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Biomedical subjects

Roshan Perera

Publications and source records attributed to Roshan Perera.

12 recordsLinked to original sources
PubMed2006

A genetically encoded infrared probe.

An orthogonal tRNA/aminoacyl-tRNA synthetase pair has been evolved that makes it possible to selectively and efficiently incorporate para-cyanophenylalanine (pCNPhe) into proteins in E. coli at sites specified by the amber nonsense codon, TAG. Substitution of pCNPhe for histidine-64 in myoglobin (Mb) affords a sensitive vibrational probe of ligand binding. This methodology provides a useful infrared reporter of protein structure, biomolecular interactions, and conformational changes.

Models, Molecular↗
PubMed2006

Panning for gold: an evidence-based tool for assessment of performance indicators in primary health care.

It is important that debate occurs between theorists, policy makers, clinicians and service end-users to develop agreement over suitable and appropriate indicators for primary health care. A formal accounting of the relative strengths and weaknesses of any proposed indicator will enable sector commentators from a variety of viewpoints to discuss the relative merits of individual indicators, to understand the political and pragmatic reasons for their inclusion in any set of indicators and to trace the likely organisational impact of any given indicator. This paper details the development of an indicator appraisal tool that combines the assessment of scientific evidence with contextual considerations from the perspective of both the policy environment and the primary health care sector. The use of the tool is discussed in the context of the proposed national implementation of a set of performance indicators in New Zealand.

Evidence-Based Medicine↗
PubMed2006

Rapid freeze-quench ENDOR study of chloroperoxidase compound I: the site of the radical.

The classical heme-monooxygenase active intermediate, compound I (Cpd-I), incorporates a heme which is oxidized by two equivalents above the resting ferric state, one equivalent associated with a ferryl center, [Fe=O]2+ (FeS = 1), and the other with an active-site radical (RS = 1/2). Theoretical calculations on models of a Cpd-I with a thiolato axial ligand have presented divergent views about its electronic structure. In one picture, the radical is on the porphyrin; in the other, it is on the sulfur. In this report, ENDOR spectroscopy answers the question, does Cpd-I of the enzyme chloroperoxidase contain a porphyrin pi-cation radical or an iron-bound cysteinyl radical: the radical is predominantly on the porphyrin, with spin density on sulfur having an upper bound, rhoS </= rhoSmax approximately 0.23. We further suggest that the same answer applies to Cpd-I of cytochromes P450.

Chloride Peroxidase↗
PubMed2006

Structures of thiolate- and carboxylate-ligated ferric H93G myoglobin: models for cytochrome P450 and for oxyanion-bound heme proteins.

Crystal structures of the ferric H93G myoglobin (Mb) cavity mutant containing either an anionic proximal thiolate sulfur donor or a carboxylate oxygen donor ligand are reported at 1.7 and 1.4 A resolution, respectively. The crystal structure and magnetic circular dichroism spectra of the H93G Mb beta-mercaptoethanol (BME) thiolate adduct reveal a high-spin, five-coordinate complex. Furthermore, the bound BME appears to have an intramolecular hydrogen bond involving the alcohol proton and the ligated thiolate sulfur, mimicking one of the three proximal N-H...S hydrogen bonds in cytochrome P450. The Fe is displaced from the porphyrin plane by 0.5 A and forms a 2.41 A Fe-S bond. The Fe(3+)-S-C angle is 111 degrees , indicative of a covalent Fe-S bond with sp(3)-hybridized sulfur. Therefore, the H93G Mb.BME complex provides an excellent protein-derived structural model for high-spin ferric P450. In particular, the Fe-S bond in high-spin ferric P450-CAM has essentially the same geometry despite the constraints imposed by covalent linkage of the cysteine to the protein backbone. This suggests that evolution led to the geometric optimization of the proximal Fe-S(cysteinate) bond in P450. The crystal structure and spectral properties of the H93G Mb acetate adduct reveal a high-spin, six-coordinate complex with proximal acetate and distal water axial ligands. The distal His-64 forms a hydrogen bond with the bound water. The Fe-acetate bonding geometry is inconsistent with an electron pair along the Fe-O bond as the Fe-O-C angle is 152 degrees and the Fe is far from the plane of the acetate. Thus, the Fe-O bonding is ionic. The H93G Mb cavity mutant has already been shown to be a versatile model system for the study of ligand binding to heme proteins; this investigation affords the first structural evidence that nonimidazole exogenous ligands bind in the proximal ligation site.

Binding Sites↗
PubMed2005

Cryoreduction EPR and 13C, 19F ENDOR study of substrate-bound substates and solvent kinetic isotope effects in the catalytic cycle of cytochrome P450cam and its T252A mutant.

We recently used cryoreduction EPR/ENDOR techniques to show that a substrate can modulate the properties of both the monooxygenase active-oxygen intermediates and of the proton-delivery network which encompasses them. In the present report we use Q-band pulsed 19F ENDOR (Mims 3-pulse sequence) to examine the substrate binding geometries of camphor, through use of the 5,5'--difluorocamphor, and 13C ENDOR to examine the binding of 5-methylenyl camphor labeled with 13C at C11. These probes are examined in multiple states of the catalytic cycle of P450cam and its T252A mutant. As part of this investigation we further report a new cryoreduction reaction, the reduction of a ferroheme to the EPR-visible Fe(I) state, and use it to probe the substrate binding to the EPR-silent ferroheme state. Finally we report the solvent kinetic isotope effect on the decay of the camphor complex of the hydroperoxo-ferric intermediate, the first such measurement on an individual step within the P450cam reaction cycle. Following reduction of oxyferrous-P450cam, this step is the rate-limiting step in camphor hydroxylation, and its solv-KIE of 1.8 at 190 K establishes that it involves activation of the hydroperoxo moiety by transfer of the 'second' proton of catalysis. We suggest that the finding that the heme pocket can exist in multiple substates, including multiple substrate binding locations, even in P450cam, along with the established possibility that the hydroperoxo-ferriheme intermediate can react with substrate, may explain the formation of multiple products by P450s.

Alanine↗
PubMed2005

Subzero-temperature stabilization and spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome P450 BM3 (CYP102) oxygenase domain and holoenzyme.

We describe herein for the first time the formation and spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome P450 BM3 (CYP102) holoenzyme and heme domain (BMP) at -55 degrees C using a 70/30 (v/v) glycerol/buffer cryosolvent. The choice of buffer is a crucial factor with Tris [tris(hydroxymethyl)aminomethane] buffer being significantly more effective than phosphate. The oxyferrous complexes have been characterized with magnetic circular dichroism spectroscopy and the resulting spectra compared to those of the more well-characterized oxyferrous cytochrome P450-CAM. The formation of a stable substrate-bound oxyferrous CYP BM3 holoenzyme, despite the fact that it has the necessary reducing equivalents for turnover, indicates that electron transfer from the flavin domain to the oxyferrous center is very slow at this temperature. The ability to prepare stable homogeneous oxyferrous derivatives of both BMP and the CYP BM3 holoenzyme will enable these species to be used as starting materials for mechanistic investigation of dioxygen activation.

Bacterial Proteins↗
PubMed2005

The influence of substrate on the spectral properties of oxyferrous wild-type and T252A cytochrome P450-CAM.

To probe whether the nature of the substrate can directly influence the spectral properties of oxyferrous cytochrome P450-CAM, the complex has been investigated in the absence and in the presence of the natural substrate (1R)-camphor (camphor) and of several camphor analogs. The oxyferrous complex of T252A P450-CAM, a mutant lacking the hydroxyl group that forms a hydrogen bond to the heme iron-coordinated dioxygen, has also been studied to gauge the influence of this hydrogen bond. UV-visible absorption and magnetic circular dichroism (MCD) spectra of these oxyferrous adducts prepared and stabilized at -40 degrees C in 60% (v/v) ethylene glycol are generally similar, exhibiting absorption bands at approximately 355, approximately 420, approximately 554, and approximately 585 nm (shoulder) and a characteristic MCD trough at approximately 585 nm. The MCD spectrum of camphor-bound oxyferrous P450-CAM is similar to that of the substrate-free oxyferrous enzyme, but the spectrum of the oxyferrous enzyme differs detectably in the presence of substrate analogs. The spectra of the oxyferrous T252A mutant and wild-type enzyme are overall similar except for Soret band position blue shifts by 2-6 nm for the mutant. 5-Methylenylcamphor (epoxidation substrate) appears to have an anomalous binding mode for the mutant compared with that for the wild-type enzyme. The present results indicate that the structures of the camphor analogs can sensitively influence the physical (spectroscopic) properties of the P450 dioxygen complex and could also affect its reactivity. The ability of substrate to modulate the reactivity of P450 intermediates could be a relevant factor in explaining the remarkable diversity of reactions catalyzed by the enzyme.

Binding Sites↗
PubMed2005

Substrate modulation of the properties and reactivity of the oxy-ferrous and hydroperoxo-ferric intermediates of cytochrome P450cam as shown by cryoreduction-EPR/ENDOR spectroscopy.

EPR/ENDOR studies have been carried out on oxyferrous cytochrome P450cam one-electron cryoreduced by gamma-irradiation at 77 K in the absence of substrate and in the presence of a variety of substrates including its native hydroxylation substrate, camphor (a), and the alternate substrates, 5-methylenyl-camphor (b), 5,5-difluorocamphor (c), norcamphor (d), and adamantanone (e); the equivalent experiments have been performed on the T252A mutant complexed with a and b. The present study shows that the properties and reactivity of the oxyheme and of both the primary and the annealed intermediates are modulated by a bound substrate. This includes alterations in the properties of the heme center itself (g tensor; (14)N, (1)H, hyperfine couplings). It also includes dramatic changes in reactivity: the presence of any substrate increases the lifetime of hydroperoxoferri-P450cam (2) no less than ca. 20-fold. Among the substrates, b stands out as having an exceptionally strong influence on the properties and reactivity of the P450cam intermediates, especially in the T252A mutant. The intermediate, 2(T252A)-b, does not lose H(2)O(2), as occurs with 2(T252A)-a, but decays with formation of the epoxide of b. Thus, these observations show that substrate can modulate the properties of both the monoxygenase active-oxygen intermediates and the proton-delivery network that encompasses them.

Adamantane↗
PubMed2004

EpoK, a cytochrome P450 involved in biosynthesis of the anticancer agents epothilones A and B. Substrate-mediated rescue of a P450 enzyme.

The epothilones are a new class of highly promising anticancer agents with a mode of action akin to that of paclitaxel but with distinct advantages over that drug. The principal natural compounds are epothilones A and B, which have an epoxide in the macrocyclic lactone ring, and C and D, which have a double bond instead of the epoxide group. The epoxidation of epothilones C and D to A and B, respectively, is mediated by EpoK, a cytochrome P450 enzyme encoded in the epothilone gene cluster. Here we report high-yield expression of EpoK, characterization of the protein, demonstration that the natural substrate can prevent-and even reverse-denaturation of the protein, identification of ligands and surrogate substrates, development of a high-throughput fluorescence activity assay based on the H(2)O(2)-dependent oxidation of 7-ethoxy-4-trifluoromethylcoumarin, and identification of effective inhibitors of the enzyme. These results will facilitate improvements in the yields of epothilones C and D and the engineering of EpoK to prepare novel epothilone analogues. Furthermore, the finding that the denatured enzyme is rescued by the substrate offers a potential paradigm for control of the P450 catalytic function.

Antineoplastic Agents↗
PubMed2003

Neutral thiol as a proximal ligand to ferrous heme iron: implications for heme proteins that lose cysteine thiolate ligation on reduction.

Cysteine plays a key role as a metal ligand in metalloproteins. In all well-recognized cases, however, it is the anionic cysteinate that coordinates. Several cysteinate-ligated heme proteins are known, but some fail to retain thiolate ligation in the ferrous state, possibly following protonation to form neutral cysteine. Ligation by cysteine thiol in ferrous heme proteins has not been documented. To establish spectroscopic signatures for such systems, we have prepared five-coordinate adducts of the ferrous myoglobin H94G cavity mutant with neutral thiol and thioether sulfur donors as well as six-coordinate derivatives such as with CO and, when possible, with NO and O(2). A thiol-ligated oxyferrous complex is reported, to our knowledge for the first time. Further, a bis-thioether ferrous H93G model for bis-methionine ligation, as found in Pseudomonas aeruginosa bacterioferritin heme protein, is described. Magnetic CD spectroscopy has been used due to its established ability in axial ligand identification. The magnetic CD spectra of the H93G complexes have been compared with those of ferrous H175CD235L cytochrome c peroxidase to show that its proximal ligand is neutral cysteine. We had previously reported this cytochrome c peroxidase mutant to be cysteinate-ligated in the ferric state, but the ferrous ligand was undetermined. The spectral properties of ferrous liver microsomal cytochrome P420 (inactive P450) are also consistent with thiol ligation. This study establishes that neutral cysteine can serve as a ligand in ferrous heme iron proteins, and that ferric cysteinate-ligated heme proteins that fail to retain such ligation on reduction may simply be ligated by neutral cysteine.

Circular Dichroism↗
PubMed2003

Spectroscopic characterization of five- and six-coordinate ferrous-NO heme complexes. Evidence for heme Fe-proximal cysteinate bond cleavage in the ferrous-NO adducts of the Trp-409Tyr/Phe proximal environment mutants of neuronal nitric oxide synthase.

Nitric oxide synthases (NOS) are a family of cysteine thiolate-ligated heme-containing monooxygenases that catalyze the NADPH-dependent two-step conversion of L-arginine to NO and L-citrulline. During the catalysis, a portion of the NOS heme forms an inhibitory complex with self-generated NO that is subsequently reverted back to NO-free active enzyme under aerobic conditions, suggesting a downstream regulator role of NO. Recent studies revealed that mutation of a conserved proximal tryptophan-409, which forms one of three hydrogen bonds to the heme-coordinated cysteine thiolate, to tyrosine or phenylalanine considerably increases the turnover number of neuronal NOS (nNOS). To further understand these properties of nNOS on its active site structural level, we have examined the oxygenase (heme-containing) domain of the two mutants in close comparison with that of wild-type nNOS with UV-visible absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopy. Among several oxidation and ligation states examined, only the ferrous-NO adducts of the two mutants exhibit spectra that are markedly distinct from those of parallel derivatives of the wild-type protein. The spectra of the ferrous-NO mutants are broadly similar to those of known five-coordinate ferrous-NO heme complexes, suggesting that these mutants are predominantly five coordinate in their ferrous-NO states. The present results are indicative of cleavage of the Fe-S bond in the nNOS mutants in their ferrous-NO state and imply a significant role of the conserved tryptophan in stabilization of the Fe-S bond.

Circular Dichroism↗