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A fluorophore-labeled form of the T4moD, the catalytic effector protein of the toluene 4-monooxygenase complex, was prepared by engineering the N-terminal region contains a tetraCys motif and treatment with biarsenical fluorescein. The results indicate that specialization of effector protein function include specific protein-protein interactions with [2Fe-2S] domains as well as the hydroxylase component.

The NMR solution structures of spinach acyl carrier protein (ACP) with decanoate and stearate attached to the 4'-phosphopantetheine prosthetic group is reported. The structures display the conformational flexibility of spinach ACP and indicate that way protein changes to accommodate its myriad binding partners.

Stearoyl-acyl carrier protein desaturase (delta9D) catalyzes the O(sub 2) and 2e(super -) dependent desaturation of stearoyl-acyl carrier protein (18:0-ACP) to yield obeoyl-ACP (18:1-ACP). These protein-protein interface involved in the Fd-delta9D complex is investigated by the use of chemical cross-linking, site-directed mutagenesis, steady-state kinetic approaches, and molecular docking studies.

The formation of both radical and cation intermediates during the hydroxylations of diagnostic hydrocarbon substrate probes by T4moH is implicated. The results present a divergence of radical and cation pathways for T4-moH-mediated hydroxylation that can be dissected by diagnostic substrate probe rearrangements and by changes in the source of oxygen used for substrate oxygenation.

Structure-function analysis of stearoyl-acyl carrier protein Delta(sup)9-desaturase (Delta9D)-substrate complex indicates that 18:0-acyl carrier protein tightly binds to one of the subunits of the dimeric resting enzyme but binds 30-fold weaker to the second subunit. Furthermore, the affinity of the enzyme for acyl-acyl carrier protein is dependent on the acyl chain length and determines the association-dissociation of the complex and enzyme catalysis.

The 18:0-holo-acyl carrier protein delta(sup)9 desaturase reactivity is probed using fatty acids with single oxygen atom replacing the methylene groups in a C-18 fatty acyl chain. Results demonstrate desaturation, acyloxy chain scission, and 'register-shift' in binding of substrate. Data indicate that the desaturase-catalyzed reactions may initiate at the C-10 position.

Research has been conducted on the dehydroepiandrosterone (DHEA). The effect of DHEA and the 7-oxo-DHEA on the lipid-metabolizing enzymes' catalytic activity, accumulation and expression has been investigated and the results are reported.

Research examines the catalysis of NADH- and oxygen-dependent hydroxylation of toluene to para-cresol by the toluene 4-monooxygenase. The enzyme shows high regiospecificity for para hydroxylation involving toluene and other substituted aromatic compounds. The reactions are mediated by the enzyme catalytic complex consisting of an hydroxylase and an effector protein.

Research has been conducted on the active nitril hydratase from Rhodococcus sp. R312. The study of nitril hydratase in Fe-nitrosyl and low-spin ferric complexes by Mossbauer spectroscopy has been carried out.

Research has been conducted on the toluene 4-monooxygenase from Pseudomonas mendocina that catalyzes the NADH- and dioxygen-dependent toluene hydroxylation to form p-cresol. The solution structure of this monooxygenase has been investigated and the results are discussed.

Effects of substrate binding on the diferric centers of Ricinus communis 18:0-ACP Delta(super.9) desaturase (Delta9D) have been studied using resonance Raman spectroscopy. It has been shown that complex formation produces changes in the frequencies of v(sub.S)(Fe-O-Fe) and v(sub.as) (Fe-O-Fe) consistent with a decrease in the Fe-O-Fe angle from about 123 degrees in the oxo-bridged diferric centers of the as-isolated enzyme to about 120 degrees in oxo-bridged diferric centers of the complex.

Catalysis of the NADH- and O (sub)2-dependent toluene hydroxylation by the diiron enzyme toluene 4-monooxygenase is discussed. Results include identification of five isoforms that retain catalytic activity.

Stearoyl acyl protein (delta)9 desaturase requires a diiron center to catalyze the NADPH- and O2-dependent desaturation of stearoyl acycl carrier protein (ACP) to form oleoyl-ACP. The steady-state kinetics of this reaction were investigated using (delta)9 desaturase from recombinant Ricinus communis. Results indicated that hydrophobic binding energy was used to enhance catalysis since experiments showed that the binding of shorter acyl chains apparently did not provide sufficient activation energy for efficient desaturase catalysis.

A study on diiron site-containing enzymes used optical, transient kinetic and Mossbauer techniques to further investigate the origin and nature of peroxo stearoyl-acyl carrier protein delta9-desaturase. Findings show that 18:0-acyl carrier protein binding and 4e- stearoyl-acyl carrier protein delta9-desaturase leads to peroxo stearoyl-acyl carrier protein delta9-desaturase, which contains diiron clusters with spectroscopically distinguishable iron sites, and can be reversibly photodissociated to produce O2 and diferrous clusters.

Optical and resonance Raman spectroscopies were conducted to determine the intermediate conformation of stearoyl-acyl carrier protein Delta9 desaturase. Results reveal that the addition of four electrons converts the native conformation to a peroxodiferric intermediate similar to ribonucleotide reductase. Decay involves the release of two electrons during the reversion to the diferric state. The stoichiometry of the desaturase reaction corresponds to that of an oxidase reaction.

The regiospecificity of aromatic hydroxylation by the natural isoform of the toluene 4-monooxygenase as well as the effect of the changes in the amino acids within the active site on the configuration of the enzyme were determined. The utilization of a series of production distribution as probes to monitor the changes revealed that radical changes in the toluene product distribution were present following conformational alteration in the Q141C and F205I. These changes affect the regiospecificity of the toluene 4-monooxygenase.

Mossbauer, optical, and EPR measurements of enzyme complexes, catalyzing monooxygenation reactions at various sites on the toluene molecule, show that toluene-4-monooxygenase (T4MO) from Pseudomonas mendocina KR1 is a multicomponent monooxygenase complex. A catalytically active form of the T4MO complex is generated by the expression of the tmoA-F gene cluster from P. mendocina KR1 in Escherichia coli BL21 (DE3). In vitro reconstitution of T4MO catalytic activity requires purified forms of a diiron hydroxylase, a Reiske-type ferredoxin, an effector protein, and an NADH oxidoreductase.

Reaction of H2O2 with (mu-oxo)diiron(III) TPA complex, where TPA is tris(2-pyridylmethyl)-amine), results in a dinuclear complex. Spectroscopic studies and elemental analysis reveal the complex to be (Fe2(O)2(5-Me-TPA)2)-(ClO4)3. The zero-field splitting of an S = 3/2 ground state of the complex is large and nearly axial. Magnetic susceptibility and x-ray absorption K-edge spectroscopic analyses confirm the complex to be dinuclear and high-valent.

Analysis of the deduced amino acid sequences for the membrane desaturases from mammals, fungi, insects, higher plants, and cyanobacteria reveals three conserved primary sequence regions containing HX(3 or 4)H, HX(2 or 3)HH, and HX(2 or 3)HH and up to three hydrophobic regions, the length of which can span the membrane bilayer twice. The positioning of the conserved His-containing regions with respect to these potential membrane spanning domains suggests that a related protein fold for the membrane fatty acid desaturases and hydrocarbon hydroxylases. A functional analysis of the conserved His residues of Delta9 desaturase by site-directed mutageneis indicates that they are essential for the catalytic function of the enzyme and that they serve as ligands for the iron atom(s) in these enzymes.

X-ray crystallographic analysis of the primary sequence motif- and the structural differences of stearoyl-ACP Delta9 desaturases from plants, that contain oxo- or hydroxo-bridged diiron clusters, permits the classification of these soluble diiron-oxo proteins into two groups, I and II. Resonance Raman spectroscopy helps detect symmetric and asymmetric vibrational modes in the castor stearoyl-ACP Delta9 desaturase, which are typical of oxo-bridged diiron clusters that are triply bridged in the diferric state. The conserved amino acid residues of the class II diiron-oxo proteins serve as iron cluster ligands and are involved in a hydrogen-bonding network, O2 binding and activation.

A combination of electrochemical, EPR and Mossbauer spectroscopic studies conducted on reductive reactions of methane monooxygenase hydroxylase (MMOH) in the presence of MMO-reductase, MMOB and propylene, yields implicit potential values for each of MMOH's redox states. MMOH from Methylosinus (M) trichosporium OB3b differs in the two primary redox values of MMOH obtained from the enzyme M. capsulatus. Redox-dependent structural variations in MMOH relate to MMOH affinity for complex formation.

An analysis of the geometric and electronic structure of the binuclear Fe(II) active site of the reduced hydroxylase unit of methane monooxygenase (MMOH), involves the use of a combination of circular dichroism and magnetic circular dichroism spectroscopies. Two five-coordinate ferrous centers having distinct geometries are evident for the reduced MMOH.

The first homogeneous coordination compound to show a high-spin iron (IV) configuration results from EPR studies of integer and Mossbauer spin of (FeIVCl(eta4-MAC*)) that reveal Mossbauer spectra with magnetic hyperfine designs characteristic of complexes possessing integer electronic spin. The redox states of (FeIIICl(eta4-MAC*))2- and (FeIVCl(eta4-MAC*))- probably have considerable covalency, and studies of their high-field Mossbauer spectra show significantly reduced magnetic hyperfine interaction.

Spectroscopic studies of the active-site diiron cluster of the hydroxylase component of methane monooxygenase probe into the source of its ability to catalyze the oxidation even of stable hydrocarbons. The studies reveal weak exchange coupling of the diferric state, sensitivity of the spectral signals to the binding of products nd inhibitors, ferromagnetic coupling of the irons in the diferrous state and structurally differentiated iorns in the two subsites of the cluster in each of the three accessible redox states. The implications of these results are discussed.

The active site ligation of the isopenicillin N synthase (IPNS) substrate complex was examined. The Mossbauer, electron paramagnetic resonance and optical spectra of both the wild-type and mutant enzymes and their complexes with (L-alpha-amino-sigma-adipoyl)-L-cysteinyl-D-valine (ACV) and nitric oxide (NO) showed that the two endogenous cysteines of IPNS are not iron ligands. Rather, it was found that an active site iron is simultaneously coordinated by the thiolate moiety of ACV, water and NO in the IPNS.ACV.NO complex.

Various spectroscopic methods were utilized to understand the important roles of both resting and substrate-bound forms of cysteine dioxygenase (CDO), a mononuclear non-heme Fe-dependent dioxygenase in the Fe(II) and Fe(III) states in the catalytic mechanism of the enzyme. The results provide a more complete description of several catalytically relevant species and provide support for a reaction mechanism similar to that established for many structurally related 2-His-1-carboxylate Fe(II)-dependent dioxygenases.

Complementary chemical and NMR methods are used to better understand the effect of fatty acid length on the dynamics of acyl carrier protein involved in fatty acid biosynthesis. The acyl-ACP offers an interesting system for future studies of complex protein motions on the micro- and millisecond time scales.

Toluene 4-monooxygenase has a peroxide-shunt reaction that is much slower than catalysis driven by biological electron transfer. The studies have provided a structural benchmark for how activated intermediates are formed and dispatched during diiron hydroxylase catalysis.

Studies were conducted to gain insight into kinetic, biochemical, and structural role of active site residue Thr-201 in toluene 4-monooxygenase hydroxylase (T4moH) towards stabilization of a peroxo-level intermediate during enzyme catalysis. Both isoforms of enzyme T201 T4moH were tightly coupled during steady-state catalysis with saturating toluene and other optimal substrates and exhibited near-identical kinetic parameters.

Several techniques are employed to study the geometric, as well as the electronic structures of the binuclear nonheme ferrous active site of toluene-4-monooxygenase. The geometry of the iron center is shown to change completely by the binding of the effector protein.

Electron paramagnetic resonance (EPR) spectroscopy and density functional theory (DFT) methods are used for probing many aspects of the proposed mammalian cysteine dioxygenase (CDO) catalytic cycle. A model is proposed for the substrate-bound CDO active site.

The contribution of a specialized Rieske-type ferrodoxin to the function of the four-protein toluene 4-monooxygenase complex is examined. Results suggest that two different faces of T4moC may be involved in protein-protein interactions during catalysis.

Rv3230c is expressed as a partially soluble protein in Escherichia coli and recombinant DesA3 is expressed in Mycobacterium smegmatis as a catalytical active membrane protein in order to investigate if Rv230c from Mycobacterium tuberculosis H37Rv is a biologically relevant electron transfer partner for DesA3 from the same pathogen. Evidence was observed to the effect that Rv3230c from Mycobacterium tuberculosis H37Rv is a biologically relevant electron transfer partner for DesA3 from the same pathogen.

The crystal structures for native T4moD and two engineered variants with either four or 10 residues removed from the N-terminal at 2.1-, 1.7-, and 1.9-angstrom resolution are reported. The relative insensitivity to changes in the N-terminal region distinguishes T4moD from the MmoB homologues, which each require the approximately 33 residue N-terminal region for catalytic activity.

The reactions of the multiprotein diiron enzyme complex with stearoyl-acyl carrier protein are discussed. The changes in diiron enzymes in electronic configuration at metal centers are carried out by spectroscopic methods.

Results discuss the reactions of soluble stearoyl-ACP delta9 desaturase with 9- and 10-thiastearoyl-ACPs, which indicate that the primary reaction pathway for delta9D with 9-thiastearoyl-ACP is not sulfoxidation but is an acyl chain cleavage between S-9 and C-10. hese reactions are different from those previously reported for the integral membrane stearoyl-CoA desaturase with thistearoyl CoAs.

Results show that reduced [2Fe-2S] ferredoxin catalytically donates electrons during the conversion of stearoyl-acyl carrier protein 18:0 into 18:1 stearoyl-acyl carrier protein. Data indicate that the conversion rate is about 95% per delta9 desaturase active site in a single turnover depending upon the experimental conditions. The reductions of the prereduced enzyme-substrate complex with oxygen yield product at approximately 90% per subunit, while in the oxidized enzyme substrate complex, the product yield is slow.

The evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil and symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology. [ABSTRACT FROM AUTHOR]

Diferric complex with a (mu-oxo)(mu-hydroxo) bridge is prepared by terminal ligand manipulation. X-ray crystallographic studies of ((Fe2O(OH)6TLA)2)(CLO4)3 show two unique molecules in a unit cell. These molecules have an inversion center of the Fe2O2 core. This diferric complex has a mild oxidative property, attributed to its structure.

A study was done on the synthesis of an intermediate-spin iron(IV) complex of a strong pi-acid ligand. It was shown that the geometry around the iron atom of Fe(eta4-1)(CNtBu) is an axially distorted octahedron. The average Fe-C distance is 1.969 angstroms while the average Fe-N distance is 1.910 angstroms. A study of the iron-isocyanide complexes show that the pi-acid character of the isocyanides is not strongly expressed.

β-mannanase SACTE_2347 from cellulolytic Streptomyces sp. SirexAA-E is abundantly secreted into the culture medium during growth on cellulosic materials. The enzyme is composed of domains from the glycoside hydrolase family 5 (GH5), fibronectin type-III (Fn3), and carbohydrate binding module family 2 (CBM2). After secretion, the enzyme is proteolyzed into three different, catalytically active variants with masses of 53, 42 and 34 kDa corresponding to the intact protein, loss of the CBM2 domain, or loss of both the Fn3 and CBM2 domains. The three variants had identical N-termini starting with Ala51, and the positions of specific proteolytic reactions in the linker sequences separating the three domains were identified. To conduct biochemical and structural characterizations, the natural proteolytic variants were reproduced by cloning and heterologously expressed in Escherichia coli. Each SACTE_2347 variant hydrolyzed only β-1,4 mannosidic linkages, and also reacted with pure mannans containing partial galactosyl- and/or glucosyl substitutions. Examination of the X-ray crystal structure of the GH5 domain of SACTE_2347 suggests that two loops adjacent to the active site channel, which have differences in position and length relative to other closely related mannanases, play a role in producing the observed substrate selectivity. [ABSTRACT FROM AUTHOR]

The (Fe(II)Fe(II)) centers in reduced Delta9-desaturase and its substrate-bound form (stearoyl-acyl carrier protein Delta9-desaturase) were studied through a combination of the excited spectral techniques of circular dichroism and magnetic circular dichroism. Findings provide the first direct proof that the stearoyl-acyl carrier protein binding strongly perturbs the active site, establishing an additional open coordination position that correlates with improved dioxygen reactivity.

Selective isotope labeling was employed to analyze the origin of the paramagnetically shifed 1H, 2H and 15N resonances of T4MOC, a soluble Rieske ferredoxin component of toluene 4-monooxygenase. The paramagnetic H NMR signals from T4MOC differ from those of the plant-type and vertebrate ferredoxins, and assignments of these signals to residue and atom type in T4MOC question particular 1H NMR assignments made for the Xanthobacter Rieske center. The pattern of H-bonding to the iron-sulfur cluster of T4MOC is discussed.

Multicomponent monooxygenases, which carry out a variety of highly specific hydroxylation reactions, are of great interest as potential biocatalysts in a number of applications. These proteins share many similarities in structure and show a marked increase in 02 reactivity upon addition of an effector component. In this study, circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field (VTVH) MCD have been used to gain spectroscopic insight into the Fe(II)Fe(II) active site in the hydroxylase component of Toluene-4 monoxygenase (T4moH) and the complex of T4moH bound by its effector protein, T4moD. These results have been correlated to spectroscopic data and density functional theory (DFT) calculations on MmoH and its interaction with MmoB. Together, these data provide further insight into the geometric and electronic structure of these biferrous active sites and, in particular, the perturbation associated with component BID binding. It is found that binding of the effector protein changes the geometry of one iron center and orientation of its redox active orbital to accommodate the binding of O2 in a bridged structure for efficient 2-electron transfer that can form a peroxo intermediate. [ABSTRACT FROM AUTHOR]