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[Display omitted] Many economically important biosyntheses incorporate regiospecific and stereospecific oxidations at unactivated carbons. Such oxidations are commonly catalyzed by cytochrome P450 monooxygenases, heme-containing enzymes that activate molecular oxygen while selectively binding and orienting the substrate for reaction. Despite the plethora of P450-catalyzed reactions, the P450 fold is highly conserved, and static structures are often insufficient for characterizing conformational states that contribute to specificity. High-resolution solution nuclear magnetic resonance (NMR) offers insights into dynamic processes and conformational changes that are required of a P450 in order to attain the combination of specificity and efficiency required for these reactions. [ABSTRACT FROM AUTHOR]

Cytochrome P450 cam (CYP101A1) catalyzes the stereospecific 5- exo hydroxylation of d -camphor by molecular oxygen. Previously, residual dipolar couplings measured for backbone amide 1 H– 15 N correlations in both substrate-free and bound forms of CYP101A1 were used as restraints in soft annealing molecular dynamic simulations in order to identify average conformations of the enzyme with and without substrate bound. Multiple substrate-dependent conformational changes remote from the enzyme active site were identified, and site-directed mutagenesis and activity assays confirmed the importance of these changes in substrate recognition. The current work makes use of perturbation response scanning (PRS) and umbrella sampling molecular dynamic of the residual dipolar coupling-derived CYP101A1 structures to probe the roles of remote structural features in enforcing the regio- and stereospecific nature of the hydroxylation reaction catalyzed by CYP101A1. An improper dihedral angle Ψ was defined and used to maintain substrate orientation in the CYP101A1 active site, and it was observed that different values of Ψ result in different PRS response maps. Umbrella sampling methods show that the free energy of the system is sensitive to Ψ, and bound substrate forms an important mechanical link in the transmission of mechanical coupling through the enzyme structure. Finally, a qualitative approach to interpreting PRS maps in terms of the roles of secondary structural features is proposed. [ABSTRACT FROM AUTHOR]

Acireductone dioxygenase (ARD) from the methionine salvage pathway (MSP) is a unique enzyme that exhibits dual chemistry determined solely by the identity of the divalent transition-metal ion (Fe2+ or Ni2+) in the active site. The Fe2+-containing isozyme catalyzes the on-pathway reaction using substrates 1,2-dihydroxy-3-keto-5-methylthiopent-1-ene (acireductone) and dioxygen to generate formate and the ketoacid precursor of methionine, 2-keto-4-methylthiobutyrate, whereas the Ni2+-containing isozyme catalyzes an off-pathway shunt with the same substrates, generating methylthiopropionate, carbon monoxide, and formate. The dual chemistry of ARD was originally discovered in the bacterium Klebsiella oxytoca, but it has recently been shown that mammalian ARD enzymes (mouse and human) are also capable of catalyzing metal-dependent dual chemistry in vitro. This is particularly interesting, since carbon monoxide, one of the products of off-pathway reaction, has been identified as an antiapoptotic molecule in mammals. In addition, several biochemical and genetic studies have indicated an inhibitory role of human ARD in cancer. This comprehensive review describes the biochemical and structural characterization of the ARD family, the proposed experimental and theoretical approaches to establishing mechanisms for the dual chemistry, insights into the mechanism based on comparison with structurally and functionally similar enzymes, and the applications of this research to the field of artificial metalloenzymes and synthetic biology. [ABSTRACT FROM AUTHOR]

Acireductone dioxygenase (ARD) from the methionine salvage pathway of Klebsiella oxytoca is the only known naturally occurring metalloenzyme that catalyzes different reactions in vivo based solely on the identity of the divalent transition metal ion (Fe2+ or Ni2+) bound in the active site. The ironcontaining isozyme catalyzes the cleavage of substrate 1,2-dihydroxy-3-keto-5-(thiomethyl)pent-1-ene (acireductone) by O2 to formate and the ketoacid precursor of methionine, whereas the nickelcontaining isozyme uses the same substrates to catalyze an off-pathway shunt to form methylthiopropionate, carbon monoxide and formate. This dual chemistry was recently demonstrated in vitro by ARD from Mus musculus (MmARD), providing the first example of a mammalian ARD exhibiting metal-dependent catalysis. We now show that human ARD (HsARD) is also capable of metaldependent dual chemistry. Recombinant HsARD was expressed and purified to obtain a homogeneous enzyme with a single transition metal ion bound. As with MmARD, the Fe2+-bound HsARD shows the highest activity and catalyzes on-pathway chemistry, whereas Ni2+, Co2+ or Mn2+ forms catalyze off-pathway chemistry. The thermal stability of the HsARD isozymes is a function of the metal ion identity, with Ni2+-bound HsARD being the most stable followed by Co2+ and Fe2+, and Mn2+-bound HsARD being the least stable. As with the bacterial ARD, solution NMR data suggest that HsARD isozymes can have significant structural differences depending upon the metal ion bound. [ABSTRACT FROM AUTHOR]

R5823 --> 539.9 --> Human cytochrome P450 (CYP) enzymes are versatile, dynamic, and essential to many aspects of human health. The catalytic versatility of this superfamily of enzymes is driven by structural dynamics, which are often elusive to static structural techniques. For this reason, solution nuclear magnetic resonance (NMR) is likely to provide useful structural information orthogonal to X‐ray crystallography. The challenges of human P450 NMR studies are two‐pronged: 1) Human P450 enzymes are membrane‐bound, and often aggregate or precipitate in aqueous environments, and 2) P450 enzymes contain a paramagnetic heme iron buried within the active site, leading to decreased intensity of NMR correlations corresponding to nearby amino acid residues. We have overcome both obstacles with the human steroidogenic P450 enzyme CYP17A1 in complex with a novel substrate derivative, 3‐formyl pregnane‐20‐isonitrile1. The steroid skeleton of this ligand is similar to that of CYP17A1 endogenous substrates and therapeutics, but in this case the C20 isonitrile functional group directly coordinates both the oxidized (Fe3+) and the reduced (Fe2+) diamagnetic states of the heme iron. As a result, this compound both stabilizes CYP17A1 and suppresses paramagnetic relaxation and will facilitate application of multidimensional solution NMR to CYP17A1. The dissociation constant (Kd) and inhibition (IC50) of this isonitrile have been determined and compared to the prostate cancer drug abiraterone, which is the tightest‐binding CYP17A1 inhibitor to date. Structural characterization of this complex by solution NMR is ongoing, but this strategy should open the door to new methods of structural analysis of dynamic human P450 enzymes. [ABSTRACT FROM AUTHOR]

The camphor hydroxylase cytochrome P450[sub 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[sub 4]Fe[sub 2]S[sub 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 ¹H, [sup 15]N, and [sup 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. [ABSTRACT FROM AUTHOR]

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 Å 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. [ABSTRACT FROM AUTHOR]

Two dioxygenases (ARD and ARD′) were cloned from Klebsiella pneumoniae that catalyze different oxidative decomposition reactions of an advanced aci-reductone intermediate, CH[SUB3]SCH[SUB2]CH[SUB2]COCH(OH)=CH(OH) (I), in the methionine salvage pathway. The two enzymes are remarkable in that they have the same polypeptide sequence but bind different metal ions (Ni[SUP2+] and Fe[SUP2+], respectively). ARD converts I to CH[SUB3]SCH[SUB2]CH[SUB2]COOH, CO, and HCOOH. ARD′ converts I to CH[SUB3]SCH[SUB2]CH[SUB2]COCOOH and HCOOH. Kinetic analyses suggest that both ARD and ARD′ have ordered sequential mechanisms. A model substrate (II), a dethio analogue of I, binds to the enzyme first as evidenced by its λ[SUBmax] red shift upon binding. The dianion formation from II causes the same λ[SUBmax] red shift, suggesting that II bind to the enzyme as a dianion. The electron-rich II dianion likely reacts with O[SUB2] to form a peroxide anion intermediate. Previous [SUP18]O[SUB2] and [SUP14]C tracer experiments established that ARD incorporates [SUP18]O[SUB2] into C[SUB1] and C[SUB3] of II and C[SUB2] is released as CO. ARD′ incorporates [SUP18]O[SUB2] into C[SUB1] and C[SUB2] of II. The product distribution seems to necessitate the formation of a five-membered cyclic peroxide intermediate for ARD and a four-membered cyclic peroxide intermediate for ARD′. A model chemical reaction demonstrates the chemical and kinetic competency of the proposed five-membered cyclic peroxide intermediate. The breakdown of the four-membered and five-membered cyclic peroxide intermediates gives the ARD′ and ARD products, respectively. The nature of the metal ion appears to dictate the attack site of the peroxide anion and, consequently, the different cyclic peroxide intermediates and the different oxidative cleavages of II. A cyclopropyl substrate analogue inactivates both enzymes after multiple turnovers, providing evidence that a radical... [ABSTRACT FROM AUTHOR]

Studies the redox-dependent conformation selection in a Cys[sub 4]Fe[sub 2]S[sub 2] ferredoxin from Pseudomonas putida. Stability, metal cluster incorporation and spectroscopic characterization of mutants; Time scales of affected motions; Evidence for redox-dependent conformational changes from hyperfine-shifted [sup 15]N and [sup 13]C resonances.

Presents a refined model for the solution structure of oxidized putidaredoxin (Pdx o), a Cys4Fe2S2 ferredoxin. Diamagnetic 15N and 13C resonances of Pdx o; Improvement of precision of restraints on the nuclear Overhauser effect; Structure of metal binding site.

Examines the structural constraints on the C-terminal region of the globular protein putidaredoxin and describes structural features that are likely are to be located in or near the protein-protein interface in the diprotein complex. Differences in hyperfine broadening of nuclear magnetic resonances; Chemical shift changes.

Presents a model for the solution structure of oxidized putidaredoxin (Pdx), a globular protein, as determined by homonuclear nuclear magnetic resonance methods. Features of the Pdx solution structure model; Limitations on the model.

Examines the influence of ion pairing on the physical and chemical behavior of ionic solutions using interionic nuclear Overhauser effects (NOEs). Evaluation on the time average structure of ion pairs; Overall motion of the ion pair; Relation of the orientation of the ion on the time scale of picoseconds.

The article informs that Cytochrome CYP101 catalyzes the 5-exo-hydroxylation of camphor 1, the first step of catabolism of 1 by the soil bacterium Pseudomonas putida. Two electrons are required for turnover, and the second reduction, of the Fe(II) CYP101 ternary complex, is the rate-limiting step under physiological conditions. The ferredoxin putidaredoxin (Pdx) is the biological effector and reductant of CYP101. Structural perturbations have been observed spectroscopically in the CYP101 active site upon binding of Pdx, and it has been proposed that these perturbations are involved in the effector and/or electron-transfer activity of Pdx.

Multiple factors involving the methionine salvage pathway (MSP) and polyamine biosynthesis have been found to be involved in cancer cell proliferation, migration, invasion and metastasis. This review summarizes the relationships of the MSP enzyme acireductone dioxygenase (ARD), the ADI1 gene encoding ARD and other gene products (ADI1GP) with carcinomas and carcinogenesis. ARD exhibits structural and functional differences depending upon the metal bound in the active site. In the penultimate step of the MSP, the Fe2+ bound form of ARD catalyzes the on-pathway oxidation of acireductone leading to methionine, whereas Ni2+ bound ARD catalyzes an off-pathway reaction producing methylthiopropionate and carbon monoxide, a biological signaling molecule and anti-apoptotic. The relationship between ADI1GP, MSP and polyamine synthesis are discussed, along with possible role(s) of metal in modulating the cellular behavior of ADI1GP and its interactions with other cellular components. [ABSTRACT FROM AUTHOR]

The author discusses the αsynuclein (αSyn) tetramer. Topics discussed include association of in vivo multimerization state of the neuronal protein αSyn with neurotoxicity, identification αSyn as a component of Alzheimer's plaques from human brain tissue, and insoluble which serves a hallmark for Parkinson's disease (PD).

The author discusses aspects of analyzing the self-organization of enzymes in a membrane. He mentions their dynamic structures which respond to environmental changes as well as the impact of cofactors, substrate and ligand oxidation states on their oligomerization. An overview of the study on the structural complex of sterol 14α-demethylase (CYP51) is also presented.

Alkyl isonitriles, R—NC, have previously been shown to ligate the heme (haem) iron of cytochromes P450 in both accessible oxidation states (ferrous, Fe2+, and ferric, Fe3+). Herein, the preparation of four steroid-derived isonitriles and their interactions with several P450s, including the steroidogenic CYP17A1 and CYP106A2, as well as the more promiscuous drug metabolizers CYP3A4 and CYP2D6, is described. It was found that successful ligation of the heme iron by the isonitrile functionality for a given P450 depends on both the position and stereochemistry of the isonitrile on the steroid skeleton. Spectral studies indicate that isonitrile ligation of the ferric heme is stable upon reduction to the ferrous form, with reoxidation resulting in the original complex. A crystallographic structure of CYP17A1 with an isonitrile derived from pregnanalone further confirmed the interaction and identified the absolute stereochemistry of the bound species. all: The inhibition of cytochrome P450 (CYP) enzymes is central to many therapeutics, however, achieving selectivity across CYP enzymes remains challenging. Here, the authors develop isonitrile-harboring steroids as selective steroidogenic CYP heme iron ligands. [ABSTRACT FROM AUTHOR]

Studies the conversion of a Fe2S2 ferredoxin into a Ga3+ rubredoxin. Synthesis; Structure; Properties; Reaction schemes; Products.

The two acireductone dioxygenase (ARD) isozymes from the methionine salvage pathway of Klebsiella oxytoca are the only known pair of naturally occurring metalloenzymes with distinct chemical and physical properties determined solely by the identity of the divalent transition metal ion (Fe2+ or Ni2+) in the active site. We now show that this dual chemistry can also occur in mammals. ARD from Mus musculus (MmARD) was studied to relate the metal ion identity and three-dimensional structure to enzyme function. The iron-containing isozyme catalyzes the cleavage of 1,2-dihydroxy-3-keto-5-(thiomethyl)pent-1-ene (acireductone) by O2 to formate and the ketoacid precursor of methionine, which is the penultimate step in methionine salvage. The nickel-bound form of ARD catalyzes an off-pathway reaction resulting in formate, carbon monoxide (CO), and 3-(thiomethyl) propionate. Recombinant MmARD was expressed and purified to obtain a homogeneous enzyme with a single transition metal ion bound. The Fe2+-bound protein, which shows about 10-fold higher activity than that of others, catalyzes on-pathway chemistry, whereas the Ni2+, Co2+, or Mn2+ forms exhibit off-pathway chemistry, as has been seen with ARD from Klebsiella. Thermal stability of the isozymes is strongly affected by the metal ion identity, with Ni2+-bound MmARD being the most stable, followed by Co2+ and Fe2+, and Mn2+-bound ARD being the least stable. Ni2+- and Co2+-bound MmARD were crystallized, and the structures of the two proteins found to be similar. Enzyme-ligand complexes provide insight into substrate binding, metal coordination, and the catalytic mechanism. [ABSTRACT FROM AUTHOR]

Abstract: The 58-kDa complex formed between the [2Fe–2S] ferredoxin, putidaredoxin (Pdx), and cytochrome P450cam (CYP101) from the bacterium Pseudomonas putida has been investigated by high-resolution solution NMR spectroscopy. Pdx serves as both the physiological reductant and effector for CYP101 in the enzymatic reaction involving conversion of substrate camphor to 5-exo-hydroxycamphor. In order to obtain an experimental structure for the oxidized Pdx–CYP101 complex, a combined approach using orientational data on the two proteins derived from residual dipolar couplings and distance restraints from site-specific spin labeling of Pdx has been applied. Spectral changes for residues in and near the paramagnetic metal cluster region of Pdx in complex with CYP101 have also been mapped for the first time using 15N and 13C NMR spectroscopy, leading to direct identification of the residues strongly affected by CYP101 binding. The new NMR structure of the Pdx–CYP101 complex agrees well with results from previous mutagenesis and biophysical studies involving residues at the binding interface such as formation of a salt bridge between Asp38 of Pdx and Arg112 of CYP101, while at the same time identifying key features different from those of earlier modeling studies. Analysis of the binding interface of the complex reveals that the side chain of Trp106, the C-terminal residue of Pdx and critical for binding to CYP101, is located across from the heme-binding loop of CYP101 and forms non-polar contacts with several residues in the vicinity of the heme group on CYP101, pointing to a potentially important role in complex formation. [Copyright &y& Elsevier]

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 Pdxr 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 µM 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 CYP 101 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 CYP 101-bound camphor upon addition of cytochrome b5, a phenomenon also associated with the formation of the CYP101Pdx complex, albeit with larger perturbations [Wei, J. Y., et al. (2005) J. Am. Chem. Soc. 127, 6974-69761. 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. [ABSTRACT FROM AUTHOR]

Adrenodoxin (Adx) belongs to the family of Cys[sub 4]Fe[sub 2]S[sub 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[sub 4]Fe[sub 2]S[sub 2] cluster. In bovine Adx the side chain of this residue, His 56, is involved in a hydrogenbonding 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[sup o]) is more dynamic than reduced Adx (Adx[sup 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[sup o]) and reduced (Adx[sup r]) adrenodoxin were probed by measurement of [sup 15]N relaxation parameters. Significant differences between [sup 15]N R[sub 2] rates were observed for all residues that could be measured, with those rates being faster in Adx[sup o] than in Adx[sup r]. Two mutations of His 56, H56R and H56Q, were also characterized. No systematic redox-dependent differences between [sup 15]N R[sub 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 redoxdependent changes are smaller in these mutants than in the wild-type Adx. [ABSTRACT FROM AUTHOR]

Abstract A strategy for elucidating sequence determinants of function in the class of cytochrome P450 (CYP) enzymes that catalyze the first steps of terpene metabolism in wild microbiomes is described. Wild organisms that can use camphor, terpineol, pinene and limonene were isolated from soils rich in coniferous waste. Cell free extracts and growth beers were analyzed by gas chromatography/mass spectrometry to identify primary oxidative metabolites. For one organism, Pseudomonas nitroreducens TPJM, a cytochrome P450 (CYP108B1) isolated from cell free extracts was demonstrated to catalyze the oxidation of α-terpineol in assays combining the native ferredoxin and putidaredoxin reductase, and the resulting oxidation products identified by gas chromatography/mass spectrometry. Shotgun sequencing of Pn TPJM identified four candidate P450 genes, including an apparently fragmentary gene with a high degree of homology with the known enzyme CYP108 (P450 terp). Graphical abstract Structural model of a novel cytochrome P450 (CYP108B1), with identified metabolic products of terpineol from the organism in which P450 TPJM is found. [ABSTRACT FROM AUTHOR]

CYP106A2 (cytochrome P450 meg) is a bacterial enzyme originally isolated from B. megaterium , and has been shown to hydroxylate a wide variety of substrates, including steroids. The regio- and stereochemistry of CYP106A2 hydroxylation has been shown to be dependent on a variety of factors, and hydroxylation often occurs at more than one site and/or with lack of stereospecificity for some substrates. Comprehensive backbone 15N, 1H and 13C resonance assignments based on multidimensional nuclear magnetic resonance (NMR) experiments performed with uniform and selective isotopically labeled CYP106A2 samples are reported herein, and broadening and splitting of resonances assigned to regions of the enzyme shown to be affected by substrate binding in other P450 enzymes indicate that substrate binding does not reduce structural heterogeneity as has been observed previously in P450 enzymes CYP101A1 and MycG. Paramagnetic relaxation enhancement (PRE) due to proximity between substrate protons and the heme iron were measured for three different substrates, and the relatively uniform nature of the PREs support the proposal that multiple substrate binding modes are occupied at saturating substrate concentrations. Left) Two equal energy orientations of abietic acid in the CYP106A2 active site. Structure in gray sticks shows distance between hydroxylation site and heme. Structure in orange lines places the carboxylate close to the heme. Right) Structure of CYP106A2 with residues most affected by heterogeneity highlighted in red. [Display omitted] • Comprehensive backbone NMR assignments for CYP106A2. • Multiple substrate binding modes give rise to heterogeneity in NMR spectra. • Most affected regions are those involved in substrate binding. • Paramagnetic relaxation patterns support multiple substrate binding orientations. [ABSTRACT FROM AUTHOR]

Investigates the heteronuclear and homonuclear multiple quantum coherence detection in paramagnetic proteins. Identification of the paramagnetic region of acireductone dioxygenase (ARD); Reduction of coherence transfer by electron-nuclear spin relaxation; Detection of nickel in ARD.

The majority of characterized cytochrome P450 enzymes in actinomycete secondary metabolic pathways are strictly substrate-, regio-, and stereo-specific. Examples of multifunctional biosynthetic cytochromes P450 with broader substrate and regio-specificity are growing in number and are of particular interest for biosynthetic and chemoenzymatic applications. MycG is among the first P450 monooxygenases characterized that catalyzes both hydroxylation and epoxidation reactions in the final biosynthetic steps, leading to oxidative tailoring of the 16-membered ring macrolide antibiotic mycinamicin II in the actinomycete Micromonospora griseorubida. The ordering of steps to complete the biosynthetic process involves a complex substrate recognition pattern by the enzyme and interplay between three tailoring modifications as follows: glycosylation, methylation, and oxidation. To understand the catalytic properties of MycG, we structurally characterized the ligand-free enzyme and its complexes with three native metabolites. These include substrates mycinamicin IV and V and their biosynthetic precursor mycinamicin III, which carries the monomethoxy sugar javose instead of the dimethoxylated sugar mycinose. The two methoxy groups of mycinose serve as sensors that mediate initial recognition to discriminate between closely related substrates in the post-polyketide oxidative tailoring of mycinamicin metabolites. Because x-ray structures alone did not explain the mechanisms of macrolide hydroxylation and epoxidation, paramagnetic NMR relaxation measurements were conducted. Molecular modeling based on these data indicates that in solution substrate may penetrate the active site sufficiently to place the abstracted hydrogen atom of mycinamicin IV within 6 Å of the heme iron and ∼4 Å of the oxygen of iron-ligated water. [ABSTRACT FROM AUTHOR]

Removal of substrate (+)-camphor from the active site of cytochrome P450cam (CYP101A1) results in nuclear magnetic resonance-detected perturbations in multiple regions of the enzyme. The 1H-15N correlation map of substrate-free diamagnetic Fe(II) CO-bound CYP101A permits these perturbations to be mapped onto the solution structure of the enzyme. Residual dipolar couplings (RDCs) were measured for 15N-1H amide pairs in two independent alignment media for the substrate-free enzyme and used as restraints in solvated molecular dynamics (MD) simulations to generate an ensemble of best-fit structures of the substrate-free enzyme in solution. Nuclear magnetic resonance-detected chemical shift perturbations reflect changes in the electronic environment of the NH pairs, such as hydrogen bonding and ring current shifts, and are observed for residues in the active site as well as in hinge regions between secondary structural features. RDCs provide information about relative orientations of secondary structures, and RDC-restrained MD simulations indicate that portions of a β-rich region adjacent to the active site shift so as to partially occupy the vacancy left by removal of the substrate. The accessible volume of the active site is reduced in the substrate-free enzyme relative to the substrate-bound structure calculated using the same methods. Both symmetric and asymmetric broadening of multiple resonances observed upon substrate removal as well as localized increased errors in RDC fits suggest that an ensemble of enzyme conformations are present in the substrate-free form. [ABSTRACT FROM AUTHOR]

A heterologously expressed form of the human Parkinson disease-associated protein α-synuclein with a 10-residue N-terminal extension is shown to form a stable tetramer in the absence of lipid bilayers or micelles. Sequential NMR assignments, intramonomer nuclear Overhauser effects, and circular dichroism spectra are consistent with transient formation of α-helices in the first 100 N-terminal residues of the 140-residue α-synuclein sequence. Total phosphorus analysis indicates that phospholipids are not associated with the tetramer as isolated, and chemical cross-linking experiments confirm that the tetramer is the highest-order oligomer present at NMR sample concentrations. Image reconstruction from electron micrographs indicates that a symmetric oligomer is present, with three- or fourfold symmetry. Thermal unfolding experiments indicate that a hydrophobic core is present in the tetramer. A dynamic model for the tetramer structure is proposed, based on expected close association of the amphipathic central helices observed in the previously described micelle-associated "hairpin" structure of α-synuclein. [ABSTRACT FROM AUTHOR]

Heterotrimeric G protein complexes are conserved from plants to mammals, but the complexity of each system varies. Arabidopsis thaliana contains one Gα, one Gβ (AGB1), and at least three Gγ subunits, allowing it to form three versions of the heterotrimer. This plant model is ideal for genetic studies because mammalian systems contain hundreds of unique heterotrimers. The activation of these complexes promotes interactions between both the Gα subunit and the Gβ dimer with enzymes and scaffolds to propagate signaling to the cytoplasm. However, although effectors of Gα and Gβ are known in mammals, no Gβ effectors were previously known in plants. Toward identifying AGB1 effectors, we genetically screened for dominant mutations that suppress Gβ-null mutant (agb1-2) phenotypes. We found that overexpression of acireductone dioxygenase 1 (ARD1) suppresses the 2-day-old etiolated phenotype of agb1-2. ARD1 is homologous to prokaryotic and eukaryotic ARD proteins; one function of ARDs is to operate in the methionine salvage pathway. We show here that ARD1 is an active metalloenzyme, and AGB1 and ARD1 both control embryonic hypocotyl length by modulating cell division; they also may contribute to the production of ethylene, a product of the methionine salvage pathway. ARD1 physically interacts with AGB1, and ARD enzymatic activity is stimulated by AGB1 in vitro. The binding interface on AGB1 was deduced using a comparative evolutionary approach and tested using recombinant AGB1 mutants. A possible mechanism for AGB1 activation of ARD1 activity was tested using directed mutations in a loop near the substrate-binding site. [ABSTRACT FROM AUTHOR]

Residual dipolar couplings (RDCs) were used as restraints in fully solvated molecular dynamics simulations of reduced substrate- and carbonmonoxy-bound cytochrome P450cam (CYP101A1), a 414-residue soluble monomeric heme-containing camphor monooxygenase from the soil bacterium Pseudomonas putida. The 1DNH residual dipolar couplings used as restraints were measured in two independent alignment media. A soft annealing protocol was used to heat the starting structures while incorporating the RDC restraints. After production dynamics, structures with the lowest total violation energies for RDC restraints were extracted to identify ensembles of conformers accessible to the enzyme in solution. The simulations result in substrate orientations different from that seen in crystallographic structures and a more open and accessible enzyme active site and largely support previously reported differences between the open and closed states of CYP101A1. [ABSTRACT FROM AUTHOR]

Helicobacter pylori, a pathogen that colonizes the human stomach, requires the nickel-containing metalloenzymes urease and NiFe-hydrogenase to survive this low pH environment. The maturation of both enzymes depends on the metallochaperone, HypA. HypA contains two metal sites, an intrinsic zinc site and a low-affinity nickel binding site. X-ray absorption spectroscopy (XAS) shows that the structure of the intrinsic zinc site of HypA is dynamic and able to sense both nickel loading and pH changes. At pH 6.3, an internal pH that occurs during acid shock, the zinc site undergoes unprecedented ligand substitutions to convert from a Zn(Cys)4 site to a Zn(His)2(Cys)2 site. NMR spectroscopy shows that binding of Ni(II) to HypA results in paramagnetic broadening of resonances near the N-terminus. NOEs between the β-CH2 protons of Zn cysteinyl ligands are consistent with a strand-swapped HypA dimer. Addition of nickel causes resonances from the zinc binding motif and other regions to double, indicating more than one conformation can exist in solution. Although the structure of the high-spin, 5-6 coordinate Ni(II) site is relatively unaffected by pH, the nickel binding stoichiometry is decreased from one per monomer to one per dimer at pH = 6.3. Mutation of any cysteine residue in the zinc binding motif results in a zinc site structure similar to that found for holo-WT-HypA at low pH and is unperturbed by the addition of nickel. Mutation of the histidines that flank the CXXC motifs results in a zinc site structure that is similar to holo-WT-HypA at neutral pH (Zn(Cys)4) and is no longer responsive to nickel binding or pH changes. Using an in vitro urease activity assay, it is shown that the recombinant protein is sufficient for recovery of urease activity in cell lysate from a HypA deletion mutant, and that mutations in the zinc-binding motif result in a decrease in recovered urease activity. The results are interpreted in terms of a model wherein HypA controls the flow of nickel traffic in the cell in response to nickel availability and pH. [ABSTRACT FROM AUTHOR]

Local protein backbone dynamics of the camphor hydroxylase cytochrome P450cam (CYP101) depend upon the oxidation and ligation state of the heme iron. ¹H-15N correlation nuclear magnetic resonance experiments were used to compare backbone dynamics of oxidized and reduced forms of this 414-residue metalloenzyme via hydrogen--deuterium exchange kinetics (H--D exchange) and 15N relaxation measurements, and these results are compared with previously published results obtained by H--D exchange mass spectrometry. In general, the reduced enzyme exhibits lower-amplitude motions of secondary structural features than the oxidized enzyme on all of the time scales accessible to these experiments, and these differences are more pronounced in regions of the enzyme involved in substrate access to the active site (B' helix and β3 and β5 sheets) and binding of putidaredoxin (C and L helices), the iron--sulfur protein that acts as the effector and reductant of CYP101 in vivo. These results are interpreted in terms of local structural effects of changes in the heme oxidation state, and the relevance of the observed effects to the enzyme mechanism is discussed. [ABSTRACT FROM AUTHOR]

Abstract: Experimental evidence has been provided for a functionally relevant cis–trans isomerization of the Ile88–Pro89 peptide bond in cytochrome P450cam (CYP101). The isomerization is proposed to be a key element of the structural reorganization leading to the catalytically competent form of CYP101 upon binding of the effector protein putidaredoxin (Pdx). A detailed comparison of the results of molecular dynamics simulations on the cis and trans conformations of substrate- and carbonmonoxy-bound ferrous CYP101 with sequence-specific Pdx-induced structural perturbations identified by nuclear magnetic resonance is presented, providing insight into the structural and dynamic consequences of the isomerization. The mechanical coupling between the Pdx binding site on the proximal face of CYP101 and the site of isomerization is described. [Copyright &y& Elsevier]

Summary: The two-protein complex between putidaredoxin (Pdx) and cytochrome P450cam (CYP101) is the catalytically competent species for camphor hydroxylation by CYP101. We detected a conformational change in CYP101 upon binding of Pdx that reorients bound camphor appropriately for hydroxylation. Experimental evidence shows that binding of Pdx converts a single X-proline amide bond in CYP101 from trans or distorted trans to cis. Mutation of proline 89 to isoleucine yields a mixture of both bound camphor orientations, that seen in Pdx-free and that seen in Pdx-bound CYP101. A mutation in CYP101 that destabilizes the cis conformer of the Ile 88-Pro 89 amide bond results in weaker binding of Pdx. This work provides direct experimental evidence for involvement of X-proline isomerization in enzyme function. [Copyright &y& Elsevier]

Ilie two acirecluctone dioxygenase (AR])) isozymes from the methionine salvage pathway of Kiebsiella ATCC 8724 present an unusual case in which two enzymes with different structures and distinct activities toward their common substrates (1 ,2-dihydroxy-3-oxo-5-(methylthio)pent- I -ene and dioxygen) are derived from the same polypeptide chain. Structural and functional differences between the two isozymes are determined by the type of M2~ metal ion bound in the active site. The Ni2~-bound NiARD catalyzes an off-pathway shunt from the methionine salvage pathway leading to the production of formate, methylthiopropionate, and carbon monoxide, while the Fe2~-bound FeARD' catalyzes the on-pathway formation of methionine precursor 2-keto-4-methylthiobutyrate and formate. Four potential protein-based metal ligands were identified by sequence homology and structural considerations. Based on the results of site-directed mutagenesis experiments, X-ray absorption spectroscopy (XAS), and isothermal calorimetry measurements, it is concluded that the same four residues, His96, His98, G1u102 and His 140, provide the protein-based ligands for the metal in both the Ni- and Fe-containing forms of the enzyme, and subtle differences in the local backbone conformations trigger the observed structural and functional differences between the FeARD' and NiARD isozymes. Furthermore, both forms of the enzyme bind their respective metals with pseudo-octahedral geometry, and both may lose a histidine ligand upon binding of substrate under anaerobic conditions. However, mutations at two conserved nonligand acidic residues, G1u95 and Glu 100, result in low metal contents for the mutant proteins as isolated, suggesting that some of the conserved charged residues may aid in transfer of metal from in vivo sources or prevent the loss of metal to stronger chelators. The Glu 100 mutant reconstitutes readily but has low activity. Mutation of AsplOl results in an active enzyme that incorporates metal in vivo but shows evidence of mixed forms. [ABSTRACT FROM AUTHOR]

Abstract: Backbone dynamics of the camphor monoxygenase cytochrome P450cam (CYP101) as a function of oxidation/ligation state of the heme iron were investigated via hydrogen/deuterium exchange (H/D exchange) as monitored by mass spectrometry. Main chain amide NH hydrogens can exchange readily with solvent and the rate of this exchange depends upon, among other things, dynamic fluctuations in local structural elements. A fluxional region of the polypeptide will exchange more quickly with solvent than one that is more constrained. In most regions of the enzyme, exchange rates were similar between oxidized high-spin camphor-bound and reduced camphor- and CO-bound CYP101 (CYP-S and CYP-S-CO, respectively). However, in regions of the protein that have previously been implicated in substrate access by structural and molecular dynamics investigations, the reduced enzyme shows significantly slower exchange rates than the oxidized CYP-S. This observation corresponds to increased flexibility of the oxidized enzyme relative to the reduced form. Structural features previously found to be perturbed in CYP-S-CO upon binding of the biologically relevant effector and reductant putidaredoxin (Pdx) as determined by nuclear magnetic resonance are also more protected from exchange in the reduced state. To our knowledge, this study represents the first experimental investigation of backbone dynamics within the P450 family using this methodology. [Copyright &y& Elsevier]

We have previously identified an androgen-responsive gene in rat prostate that shares homology with the acireductone dioxygenase (ARD/ARD') family of metalbinding enzymes involved in methionine salvage. We found that the gene, aci-reductone dioxygenase 1 (ADI1), was downregulated in prostate cancer cells, whereas enforced expression of rat Adi1 in these cells caused apoptosis. Here we report the characterization of human ADI1 in prostate cancer. Androgens induced ADI1 expression in human prostate cancer LNCaP cells, which was not blocked by cycloheximide, indicating that ADI1 is a primary androgen-responsive gene. In human benign prostatic hyperplasia specimens, epithelial cells expressed ADI1. Immunohistochemistry of prostate tumor tissue microarrays showed that benign regions expressed more ADI1 than tumors, suggesting a suppressive role for ADI1 in prostate cancer. Bacterial lysates containing recombinant ADI1 produced a five-fold increase in aci-reductone decay over controls, demonstrating that ADI1 has ARD activity. We generated point mutations at key residues in the metal-binding site of ADI1 to disrupt ARD function, and we found that these mutations did not affect intracellular localization, apoptosis, or colony formation suppression in human prostate cancer cells. Collectively, these observations argue that ADI1 may check prostate cancer progression through apoptosis and that this activity does not require metal binding. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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 β-sandwich fold of ARD, but a structural entropy switch increases order at one end of a two-helix system that bisects the β-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. [Copyright &y& Elsevier]

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 Fe2+ 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. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

Investigates the functional domains of vertebrate-type ferredoxins. Structural characterization of the acidic protein; Mechanical transmission of the iron-sulfur binding loop to the C-terminal domain; Oxidative metabolism of the ferredoxins.