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Thomas C Pochapsky

Publications and source records attributed to Thomas C Pochapsky.

14 recordsLinked to original sources
PubMed2006

Specific effects of potassium ion binding on wild-type and L358P cytochrome P450cam.

The camphor monoxygenase cytochrome P450cam (CYP101) requires potassium ion (K+) to drive formation of the characteristic high-spin state of the heme Fe+3 upon substrate binding. Amide 1H, 15N correlations in perdeuterated [U-15N] CYP101 were monitored as a function of K+ concentration by 2D-TROSY-HSQC in both camphor-bound oxidized (CYP-S) and camphor- and CO-bound reduced CYP101 (CYP-S-CO). In both forms, K+-induced spectral perturbations are detected in the vicinity of the K+ binding site proposed from crystallographic structures, but are larger and more widespread structurally in CYP-S than in CYP-S-CO. In CYP-S-CO, K+-induced perturbations occur primarily near the proposed K+ binding site in the B-B' loop and B' helix, which are also perturbed by binding of effector, putidaredoxin (Pdx). The spectral effects of K+ binding in CYP-S-CO oppose those observed upon Pdxr titration. However, Pdxr titration of CYP-S-CO in the absence of K+ results in multiple conformations. The spin-state equilibrium in the L358P mutant of CYP101 is more sensitive to K+ concentration than WT CYP101, consistent with a hypothesis that L358P preferentially populates conformations enforced by Pdx binding in WT CYP101. Thallium(I), a K+ mimic, minimizes the effects of Pdx titration on the NMR spectrum of CYP-S-CO, but is competent to replace K+ in driving the formation of high-spin CYP-S. These observations suggest that the role of K+ is to stabilize conformers of CYP-S that drive the spin-state change prior to the first electron transfer, and that K+ stabilizes the CYP-S-CO conformer that interacts with Pdx. However, upon binding of Pdx, further conformational changes occur that disfavor K+ binding.

Camphor 5-Monooxygenase↗
PubMed2006

One protein, two enzymes revisited: a structural entropy switch interconverts the two isoforms of acireductone dioxygenase.

Acireductone dioxygenase (ARD) catalyzes different reactions between O2 and 1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene (acireductone) depending upon the metal bound in the active site. Ni2+ -ARD cleaves acireductone to formate, CO and methylthiopropionate. If Fe2+ is bound (ARD'), the same substrates yield methylthioketobutyrate and formate. The two forms differ in structure, and are chromatographically separable. Paramagnetism of Fe2+ renders the active site of ARD' inaccessible to standard NMR methods. The structure of ARD' has been determined using Fe2+ binding parameters determined by X-ray absorption spectroscopy and NMR restraints from H98S ARD, a metal-free diamagnetic protein that is isostructural with ARD'. ARD' retains the beta-sandwich fold of ARD, but a structural entropy switch increases order at one end of a two-helix system that bisects the beta-sandwich and decreases order at the other upon interconversion of ARD and ARD', causing loss of the C-terminal helix in ARD' and rearrangements of residues involved in substrate orientation in the active site.

Binding Sites↗
PubMed2006

Comparison of the complexes formed by cytochrome P450cam with cytochrome b5 and putidaredoxin, two effectors of camphor hydroxylase activity.

Structural perturbations in cytochrome P450cam (CYP101) induced by the soluble fragment of cytochrome b5, a nonphysiological effector of CYP101, were investigated by NMR spectroscopy and compared with the perturbations induced by the physiological reductant and effector putidaredoxin (Pdx). Chemical shifts of perdeuterated [U-15N]CYP101 backbone amide (NH) resonances were monitored as a function of cytochrome b5 concentration by 1H-15N TROSY-HSQC experiments. The association of cytochrome b5 with the reduced CYP101-camphor-carbon monoxide complex (CYP-S-CO) perturbs many of the same resonances that Pdx does, including regions of the CYP101 molecule implicated in substrate access and orientation. The perturbations are smaller in magnitude than those observed with Pdx(r) due to a lower binding affinity (a Kd of 13 +/- 3 mM, for the reduced cytochrome b5-CYP-S-CO complex compared to a Kd of 26 +/- 12 microM for the Pdx-CYP-S-CO complex). The results are in accord with our previous suggestion that the observed perturbations are related to effector activity and support the proposal that the primary role of the effector is to populate the active conformation of CYP101 to prevent uncoupling [Pochapsky, S. S., et al. (2003) Biochemistry 42, 5649-5656]. A titratable perturbation is observed at the 1H resonance of the 8-CH3 group of CYP101-bound camphor upon addition of cytochrome b5, a phenomenon also associated with the formation of the CYP101 x Pdx complex, albeit with larger perturbations [Wei, J. Y., et al. (2005) J. Am. Chem. Soc. 127, 6974-6976]. The effector activity of the particular rat cytochrome b5 construct used for NMR studies was confirmed by monitoring the enzymatic turnover that yielded 5-exo-hydroxycamphor using gas chromatography and mass spectrometry. Finally, the common features of the perturbations observed in the NMR spectra of the two complexes are discussed, and their relevance to effector activity is considered.

Amino Acid Sequence↗
PubMed2006

A refined model for the structure of acireductone dioxygenase from Klebsiella ATCC 8724 incorporating residual dipolar couplings.

Acireductone dioxygenase (ARD) from Klebsiella ATCC 8724 is a metalloenzyme that is capable of catalyzing different reactions with the same substrates (acireductone and O2) depending upon the metal bound in the active site. A model for the solution structure of the paramagnetic Ni2+-containing ARD has been refined using residual dipolar couplings (RDCs) measured in two media. Additional dihedral restraints based on chemical shift (TALOS) were included in the refinement, and backbone structure in the vicinity of the active site was modeled from a crystallographic structure of the mouse homolog of ARD. The incorporation of residual dipolar couplings into the structural refinement alters the relative orientations of several structural features significantly, and improves local secondary structure determination. Comparisons between the solution structures obtained with and without RDCs are made, and structural similarities and differences between mouse and bacterial enzymes are described. Finally, the biological significance of these differences is considered.

Bacterial Proteins↗
PubMed2005

Detection of a high-barrier conformational change in the active site of cytochrome P450cam upon binding of putidaredoxin.

The orientation of the substrate camphor in the active site of reduced CO-bound cytochrome P450cam (CYP101) as a function of reduced putidaredoxin (Pdxr) addition has been examined by NMR using perdeuterated CYP101 and perdeuterated Pdx as well as isotopically labeled d-camphor. This permits the 1H resonances of CYP101-bound camphor to be observed without interference from the signals of CYP101 or Pdx and confirms assignments of the methyl signals of camphor in the bound form. The Cys4Fe2S2 ferredoxin Pdx is the physiological redox partner and effector of CYP101. The addition of Pdx to the reduced CYP101-camphor-CO complex results in a conformational selection that is slow on the chemical shift time scale with spectral effects observed primarily at the 8-CH3 group of the camphor. The camphor signals are ring current shifted by the heme, and for the 9- and 10-CH3 resonances, these shifts are reasonably well predicted by ring current calculations from the crystal structure of CO-bound CYP101. However, in the absence of Pdx, the 8-CH3 resonance of CYP101-bound camphor is observed at considerably higher field than predicted. Dynamic simulations using ring current shift restraints generated a structure with low chemical shift violations in which the hydrogen bond between the camphor carbonyl oxygen and the OH of Tyr96 is lost, and an expansion of the active site takes place that permits reorientation of the camphor within the active site.

Binding Sites↗
PubMed2005

The immediate-early ethylene response gene OsARD1 encodes an acireductone dioxygenase involved in recycling of the ethylene precursor S-adenosylmethionine.

Methylthioadenosine (MTA) is formed as a by-product of ethylene biosynthesis from S-adenosyl-L-methionine (AdoMet). The methionine cycle regenerates AdoMet from MTA. In two independent differential screens for submergence-induced genes and for 1-aminocyclopropane-1-carboxylic acid (ACC)-induced genes from deepwater rice (Oryza sativa L.) we identified an acireductone dioxygenase (ARD). OsARD1 is a metal-binding protein that belongs to the cupin superfamily. Acireductone dioxygenases are unique proteins that can acquire two different activities depending on the metal ion bound. Ectopically expressed apo-OsARD1 preferentially binds Fe(2+) and reconstituted Fe-OsARD1 catalyzed the formation of 2-keto-pentanoate and formate from the model substrate 1,2-dihydroxy-3-ketopent-1-ene and dioxygen, indicating that OsARD1 is capable of catalyzing the penultimate step in the methionine cycle. Two highly homologous ARD genes were identified in rice. OsARD1 mRNA levels showed a rapid, early and transient increase upon submergence and after treatment with ethylene-releasing compounds. The second gene from rice, OsARD2, is constitutively expressed. Accumulation of OsARD1 transcript was observed in the same internodal tissues, i.e. the meristem and elongation zone, which were previously shown to synthesize ethylene. OsARD1 transcripts accumulated in the presence of cycloheximide, an inhibitor of protein synthesis, indicating that OsARD1 is a primary ethylene response gene. Promoter analysis suggests that immediate-early regulation of OsARD1 by ethylene may involve an EIN3-like transcription factor. OsARD1 is induced by low levels of ethylene. We propose that early feedback activation of the methionine cycle by low levels of ethylene ensures the high and continuous rates of ethylene synthesis required for long-term ethylene-mediated submergence adaptation without depleting the tissue of AdoMet.

Amino Acid Sequence↗
PubMed2004

Analogs of 1-phosphonooxy-2,2-dihydroxy-3-oxo-5-(methylthio)pentane, an acyclic intermediate in the methionine salvage pathway: a new preparation and characterization of activity with E1 enolase/phosphatase from Klebsiella oxytoca.

The methionine salvage pathway allows the in vivo recovery of the methylthio moiety of methionine upon the formation of methylthioadenosine (MTA) from S-adenosylmethionine (SAM). The Fe(II)-containing form of acireductone dioxygenase (ARD) catalyzes the penultimate step in the pathway in Klebsiella oxytoca, the oxidative cleavage of the acireductone 1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene (2) by dioxygen to give formate and 2-oxo-4-(methylthio)butyrate (3). The Ni(II)-bound form (Ni-ARD) catalyzes an off-pathway shunt, forming 3-(methylthio)propionate (4), carbon monoxide, and formate. Acireductone 2 is formed by the action of another enzyme, E1 enolase/phosphatase, on precursor 1-phosphonooxy-2,2-dihydroxy-3-oxo-5-methylthiopentane (1). Simple syntheses of several analogs of 1 are described, and their activity as substrates for E1 enolase/phosphatase characterized. A new bacterial overexpression system and purification procedure for E1, a member of the haloacid dehalogenase (HAD) superfamily, is described, and further characterization of the enzyme presented.

Amino Acid Sequence↗
PubMed2003

A conserved histidine in vertebrate-type ferredoxins is critical for redox-dependent dynamics.

Adrenodoxin (Adx) belongs to the family of Cys(4)Fe(2)S(2) vertebrate-type ferredoxins that shuttle electrons from NAD(P)H-dependent reductases to cytochrome P450 enzymes. The vertebrate-type ferredoxins contain a conserved basic residue, usually a histidine, adjacent to the third cysteine ligand of the Cys(4)Fe(2)S(2) cluster. In bovine Adx the side chain of this residue, His 56, is involved in a hydrogen-bonding network within the domain of Adx that interacts with redox partners. It has been proposed that this network acts as a mechanical link between the metal cluster binding site and the interaction domain, transmitting redox-dependent conformational or dynamical changes from the cluster binding loop to the interaction domain. H/D exchange studies indicate that oxidized Adx (Adx(o)) is more dynamic than reduced Adx (Adx(r)) on the kilosecond time scale in many regions of the protein, including the interaction domain. Dynamical differences on picosecond to nanosecond time scales between the oxidized (Adx(o)) and reduced (Adx(r)) adrenodoxin were probed by measurement of (15)N relaxation parameters. Significant differences between (15)N R(2) rates were observed for all residues that could be measured, with those rates being faster in Adx(o) than in Adx(r). Two mutations of His 56, H56R and H56Q, were also characterized. No systematic redox-dependent differences between (15)N R(2) rates or H/D exchange rates were observed in either mutant, indicating that His 56 is required for the redox-dependent behavior observed in WT Adx. Comparison of chemical shift differences between oxidized and reduced H56Q and H56R Adx confirms that redox-dependent changes are smaller in these mutants than in the wild-type Adx.

Animals↗
PubMed2003

A model for effector activity in a highly specific biological electron transfer complex: the cytochrome P450(cam)-putidaredoxin couple.

The camphor hydroxylase cytochrome P450(cam) (CYP101) catalyzes the 5-exo hydroxylation of camphor in the first step of camphor catabolism by Pseudomonas putida. CYP101 forms a specific electron transfer complex with its physiological reductant, the Cys(4)Fe(2)S(2) ferredoxin putidaredoxin (Pdx). Pdx, along with other proteins and small molecules, has also been shown to be an effector for turnover by CYP101. Multidimensional nuclear magnetic resonance (NMR) techniques have been used to make extensive sequential (1)H, (15)N, and (13)C resonance assignments in CYP101 that permit a more complete characterization of the complex formed by CYP101 and Pdx. NMR-detected perturbations in CYP101 upon Pdx binding encompass regions of the CYP101 remote from the putative Pdx binding site, including in particular a region of the CYP101 molecule that has been implicated in substrate access to the active site via dynamical processes. A model for effector activity is proposed in which the primary role of the effector is to prevent uncoupling (formation of reduced oxo species without formation of hydroxycamphor) by enforcing conformations of CYP101 that prevent loss of substrate and/or intermediates prior to turnover. A secondary role could also be to enforce conformations that permit efficient proton transfer into the active site for coupled proton/electron transfer.

Amino Acid Sequence↗
PubMed2002

Rapid recycle (13)C',(15)N and (13)C,(13)C' heteronuclear and homonuclear multiple quantum coherence detection for resonance assignments in paramagnetic proteins: example of Ni(2+)-containing acireductone dioxygenase.

NMR resonance assignments in the vicinity of paramagnetic metals in proteins are often difficult or impossible to make using conventional 1H detected 2-D and 3-D methods due to paramagnetic line broadening. The applicability of 13Calpha{13C'} and 13C'{15N} multiple quantum coherence methods for residue-specific assignments of resonances near paramagnetic centers is described, using the Ni2+-containing enzyme acireductone dioxygenase as an example.

Dioxygenases↗
PubMed2002

XAS investigation of the structure and function of Ni in acireductone dioxygenase.

Acireductone dioxygenases (ARDs) are enzymes involved in the methionine recycle pathway, which regulates aspects of the cell cycle. Klebsiella pneumoniae produces two enzymes that share a common polypeptide sequence and differ only in the metal ion present. Reaction of acireductone (1,2-dihydroxy-3-keto-5-methylthiopentene) with Fe-ARD and dioxygen produces formate and 2-keto-4-methylthiobutanoic acid, the alpha-ketoacid precursor of methionine. Ni-ARD reacts with acireductone and dioxygen to produce methylthiopropionate, CO, and formate and does not lie on the methionine recycle pathway. An X-ray absorption spectroscopy (XAS) study of the structure of the catalytic Ni center in resting Ni-ARD enzyme and the enzyme-substrate complex is reported. This study establishes the structure of the Ni site in resting Ni-ARD as containing a six coordinate Ni site composed of O/N-donor ligands including 3-4 histidine residues, demonstrates that the substrate binds to the Ni center in a bidentate fashion by displacing two ligands, at least one of which is a histidine ligand, and provides insight into the mechanism of catalysis employed by a Ni-containing dioxygenase. Efficiently relaxed and hyperfine-shifted resonances are observed in the (1)H nuclear magnetic resonance spectrum of Ni-ARD that can be attributed to the His imidazoles ligating the paramagnetic Ni ion and are consistent with the XAS results regarding His ligation. These resonances show significant perturbation in the presence of substrate, confirming that the metal ion interacts directly with the substrate.

Catalytic Domain↗
PubMed2002

Comparison of functional domains in vertebrate-type ferredoxins.

The vertebrate-type Cys(4)Fe(2)S(2) ferredoxins are a class of small acidic proteins that typically act as electron shuttles between NAD(P)H-dependent reductases and monoxygenases, particularly cytochromes P450. Nuclear magnetic resonance assignments and detailed analysis of nuclear Overhauser effects permit the direct comparison of the functional C-terminal domains of three vertebrate-type ferredoxins, the mammalian adrenodoxin (Adx) and the bacterial ferredoxins putidaredoxin (Pdx) and terpredoxin (Tdx). In particular, homologous hydrogen-bonding networks involving a conserved basic residue (His 49 in Pdx, His 56 in Adx, Arg 49 in Tdx) are detailed. This hydrogen bond network appears to play a role in the mechanical transmission of redox-dependent conformational and dynamic changes from the iron-sulfur binding loop to the C-terminal domain. Hydrogen/deuterium exchange measurements have been made in Adx as a function of oxidation state for comparison with previous studies of Pdx and Tdx. The results of these measurements highlight the importance of the conserved basic residue in the linkage between oxidation state and protein dynamics. Finally, a series of mutations have been made in the C-terminal domain of Pdx, including one, Y51F, that disrupts the proposed hydrogen-bonding network without perturbing steric and hydrophobic interactions in the functional domain. Although the mutant is considerably destabilized with respect to wild-type Pdx, relatively unperturbed chemical shifts for residues near the site of the mutation and NOEs between water and Phe 51 suggest that the network is reconstituted with a solvent water in place of the tyrosine hydroxyl group in this mutant.

Adrenodoxin↗
PubMed2002

Modeling and experiment yields the structure of acireductone dioxygenase from Klebsiella pneumoniae.

Here we report the structure of acireductone dioxygenase (ARD), the first determined for a new family of metalloenzymes. ARD represents a branch point in the methionine salvage pathway leading from methylthioadenosine to methionine and has been shown to catalyze different reactions depending on the type of metal ion bound in the active site. The solution structure of nickel-containing ARD (Ni-ARD) was determined using NMR methods. X-ray absorption spectroscopy, assignment of hyperfine shifted NMR resonances and conserved domain homology were used to model the metal-binding site because of the paramagnetism of the bound Ni2+. Although there is no structure in the Protein Data Bank within 3 A r.m.s deviation of that of Ni-ARD, the enzyme active site is located in a conserved double-stranded b-helix domain. Furthermore, the proposed Ni-ARD active site shows significant post-facto structural homology to the active sites of several metalloenzymes in the cupin superfamily.

Amino Acid Sequence↗