Results: Thirty anesthesiology residents were surveyed, 19 of whom agreed to participate in the pilot study. One participant with incomplete responses was excluded, yielding a final cohort of 18 respondents. Most residents (56%, 10/18) reported that the COVID-19 pandemic negatively affected their residency training. The time spent on didactic training and independent studies was, nevertheless, not affected by the pandemic for 90% (16/18) of respondents. Nonetheless, 72% (13/18) of residents were less engaged during virtual lectures in comparison to in-person didactics. Important limitations included distraction from the physical environment (67%, 12/18), internet instability (67%, 12/18), less obligation to participate (44%, 8/18), technical difficulty and unmuted microphones (33%, 6/18, each), and people speaking over each other (28%, 5/18). Despite these limitations, most residents stated that they would like to keep a combination of virtual didactics including live Zoom lectures (56%, 10/18), prerecorded web didactics (56%, 10/18), and virtual ground rounds via Zoom (50%, 9/18) as the "new normal.", Conclusions: Despite important limitations listed in this report, anesthesia residents would like to keep a combination of virtual lectures and presentations as the new normal after the COVID-19 pandemic., (©Ala Nozari, Shivali Mukerji, Ling-Ling Lok, Qingrou Gu, Lauren Buhl, Sanjay Jain, Rafael Ortega. Originally published in JMIR Medical Education (https://mededu.jmir.org), 31.03.2022.)

Care workers in residential care settings for older adults often experience job burnout, resulting in a high turnover rate. Previous studies offered contradictory findings on technology use in the workplace and its relationship with burnout. This study aimed to explore the moderator role technology acceptance plays in the relationship between burnout and intention to leave among care workers in residential care settings in Hong Kong. The study was based on a multicenter, cross-sectional questionnaire survey. The acceptance of general, and three specific, technologies (i.e., tablets, social robots and video gaming) was measured based on the scale of the Technology Acceptance Model. Two dimensions of burnout (exhaustion and disengagement) were measured using the Oldenburg Burnout Inventory scale. Intention to leave was measured using a self-reported item. Data collection took place from July to December 2018. We analysed data from 370 care workers from seven non-private residential care homes for older people in Hong Kong. A hierarchical multiple regression approach was used for moderator analysis. The results revealed that two measures of burnout (exhaustion and disengagement) were significantly and positively associated with intention to leave. The four measures of technology acceptance were negatively associated with intention to leave. The interaction of video-gaming acceptance and exhaustion was predictive of intention to leave (standardized beta = -0.20, p = .011). Acceptance of video gaming changed the strength of the relationship between exhaustion and intention to leave among participants. No significant moderating effects were observed in the relationship between disengagement and intention to leave. We highlight the importance of integrating technology variables, especially subjective appraisal of technology, in the issues of burnout and intention to leave. These findings shed new light on policies and practices that consider implement technology in routine care in residential care settings without unanticipated negative impacts for care staff., (© 2021 John Wiley & Sons Ltd.)

Objectives: To test the effects of deploying a humanoid companion robot (Kabochan) in comparison with usual care for long-term care facilities' residents with dementia., Design: A 2-arm, randomized controlled trial with ABAB withdrawal design, lasting 32 weeks. After an 8-week baseline period, Kabochan was introduced in a nonfacilitated, individual approach with experimental-group participants (n = 52) for 8 weeks, then removed for 8 weeks, and then reintroduced for another 8 weeks. The control group (n = 51) received the usual standardized care., Setting and Participants: Seven long-term care facilities in Hong Kong. 103 residents (76% women, 87.2 ± 7.4 years) with a clinical diagnosis of dementia., Measures: Outcome assessments occurred at 5 time points: baseline (week 1) and the end of each phase (weeks 8, 16, 24, and 32). Primary outcomes were assessed with the Neuropsychiatric Inventory Questionnaire (symptom severity and caregiver distress subscales) and the Geriatric Depression Scale; secondary outcomes were measured by the Hong Kong Montreal Cognitive Assessment 5-minute Protocol, the Modified Barthel Index for Activities of Daily Living, and the Quality of Life-Alzheimer's disease scale., Results: A multivariate analysis of variance indicated a statistically significant group × time interaction for neuropsychiatric-related caregiver distress at week 16 (F = 6.72, P = .011), with a moderate effect size (η p 2  = 0.06). When Kabochan was removed in the withdrawal phase (weeks 17-24), the neuropsychiatric symptoms became more severe at week 24 for the intervention group (F = 4.68, P = .003), although the effect size was small to moderate (η p 2  = 0.04). No statistical between-group differences were found in other health outcomes., Conclusions and Implications: The Kabochan was potentially effective at reducing short-term neuropsychiatric symptoms and relevant caregiver distress for residents with dementia. An individualized care plan with continuous monitoring is required to integrate the humanoid robot into routine dementia care., (Copyright © 2020 AMDA – The Society for Post-Acute and Long-Term Care Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: Emerging technologies such as social robots have shown to be effective in reducing loneliness and agitation for older people with dementia. However, the acceptance of technology (specifically social robots) was found to be low for older people with dementia. The current understanding of changes in technology acceptance following direct exposure is limited and lacks rigorous study design. This study examined the change in technology acceptance after a direct interaction with a humanoid social robot (Kabochan) that was deployed for long-term care facilities' residents with dementia., Methods: The technology acceptance was assessed using randomised control trials for a time frame of 32 weeks. A total of 103 residents clinically diagnosed with dementia, with a mean age of 87.2 years (SD = 7.4), were recruited from seven long-term care facilities in Hong Kong and were randomly allocated to either Kabochan-engagement group or control group. Participants in the engagement group interacted with Kabochan in an individual, non-facilitated approach. The behavioural engagement with Kabochan was observed by care workers and recorded into constructive engagement and non-engagement. Questionnaire surveys were taken placed at pre- and post-exposure with Kabochan to measure attitudes and beliefs towards technology. The questionnaire was based on previous studies on technology acceptance and included attitudes towards technology, perceived usefulness, perceived ease of use, technology self-efficacy, technology anxiety, and facilitating conditions., Results: Perceived ease of use was improved at week 32 for participants who interacted with Kabochan (F = 4.239, p = 0.042) with a small effect (η p 2 = 0.043) in comparison to usual care group. Results further demonstrated that the magnitude of belief changes was related to the intensity of constructive behavioural engagement. Specifically, resident-robot behavioural engagement moderately improved attitudes towards technology (F = 11.62, p < 0.001, η p 2 = 0.11) and perceived usefulness (F = 5.75, p = 0.02, η p 2 = 0.06)., Conclusions: The study tentatively supports that exposure to Kabochan has potential for changing perceived ease of use but not for other beliefs and attitudes towards technology among long-term care residents with dementia. Direct engagement with a humanoid social robot might be promising in improving the perceived ease of use towards technology., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Diheme-containing succinate:menaquinone oxidoreductases (Sdh) are widespread in Gram-positive bacteria but little is known about the catalytic mechanisms they employ for succinate oxidation by menaquinone. Here, we present the 2.8 Å cryo-electron microscopy structure of a Mycobacterium smegmatis Sdh, which forms a trimer. We identified the membrane-anchored SdhF as a subunit of the complex. The 3 kDa SdhF forms a single transmembrane helix and this helix plays a role in blocking the canonically proximal quinone-binding site. We also identified two distal quinone-binding sites with bound quinones. One distal binding site is formed by neighboring subunits of the complex. Our structure further reveals the electron/proton transfer pathway for succinate oxidation by menaquinone. Moreover, this study provides further structural insights into the physiological significance of a trimeric respiratory complex II. The structure of the menaquinone binding site could provide a framework for the development of Sdh-selective anti-mycobacterial drugs.

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-, β-, 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.

We report a 3.5-angstrom-resolution cryo-electron microscopy structure of a respiratory supercomplex isolated from Mycobacterium smegmatis. It comprises a complex III dimer flanked on either side by individual complex IV subunits. Complex III and IV associate so that electrons can be transferred from quinol in complex III to the oxygen reduction center in complex IV by way of a bridging cytochrome subunit. We observed a superoxide dismutase-like subunit at the periplasmic face, which may be responsible for detoxification of superoxide formed by complex III. The structure reveals features of an established drug target and provides a foundation for the development of treatments for human tuberculosis., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of chemically inert carbon-hydrogen bonds in diverse endogenous and exogenous organic compounds by atmospheric oxygen. This C-H bond oxy-functionalization activity has huge potential in biotechnological applications. Class I CYPs receive the two electrons required for oxygen activation from NAD(P)H via a ferredoxin reductase and ferredoxin. The interaction of Class I CYPs with their cognate ferredoxin is specific. In order to reconstitute the activity of diverse CYPs, structural characterization of CYP-ferredoxin complexes is necessary, but little structural information is available. Here we report a structural model of such a complex (CYP199A2-HaPux) in frozen solution derived from distance and orientation restraints gathered by the EPR technique of orientation-selective double electron-electron resonance (os-DEER). The long-lived oscillations in the os-DEER spectra were well modeled by a single orientation of the CYP199A2-HaPux complex. The structure is different from the two known Class I CYP-Fdx structures: CYP11A1-Adx and CYP101A1-Pdx. At the protein interface, HaPux residues in the [Fe 2 S 2 ] cluster-binding loop and the α3 helix and the C-terminus residue interact with CYP199A2 residues in the proximal loop and the C helix. These residue contacts are consistent with biochemical data on CYP199A2-ferredoxin binding and electron transfer. Electron-tunneling calculations indicate an efficient electron-transfer pathway from the [Fe 2 S 2 ] cluster to the heme. This new structural model of a CYP-Fdx complex provides the basis for tailoring CYP enzymes for which the cognate ferredoxin is not known, to accept electrons from HaPux and display monooxygenase activity.

Biocatalytic direct monohydroxylation of anilides has been achieved on preparative scale using mutant cytochrome P450 BM3 enzymes. Representative mono- and disubstituted N-trifluoromethanesulfonyl anilides are shown to be converted in most cases to the corresponding 4-hydroxy derivatives, with substituent hydroxylation also occurring in two cases. By mutation variation, it is possible to achieve selective hydroxylation of either ring- or side-chain sites.

Cytochrome P450 (CYP) enzymes catalyze the insertion of oxygen into carbon-hydrogen bonds and have great potential for enzymatic synthesis. Application development of class I CYPs is hampered by their dependence on two redox partners (a ferredoxin and ferredoxin reductase), slowing catalysis compared to self-sufficient CYPs such as CYP102A1 (P450BM3). Previous attempts to address this have fused all three components in several permutations and geometries, with much reduced activity compared to the native system. We report here the new approach of fusing putidaredoxin reductase (PdR) to the carboxy-terminus of CYP101A1 (P450cam) via a linker peptide and reconstituting camphor hydroxylase activity with free putidaredoxin (Pdx). Initial purification of a P450cam-PdR fusion yielded 2.0% heme incorporation. Co-expression of E. coli ferrochelatase, lengthening the linker from 5 to 20 residues, and altering culture conditions for enzyme production furnished 85% heme content. Fusion co-expression with Pdx gave a functional system with comparable in vivo camphor oxidation activity as the native system. In vitro , the fused system's steady state NADH oxidation rate was two-fold faster than that of the native system. In contrast to the native system, NADH oxidation rates for the fusion enzyme showed non-hyperbolic dependence on Pdx concentration, suggesting a role for the PdR domain; these data were consistent with a kinetic model based on two-site binding of Pdx by P450cam-PdR and inactive dimer formation of the fusion. P450cam-PdR is the first example of a class I P450 fusion that exhibits significantly more favorable behavior than that of the native system.

Enzyme performance can be improved using decoy molecules or engineered variants to accelerate the activity without affecting selectivity. Here we combine a rate accelerator variant of cytochrome P450Bm3 with decoy molecules to enhance the oxidation activity of a range of small organic molecules. This combined approach offers superior biocatalytic efficiency without modifying the product distribution.

There is intense interest in late-stage catalytic C-H bond functionalization as an integral part of synthesis. Effective catalysts must have a broad substrate range and tolerate diverse functional groups. Drug molecules provide a good test of these attributes of a catalyst. A library of P450BM3 mutants developed from four base mutants with high activity for hydrocarbon oxidation produced human metabolites of a panel of drugs that included neutral (chlorzoxazone, testosterone), cationic (amitriptyline, lidocaine) and anionic (diclofenac, naproxen) compounds. No single mutant was active for all the tested drugs but multiple variants in the library showed high activity with each compound. The high conversions enabled full product characterization that led to the discovery of the new P450 reaction type of oxidative decarboxylation of an α-hydroxy carboxylic acid and the formation a protected imine from an amine, offering a novel route to α-functionalization of amines. The substrate range and varied product profiles suggest that this library of enzymes is a good basis for developing late-stage C-H activation catalysts., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An efficient formal total synthesis of (+) cortistatins A and J has been accomplished, by exploiting a highly diastereoselective intramolecular [4+3] cycloaddition of epoxy enolsilanes as the key reaction to construct rings B and C of the cortistatins in one step., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The crystal structure of the versatile CYP109B1 enzyme from Bacillus subtilis has been solved at 1.8 Å resolution. This is the first structure of an enzyme from this CYP family, whose members are prevalent across diverse species of bacteria. In the crystal structure the enzyme has an open conformation with an access channel leading from the heme to the surface. The substrate-free structure reveals the location of the key residues in the active site that are responsible for binding the substrate in the correct orientation for regioselective oxidation. Importantly, there are significant differences among these residues in members of the CYP109 and closely related CYP106 families and these likely account for the variations in substrate binding and oxidation profiles observed with these enzymes. A whole-cell oxidation biosystem was developed, which contains CYP109B1 and a phthalate family oxygenase reductase (PFOR), from Pseudomonas putida KT24440, as the electron transfer partner. This electron transfer system is able to support CYP109B1 activity resulting in the regioselective hydroxylation of both α- and β-ionone in vivo and in vitro. The PFOR is therefore a versatile electron transfer partner that is able to support the activity of CYP enzymes from other bacterium. The crystal structure of CYP109B1 has a positively charged proximal face and this explains why it can interact with PFOR and adrenodoxin which are predominantly negatively charged around their [2Fe-2S] clusters.

Rhodopseudomonas palustris HaA2 contains a gene, RPB3630, encoding a ferredoxin, HaPuxC, with an atypical CXXHXXC(X)nCP iron-sulfur cluster-binding motif. The ferredoxin gene is associated with a cytochrome P450 (CYP) monooxygenase-encoding gene, CYP194A3, an arrangement which is conserved in several strains of bacteria. Similar ferredoxin genes are found in other bacteria, such as Mycobacterium tuberculosis, where they are also associated with CYP genes. The crystal structure of HaPuxC has been solved at 2.3 Å resolution. The overall fold of this [3Fe-4S] cluster-containing ferredoxin is similar to other [3Fe-4S] and [4Fe-4S] species, with the loop around the iron-sulfur cluster more closely resembling those of [3Fe-4S] ferredoxins. The side chain of His17 from the cluster-binding motif in HaPuxC points away from the vacant site of the cluster and interacts with Glu61 and one of the sulfide ions of the cluster. This is the first cytochrome P450 electron-transfer partner of this type to be structurally characterized and will provide a better understanding of the electron-transfer processes between these ferredoxins and their CYP enzymes.

Protected cyclohexanol and cyclohex-2-enol substrates, containing benzyl ether and benzoate ester moieties, were designed to fit into the active site of the Tyr96Ala mutant of cytochrome P450cam. The protected cyclohexanol substrates were efficiently and selectively hydroxylated by the mutant enzyme at the trans C-H bond of C-4 on the cyclohexyl ring. The selectivity of oxidation of the benzoate ester protected cyclohexanol could be altered by making alternative amino acid substitutions in the P450cam active site. The addition of the double bond in the cyclohexyl ring of the benzoate ester protected cyclohex-2-enol has a debilitative effect on the activity of the Tyr96Ala mutant with this substrate. However, the Phe87Ala/Tyr96Phe double mutant, which introduces space at a different location in the active site than the Tyr96Ala mutant, was able to efficiently hydroxylate the C-H bonds of 1-cyclohex-2-enyl benzoate at the allylic C-4 position. Mutations at Phe87 improved the selectivity of the oxidation of 1-phenyl-1-cyclohexylethylene to trans-4-phenyl-ethenylcyclohexanol (92%) when compared to single mutants at Tyr96 of P450cam.

The recent crystal structures of CYP101D2, a cytochrome P450 protein from the oligotrophic bacterium Novosphingobium aromaticivorans DSM12444 revealed that both the native (substrate-free) and camphor-soaked forms have open conformations. Furthermore, two other potential camphor-binding sites were also identified from electron densities in the camphor-soaked structure, one being located in the access channel and the other in a cavity on the surface near the F-helix side of the F-G loop termed the substrate recognition site. These latter sites may be key intermediate positions on the pathway for substrate access to or product egress from the active site. Here, we show via the use of unbiased atomistic molecular dynamics simulations that despite the open conformation of the native and camphor-bound crystal structures, the underlying dynamics of CYP101D2 appear to be very similar to other CYP proteins. Simulations of the native structure demonstrated that the protein is capable of sampling many different conformational substates. At the same time, simulations with the camphor positioned at various locations within the access channel or recognition site show that movement towards the active site or towards bulk solvent can readily occur on a short timescale, thus confirming many previously reported in silico studies using steered molecular dynamics. The simulations also demonstrate how the fluctuations of an aromatic gate appear to control access to the active site. Finally, comparison of camphor-bound simulations with the native simulations suggests that the fluctuations can be of similar level and thus are more representative of the conformational selection model rather than induced fit., (Copyright © 2013 The Protein Society.)

CYP101D2 is a cytochrome P450 monooxygenase from Novosphingobium aromaticivorans which is closely related to CYP101A1 (P450cam) from Pseudomonas putida. Both enzymes selectively hydroxylate camphor to 5-exo-hydroxycamphor, and the residues that line the active sites of both enzymes are similar including the pre-eminent Tyr96 residue. However, Met98 and Leu253 in CYP101D2 replace Phe98 and Val247 in CYP101A1, and camphor binding only results in a maximal change in the spin state to 40 % high-spin. Substitutions at Tyr96, Met98 and Leu253 in CYP101D2 reduced both the spin state shift on camphor binding and the camphor oxidation activity. The Tyr96Ala mutant increased the affinity of CYP101D2 for hydrocarbon substrates including adamantane, cyclooctane, hexane and 2-methylpentane. The monooxygenase activity of the Tyr96Ala variant towards alkane substrates was also enhanced compared with the wild-type enzyme. The crystal structure of the substrate-free form of this variant shows the enzyme in an open conformation (PDB: 4DXY), similar to that observed with the wild-type enzyme (PDB: 3NV5), with the side chain of Ala96 pointing away from the heme. Despite this, the binding and activity data suggest that this residue plays an important role in substrate binding, evidencing that the enzyme probably undergoes catalysis in a more closed conformation, similar to those observed in the crystal structures of CYP101A1 (PDB: 2CPP) and CYP101D1 (PDB: 3LXI).

The modulatory role of 5-HT neurons and a number of different 5-HT receptor subtypes has been well documented in the regulation of sleep-wake cycles and hippocampal activity. A high level of 5-HT(6) receptor expression is present in the rat hippocampus. Further, hippocampal function has been shown to be modulated by both 5-HT(6) agonists and antagonists. In the current study, the potential involvement of 5-HT(6) receptors in the control of hippocampal theta rhythms and sleep-wake cycles has been investigated. Hippocampal activity was recorded by intracranial hippocampal electrodes both in anesthetized (n = 22) and in freely moving rats (n = 9). Theta rhythm was monitored in different sleep-wake states in freely moving rats and was elicited by stimulation of the brainstem reticular formation under anesthesia. Changes in theta frequency and power were analyzed before and after injection of the 5-HT(6) antagonist (SAM-531) and the 5-HT(6) agonist (EMD386088). In freely moving rats, EMD386088 suppressed sleep for several hours and significantly decreased theta peak frequency, while, in anesthetized rats, EMD386088 had no effect on theta power but significantly decreased theta frequency, which could be blocked by coadministration of SAM-531. SAM-531 alone did not change sleep-wake patterns and had no effect on theta parameters in both unanesthetized and anesthetized rats. Decreases in theta frequency induced by the 5-HT(6) receptor agonist correspond to previously described electrophysiological patterns shared by all anxiolytic drugs, and it is in line with its behavioral anxiolytic profile. The 5-HT(6) antagonist, however, failed to potentiate theta power, which is characteristic of many pro-cognitive substances, indicating that 5-HT(6) receptors might not tonically modulate hippocampal oscillations and sleep-wake patterns.

CYP238A1, one of the two P450 enzymes in the genome of Pseudomonas putida KT2440, has been produced heterologously in Escherichia coli, purified, and found to bind acyclic and cyclic terpene alcohols such as farnesol, nerolidol, linalool, and terpineol. The other P450 enzyme in this organism (gene locus: PP1950) was also produced in E. coli but no substrate has been identified from a limited screen. A phthalate family oxygenase reductase (PFOR) encoded by the PP1957 gene, just downstream of the PP1955 gene for CYP238A1, accepts electrons from the reduced form of both nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate and is able to support monooxygenase activity of CYP238A1, both in vitro and in E. coli, in which both enzymes are produced. CYP238A1 oxidizes cis- and trans-nerolidol to the 9-hydroxy product, with no evidence of attack at the olefinic double bonds. The NADH turnover rate of 170 nmol(nmol-P450)⁻¹ Min⁻¹ for CYP238A1 with cis-nerolidol as substrate at a PP1957:CYP238A1 concentration ratio of 8:1 suggests that this PFOR could function as the physiological redox partner for CYP238A1. The physiological role of CYP238A1 may be related to the PP1955 gene being part of an island/cluster of inducible genes associated with energy metabolism and response to xenobiotics., (© 2013 International Union of Biochemistry and Molecular Biology, Inc.)

The cytochrome P450 enzyme CYP199A4, from Rhodopseudomonas palustris HaA2, can efficiently demethylate 4-methoxybenzoic acid. It is also capable of oxidising a range of other related substrates. By investigating substrates with different substituents and ring systems we have been able to show that the carboxylate group and the nature of the ring system and the substituent are all important for optimal substrate binding and activity. The structures of the veratric acid, 2-naphthoic acid and indole-6-carboxylic acid substrate-bound CYP199A4 complexes reveal the substrate binding modes and the side-chain conformational changes of the active site residues to accommodate these larger substrates. They also provide a rationale for the selectivity of product oxidation. The oxidation of alkyl substituted benzoic acids by CYP199A4 is more complex, with desaturation reactions competing with hydroxylation activity. The structure of 4-ethylbenzoic acid-bound CYP199A4 revealed that the substrate is held in a similar position to 4-methoxybenzoic acid, and that the C(β) C-H bonds of the ethyl group are closer to the heme iron than those of the C(α) (3.5 vs. 4.8 Å). This observation, when coupled to the relative energies of the reaction intermediates, indicates that the positioning of the alkyl group relative to the heme iron may be critical in determining the amount of desaturation that is observed. By mutating a single residue in the active site of CYP199A4 (Phe185) we were able to convert the enzyme into a 4-ethylbenzoic acid desaturase., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A ferredoxin associated with biological Fe-S cluster assembly has been remodelled to transfer electrons to a P450 enzyme and support substrate oxidation at 80% of the physiological ferredoxin activity, opening up the possibility of tailoring ferredoxins to reconstitute the activity of P450 enzymes for which the electron transfer partner proteins are not known.

The crystal structures of the 4-methoxybenzoate bound forms of cytochrome P450 enzymes CYP199A2 and CYP199A4 from the Rhodopseudomonas palustris strains CGA009 and HaA2 have been solved. The structures of these two enzymes, which share 86% sequence identity, are very similar though some differences are found on the proximal surface. In these structures the enzymes have a closed conformation, in contrast to the substrate-free form of CYP199A2 where an obvious substrate access channel is observed. The switch from an open to a closed conformation arises from pronounced residue side-chain movements and alterations of ion pair and hydrogen bonding interactions at the entrance of the access channel. A chloride ion bound just inside the protein surface caps the entrance to the active site and protects the substrate and the heme from the external solvent. In both structures the substrate is held in place via hydrophobic and hydrogen bond interactions. The methoxy group is located over the heme iron, accounting for the high activity and selectivity of these enzymes for oxidative demethylation of the substrate. Mutagenesis studies on CYP199A4 highlight the involvement of hydrophobic (Phe185) and hydrophilic (Arg92, Ser95 and Arg243) amino acid residues in the binding of para-substituted benzoates by these enzymes.

CYP108D1 from Novosphingobium aromaticivorans DSM12444 binds a range of aromatic hydrocarbons such as phenanthrene, biphenyl and phenylcyclohexane. Its structure, which is reported here at 2.2 Å resolution, is closely related to that of CYP108A1 (P450terp), an α-terpineol-oxidizing enzyme. The compositions and structures of the active sites of these two enzymes are very similar; the most significant changes are the replacement of Glu77 and Thr103 in CYP108A1 by Thr79 and Val105 in CYP108D1. Other residue differences lead to a larger and more hydrophobic access channel in CYP108D1. These structural features are likely to account for the weaker α-terpineol binding by CYP108D1 and, when combined with the presence of three hydrophobic phenylalanine residues in the active site, promote the binding of aromatic hydrocarbons. The haem-proximal surface of CYP108D1 shows a different charge distribution and topology to those of CYP101D1, CYP101A1 and CYP108A1, including a pronounced kink in the proximal loop of CYP108D1, which may result in poor complementarity with the [2Fe-2S] ferredoxins Arx, putidaredoxin and terpredoxin that are the respective redox partners of these three P450 enzymes. The unexpectedly low reduction potential of phenylcyclohexane-bound CYP108D1 (-401 mV) may also contribute to the low activity observed with these ferredoxins. CYP108D1 appears to function as an aromatic hydrocarbon hydroxylase that requires a different electron-transfer cofactor protein.

P450(BM3) (CYP102A1), a fatty acid hydroxylase from Bacillus megaterium, has been extensively studied over a period of almost forty years. The enzyme has been redesigned to catalyse the oxidation of non-natural substrates as diverse as pharmaceuticals, terpenes and gaseous alkanes using a variety of engineering strategies. Crystal structures have provided a basis for several of the catalytic effects brought about by mutagenesis, while changes to reduction potentials, inter-domain electron transfer rates and catalytic parameters have yielded functional insights. Areas of active research interest include drug metabolite production, the development of process-scale techniques, unravelling general mechanistic aspects of P450 chemistry, methane oxidation, and improving selectivity control to allow the synthesis of fine chemicals. This review draws together the disparate research themes and places them in a historical context with the aim of creating a resource that can be used as a gateway to the field., (This journal is © The Royal Society of Chemistry 2012)

The catalytic regio- and stereoselective oxidation of aliphatic C-H bonds by using atmospheric dioxygen as the oxidant is a highly desirable reaction. Reetz and co-workers have recently reported the development of P450(BM3) mutants that oxidise testosterone with greatly increased turnover activity, total stereoselectivity and >95 % regioselectivity., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The substrate-free crystal structure of a five-mutation directed evolution variant of CYP102A1 (P450(BM3)) with generic activity-enhancing properties ("KT2") has been determined to 1.9-Å resolution. There is a close resemblance to substrate-bound structures of the wild-type enzyme (WT). The disruption of two salt bridges that link the G- and I-helices in WT causes conformational changes that break several hydrogen bonds and reduce the angle of the kink in the I-helix where dioxygen activation is thought to take place. The side-chain of a key active site residue, Phe87, is rotated in one molecule of the asymmetric unit, and the side-chains of Phe158 and Phe261 cascade into the orientations found in fatty-acid-bound forms of the enzyme. The iron is out of the porphyrin plane, towards the proximal cysteine. Unusually, the axial water ligand to the haem iron is not hydrogen-bonded to Ala264. The first electron transfer from the reductase domain to the haem domain of substrate-free KT2 is almost as fast as in palmitate-bound WT even though the reduction potential of the haem domain is only slightly more oxidising than that of substrate-free WT. However, NADPH is turned over slowly in the absence of substrate, so the catalytic cycle is gated by a step subsequent to the first electron transfer-a contrast to WT. Propylbenzene binding slightly raises the first electron transfer rate in WT but not in KT2. It is proposed that the generic rate accelerating properties of KT2 arise from the substrate-free form being in a catalytically ready conformation, such that substrate-induced changes to the structure play a less significant role in promoting the first electron transfer than in WT.

Cytochrome P450 (CYP) enzymes constitute a large family of haemoproteins that catalyze the monooxygenation of a great variety of endogenous and exogenous organic compounds. In common with other members of the CYP153 family of alkane hydroxylases, CYP153C1 from the oligotrophic bacterium Novosphingobium aromaticivorans DSM 12444 can bind linear alkanes such as heptane, octane and nonane. Here, the production, purification and crystallization of CYP153C1 and the collection of high-resolution diffraction data to 1.77 Å resolution are reported. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 61.0, b = 96.3, c = 149.8 Å, α = β = γ = 90.0°. Preliminary X-ray diffraction data analysis revealed that the asymmetric unit is most likely to contain two protein molecules.

Fatty acid binding and oxidation kinetics for wild type P450(BM3) (CYP102A1) from Bacillus megaterium have been found to display chain length-dependent homotropic behavior. Laurate and 13-methyl-myristate display Michaelis-Menten behavior while there are slight deviations with myristate at low ionic strengths. Palmitate shows Michaelis-Menten kinetics and hyperbolic binding behavior in 100 mmol/L phosphate, pH 7.4, but sigmoidal kinetics (with an apparent intercept) in low ionic strength buffers and at physiological phosphate concentrations. In low ionic strength buffers both the heme domain and the full-length enzyme show complex palmitate binding behavior that indicates a minimum of four fatty acid binding sites, with high cooperativity for the binding of the fourth palmitate molecule, and the full-length enzyme showing tighter palmitate binding than the heme domain. The first flavin-to-heme electron transfer is faster for laurate, myristate and palmitate in 100 mmol/L phosphate than in 50 mmol/L Tris (pH 7.4), yet each substrate induces similar high-spin heme content. For palmitate in low phosphate buffer concentrations, the rate constant of the first electron transfer is much larger than k (cat). The results suggest that phosphate has a specific effect in promoting the first electron transfer step, and that P450(BM3) could modulate Bacillus membrane morphology and fluidity via palmitate oxidation in response to the external phosphate concentration.

The oxidation of o-xylene by P450(BM3) from Bacillus megaterium yields, in addition to the products formed by microsomal P450s, two metabolites containing an NIH-shifted methyl group, one of which lacks the aromatic character of the substrate. The failure of the epoxide precursor of these two products to rearrange to the more stable 2,7-dimethyloxepin suggests that ring opening is P450-mediated. With m-xylene, the principal metabolite is 2,4-dimethylphenol. The partition between aromatic and benzylic hydroxylation is primarily governed by the steric prescriptions of the active site rather than by C-H bond reactivity. It is also substrate-dependent, o- and m-xylene appearing to bind to the enzyme in different orientations. The product distributions given by variants containing the F87A mutation, which creates additional space in the active site, resemble those reported for microsomal systems., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A concise, asymmetric synthesis of the pentacyclic framework of the cortistatins has been accomplished in 12 steps from commercially available starting materials, employing a highly diastereoselective intramolecular (4+3) cycloaddition of epoxy enolsilanes as the key step.

CYP101C1 from Novosphingobium aromaticivorans DSM12444 is a homologue of CYP101D1 and CYP101D2 enzymes from the same bacterium and CYP101A1 from Pseudomonas putida. CYP101C1 does not bind camphor but is capable of binding and hydroxylating ionone derivatives including α- and β-ionone and β-damascone. The activity of CYP101C1 was highest with β-damascone (k(cat)=86 s(-1)) but α-ionone oxidation was the most regioselective (98 % at C3). The crystal structures of hexane-2,5-diol- and β-ionone-bound CYP101C1 have been solved; both have open conformations and the hexanediol-bound form has a clear access channel from the heme to the bulk solvent. The entrance of this channel is blocked when β-ionone binds to the enzyme. The heme moiety of CYP101C1 is in a significantly different environment compared to the other structurally characterised CYP101 enzymes. The likely ferredoxin binding site on the proximal face of CYP101C1 has a different topology but a similar overall positive charge compared to CYP101D1 and CYP101D2, all of which accept electrons from the ArR/Arx class I electron transfer system.

The cytochrome P450 CYP101D2 from Novosphingobium aromaticivorans DSM12444 is closely related to CYP101D1 from the same bacterium and to P450cam (CYP101A1) from Pseudomonas putida. All three are capable of oxidizing camphor stereoselectively to 5-exo-hydroxycamphor. The crystal structure of CYP101D2 revealed that the likely ferredoxin-binding site on the proximal face is largely positively charged, similar to that of CYP101D1. However, both the native and camphor-soaked forms of CYP101D2 had open conformations with an access channel. In the active site of the camphor-soaked form, the camphor carbonyl interacted with the haem-iron-bound water. Two other potential camphor-binding sites were also identified from electron densities in the camphor-soaked structure: one located in the access channel, flanked by the B/C and F/G loops and the I helix, and the other in a cavity on the surface of the enzyme near the F helix side of the F/G loop. The observed open structures may be conformers of the CYP101D2 enzyme that enable the substrate to enter the buried active site via a conformational selection mechanism. The second and third binding sites may be intermediate locations of substrate entry and translocation into the active site, and provide insight into a multi-step substrate-binding mechanism.

The crystal structures of the haem domains of Ala330Pro and Ile401Pro, two single-site proline variants of CYP102A1 (P450(BM3)) from Bacillus megaterium, have been solved. In the A330P structure, the active site is constricted by the relocation of the Pro329 side chain into the substrate access channel, providing a basis for the distinctive C-H bond oxidation profiles given by the variant and the enhanced activity with small molecules. I401P, which is exceptionally active towards non-natural substrates, displays a number of structural similarities to substrate-bound forms of the wild-type enzyme, notably an off-axial water ligand, a drop in the proximal loop, and the positioning of two I-helix residues, Gly265 and His266, the reorientation of which prevents the formation of several intrahelical hydrogen bonds. Second-generation I401P variants gave high in vitro oxidation rates with non-natural substrates as varied as fluorene and propane, towards which the wild-type enzyme is essentially inactive. The substrate-free I401P haem domain had a reduction potential slightly more oxidising than the palmitate-bound wild-type haem domain, and a first electron transfer rate that was about 10 % faster. The electronic properties of A330P were, by contrast, similar to those of the substrate-free wild-type enzyme.

A new and simple strategy for the dispersion of single-wall carbon nanotubes in aqueous media is presented which does not rely on hydrophobic interactions between the polypeptidic dispersing agent and the nanotubes, and allows the surface charge of the resulting conjugate materials to be controlled.

Cytochrome P450 (CYP) enzymes of the CYP101 and CYP111 families from the oligotrophic bacterium Novosphingobium aromaticivorans DSM12444 are heme monooxygenases that receive electrons from NADH via Arx, a [2Fe-2S] ferredoxin, and ArR, a ferredoxin reductase. These systems show fast NADH turnovers (k(cat) = 39-91 s(-1)) that are efficiently coupled to product formation. The three-dimensional structures of ArR, Arx, and CYP101D1, which form a physiological class I P450 electron transfer chain, have been resolved by x-ray crystallography. The general structural features of these proteins are similar to their counterparts in other class I systems such as putidaredoxin reductase (PdR), putidaredoxin (Pdx), and CYP101A1 of the camphor hydroxylase system from Pseudomonas putida, and adrenodoxin (Adx) of the mitochondrial steroidogenic CYP11 and CYP24A1 systems. However, significant differences in the proposed protein-protein interaction surfaces of the ferredoxin reductase, ferredoxin, and P450 enzyme are found. There are regions of positive charge on the likely interaction face of ArR and CYP101D1 and a corresponding negatively charged area on the surface of Arx. The [2Fe-2S] cluster binding loop in Arx also has a neutral, hydrophobic patch on the surface. These surface characteristics are more in common with those of Adx than Pdx. The observed structural features are consistent with the ionic strength dependence of the activity.

Cytochrome P450 (CYP) enzymes of the CYP101 and CYP111 families from Novosphingobium aromaticivorans are heme monooxygenases that catalyze the hydroxylation of a range of terpenoid compounds. CYP101D1 and CYP101D2 oxidized camphor to 5-exo-hydroxycamphor. CYP101B1 and CYP101C1 oxidized beta-ionone to predominantly 3-R-hydroxy-beta-ionone and 4-hydroxy-beta-ionone, respectively. CYP111A2 oxidized linalool to 8-hydroxylinalool. Physiologically, these CYP enzymes could receive electrons from Arx, a [2Fe-2S] ferredoxin equivalent to putidaredoxin from the CYP101A1 system from Pseudomonas putida. A putative ferredoxin reductase (ArR) in the N. aromaticivorans genome, with high amino acid sequence homology to putidaredoxin reductase, has been over-produced in Escherichia coli and found to support substrate oxidation by these CYP enzymes via Arx with both high activity and coupling of product formation to NADH consumption. The ArR/Arx electron-transport chain has been co-expressed with the CYP enzymes in an E. coli host to provide in vivo whole-cell substrate oxidation systems that could produce up to 6.0 g L(-1) of 5-exo-hydroxycamphor at rates of up to 64 microM (gram of cell dry weight)(-1) min(-1). These efficient biocatalytic systems have potential uses in preparative scale whole-cell biotransformations.

CYP199A2 from Rhodopseudomonas palustris CGA009 is a heme monooxygenase that catalyzes the oxidation of para-substituted benzoic acids. CYP199A2 activity is reconstituted by a class I electron transfer chain consisting of the associated [2Fe-2S] ferredoxin palustrisredoxin (Pux) and a flavoprotein palustrisredoxin reductase (PuR). Another [2Fe-2S] ferredoxin, palustrisredoxin B (PuxB; RPA3956) has been identified in the genome. PuxB shares sequence identity and motifs with vertebrate-type ferredoxins involved in Fe-S cluster assembly but also 50% identity with Pux and it mediates electron transfer from PuR to CYP199A2, albeit with lower steady-state turnover activity: 99 nmol (nmol P450)(-1)min(-1) for 4-methoxybenzoic acid oxidation compared with 1,438 nmol (nmol P450)(-1 )min(-1) for Pux. This difference mainly arises from weak CYP199A2-PuxB binding (K (m) 34.3 vs. 0.45 microM for Pux) rather than slow electron transfer (k (cat) 19.1 vs. 37.9 s(-1) for Pux). Comparison of the 2.0-A-resolution crystal structure of the PuxB A105R mutant with other vertebrate-type, P450-associated ferredoxins revealed similar protein folds but also significant differences in some loop regions. Therefore, PuxB offers a platform for studying ferredoxin-P450 recognition in class I P450 systems. Substitution of PuxB residues at key locations with those in Pux shows that Ala42, Cys43, and Ala44 in the [2Fe-2S] cluster binding loop and Met66 are important in electron transfer from PuxB to CYP199A2, whereas Phe73 and the C-terminal Ala105 were involved in both protein binding and electron transfer.

CYP199A4 (RPB3613) from Rhodopseudomonas palustris HaA2 is a heme monooxygenase that catalyzes the hydroxylation of para-substituted benzoic acids. Monooxygenase activity of CYP199A4 can be reconstituted in a Class I electron transfer chain with an associated [2Fe-2S] ferredoxin, HaPux, (RPB3614) and the flavin-dependent reductase, HaPuR, (RPB3656) that is not associated with a CYP gene. CYP199A4 and the ferredoxin HaPux are produced in greater quantities using recombinant Escherichia coli expression systems when compared to the equivalent proteins in the closely related CYP199A2-Pux-PuR Class I system from R. palustris CGA009. HaPuR and HaPux can also replace PuR and Pux in supporting the CYP199A2 enzyme turnover with high activity. Whole-cell in vivo substrate oxidation systems for CYP199A4 and CYP199A2 with HaPux and HaPuR as the electron transfer proteins have been constructed. These E. coli systems were capable of selectively demethylating veratric acid at the para position to produce vanillic acid at rates of up to 15.3 microM (g-cdw)(-1) min(-1) and yields of up to 1.2 g L(-1).

Cytochrome P450-199A2 from Rhodopseudomonas palustris oxidizes para-substituted benzoic acids and may play a role in lignin and aromatic acid degradation pathways in the bacterium. CYP199A2 has an associated [2Fe-2S] ferredoxin, palustrisredoxin (Pux) but not a ferredoxin reductase. A genome search identified the palustrisredoxin reductase (PuR) gene. PuR was produced in Escherichia coli and shown to be a flavin-dependent protein that supports efficient electron transfer from NADH to Pux, thus reconstituting CYP199A2 monooxygenase activity (k(cat) = 37.9 s(-1) with 4-methoxybenzoic acid). The reduction of Pux by PuR shows K(m) = 4.2 microM and k(cat) = 262 s(-1) in 50 mM Tris, pH 7.4. K(m) is increased to 154 microM in the presence of 200 mM KCl, indicating the importance of ionic interactions in PuR/Pux binding. The crystal structure of PuR has been determined at 2.2 A resolution and found to be closely related to that of other oxygenase-coupled NADH-dependent ferredoxin reductases. Residues on the surface that had been proposed to be involved in ferredoxin reductase-ferredoxin binding are conserved in PuR. However, Lys328 in PuR lies over the FAD isoalloxazine ring and, together with His11 and Gln41, render the electrostatic potential of the surface more positive and may account for the greater involvement of electrostatic interactions in ferredoxin binding by PuR. Consistent with these observations the K328G mutation weakened Pux binding and virtually eliminated the dependence of PuR/Pux binding on salt concentration, thus confirming that the FAD si side surface in the vicinity of Lys328 is the ferredoxin binding site., (2009 Wiley-Liss, Inc.)

The power of proline: Bold amino acid substitutions in sensitive protein regions are frequently unproductive, while more subtle mutations can be sufficient to bring about dramatic changes. But introducing proline at the residue next to the sulfur ligand in P450(BM3) (CYP102A1) has the unexpected and desirable effect of enhancing the activity of this fatty acid hydroxylase with a broad range of non-natural substrates, as illustrated by the figure.

Using epoxy enol triethylsilanes as oxyallyl cation precursors, [4 + 3] cycloadditions with various dienes occur under catalysis by silyl triflates and acids in good yields. The intramolecular [4 + 3] cycloaddition proceeds under mild conditions and generate hydroxylated cycloadducts with high diastereoselectivity and yields. Enantiomerically pure epoxy enol silanes have been shown to give excellent yields of the optically pure cycloadduct bearing multiple stereocenters.

The development of hypoxia-selective radiopharmaceuticals for use as therapeutic and/or imaging agents is of vital importance for both early identification and treatment of cancer and in the design of new drugs. Radiotracers based on copper for use in positron emission tomography have received great attention due to the successful application of copper(II) bis(thiosemicarbazonato) complexes, such as [(60/62/64)Cu(II)ATSM] and [(60/62/64)Cu(II)PTSM], as markers for tumour hypoxia and blood perfusion, respectively. Recent work has led to the proposal of a revised mechanism of hypoxia-selective cellular uptake and retention of [Cu(II)ATSM]. The work presented here describes non-steady-state kinetic simulations in which the reported pO(2)-dependent in vitro cellular uptake and retention of [(64)Cu(II)ATSM] in EMT6 murine carcinoma cells has been modelled by using the revised mechanistic scheme. Non-steady-state (NSS) kinetic analysis reveals that the model is in very good agreement with the reported experimental data with a root-mean-squared error of less than 6% between the simulated and experimental cellular uptake profiles. Estimated rate constants are derived for the cellular uptake and washout (k(1) = 9.8 +/- 0.59 x 10(-4) s(-1) and k(2) = 2.9 +/- 0.17 x 10(-3) s(-1)), intracellular reduction (k(3) = 5.2 +/- 0.31 x 10(-2) s(-1)), reoxidation (k(4) = 2.2 +/- 0.13 mol(-1) dm(3) s(-1)) and proton-mediated ligand dissociation (k(5) = 9.0 +/- 0.54 x 10(-5) s(-1)). Previous mechanisms focused on the reduction and reoxidation steps. However, the data suggest that the origins of hypoxia-selective retention may reside with the stability of the copper(I) anion with respect to protonation and ligand dissociation. In vitro kinetic studies using the nicotimamide adenine dinucleotide (NADH)-dependent ferredoxin reductase enzyme PuR isolated from the bacterium Rhodopseudomonas palustris have also been conducted. NADH turnover frequencies are found to be dependent on the structure of the ligand and the results confirm that the proposed reduction step in the mechanism of hypoxia selectivity is likely to be mediated by NADH-dependent enzymes. Further understanding of the mechanism of hypoxia selectivity may facilitate the development of new imaging and radiotherapeutic agents with increased specificity for tumour hypoxia.

Cytochrome P450 enzymes catalyze a variety of reactions and are widely distributed in living organisms. In recent studies, the first members of five new families of cytochrome P450 enzymes have been identified, including cytochrome P450 219A1 (CYP219A1) from Novosphingobium aromaticivorans DSM 12444. It has also been reported that isolongifolen-9-one (C(15)H(22)O), a sesquiterpenoid ketone derivative, is a potential substrate for CYP219A1, inducing a >or=95% shift of the haem spin state to high spin upon binding. The CYP219A1 protein has been crystallized and single crystals have been studied by X-ray crystallography. Diffraction data were collected to 2.4 A resolution. The crystals belonged to space group P6, with unit-cell parameters a = 93.1, b = 93.1, c = 98.0 A. Preliminary X-ray diffraction data analysis revealed that the asymmetric unit contained one protein molecule.

CYP199A2, a cytochrome P450 enzyme from Rhodopseudomonas palustris, oxidatively demethylates 4-methoxybenzoic acid to 4-hydroxybenzoic acid. 4-Ethylbenzoic acid is converted to a mixture of predominantly 4-(1-hydroxyethyl)-benzoic acid and 4-vinylbenzoic acid, the latter being a rare example of CC bond dehydrogenation of an unbranched alkyl group. The crystal structure of CYP199A2 has been determined at 2.0-A resolution. The enzyme has the common P450 fold, but the B' helix is missing and the G helix is broken into two (G and G') by a kink at Pro204. Helices G and G' are bent back from the extended BC loop and the I helix to open up a clearly defined substrate access channel. Channel openings in this region of the P450 fold are rare in bacterial P450 enzymes but more common in eukaryotic P450 enzymes. The channel is hydrophobic except for the basic residue Arg246 at the entrance, which probably plays a role in the specificity of this enzyme for charged benzoates over neutral phenols and benzenes. The substrate binding pocket is hydrophobic, with Ser97 and Ser247 being the only polar residues. Computer docking of 4-ethylbenzoic acid into the active site suggests that the substrate carboxylate oxygens interact with Ser97 and Ser247, and the beta-methyl group is located over the heme iron by Phe185, the side chain of which is only 6.35 A above the iron in the native structure. This binding orientation is consistent with the observed product profile of exclusive attack at the para substituent. Putidaredoxin of the CYP101A1 system from Pseudomonas putida supports substrate oxidation by CYP199A2 at approximately 6% of the activity of the physiological ferredoxin. Comparison of the heme proximal faces of CYP199A2 and CYP101A1 suggests that charge reversal surrounding the surface residue Leu369 in CYP199A2 may be a significant factor in this low cross-activity.

The evolution of CYP102A1 variants with enhanced activity and altered specificity characteristics.