A case study: The deployment of a novel in situ fluorimeter for monitoring biological contamination within the urban surface waters of Kolkata, India.

The quality and health of many of our vital freshwater systems are poor. To tackle this with ever increasing pressures from anthropogenic and climatic changes, we must improve water quality monitoring and devise and implement more appropriate water quality parameters. Recent research has highlighted the potential for Peak T fluorescence (tryptophan-like fluorescence, TLF) to monitor microbial activity in aquatic systems. The VLux TPro (Chelsea Technologies Ltd., UK), an in situ real-time fluorimeter, was deployed in different urban freshwater bodies within Kolkata (West Bengal, India) during March 2019. This study is the first to apply this technology in surface waters within a densely populated urban area. Spot-sampling was also undertaken at 13 sampling locations enabling physicochemical analysis, bacterial enumeration and determination of nutrient (nitrate and phosphate) concentrations. This case study has demonstrated the ability of an in situ fluorimeter, VLux TPro, to successfully identify both biological contamination events and potential elevated microbial activity, related to nutrient loading, in complex surface freshwaters, without the need for expensive and time-consuming laboratory analysis.

The in situ bacterial production of fluorescent organic matter; an investigation at a species level.

Aquatic dissolved organic matter (DOM) plays an essential role in biogeochemical cycling and transport of organic matter throughout the hydrological continuum. To characterise microbially-derived organic matter (OM) from common environmental microorganisms (Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa), excitation-emission matrix (EEM) fluorescence spectroscopy was employed. This work shows that bacterial organisms can produce fluorescent organic matter (FOM) in situ and, furthermore, that the production of FOM differs at a bacterial species level. This production can be attributed to structural biological compounds, specific functional proteins (e.g. pyoverdine production by P. aeruginosa), and/or metabolic by-products. Bacterial growth curve data demonstrates that the production of FOM is fundamentally related to microbial metabolism. For example, the majority of Peak T fluorescence (> 75%) is shown to be intracellular in origin, as a result of the building of proteins for growth and metabolism. This underpins the use of Peak T as a measure of microbial activity, as opposed to bacterial enumeration as has been previously suggested. This study shows that different bacterial species produce a range of FOM that has historically been attributed to high molecular weight allochthonous material or the degradation of terrestrial FOM. We provide definitive evidence that, in fact, it can be produced by microbes within a model system (autochthonous), providing new insights into the possible origin of allochthonous and autochthonous organic material present in aquatic systems.

Mössbauer and EPR studies of the photoactivation of nitrile hydratase.

The alphabeta dimer of active nitrile hydratase from Rhodococcus sp. R312 contains one low-spin ferric ion that is coordinated by three Cys residues, two N-amide groups from the protein backbone, and one OH(-). The enzyme isolated from bacteria grown in the dark is inactive and contains the iron site as a six-coordinate diamagnetic Fe-nitrosyl complex, called NH(dark). The active state can be obtained from the dark state by photolysis of the Fe-NO bond at room temperature. Activation is accompanied by the conversion of NH(dark) to a low-spin ferric complex, NH(light), exhibiting an S = (1)/(2) EPR signal with g values of 2.27, 2.13, and 1.97. We have characterized both NH(dark) and NH(light) with Mössbauer spectroscopy. The z-axis of the 57Fe magnetic hyperfine tensor, A, of NH(light) was found to be rotated by approximately 45 degrees relative to the z-axis of the g tensor (g(z) = 1.97). Comparison of the A tensor of NH(light) with the A tensors of low-spin ferric hemes indicates a substantially larger degree of covalency for nitrile hydratase. We have also performed photolysis experiments between 2 and 20 K and characterized the photolyzed products by EPR and Mössbauer spectroscopy. Photolysis at 4.2 K in the Mössbauer spectrometer yielded a five-coordinate low-spin ferric species, NH(A), which converted back into NH(dark) when the sample was briefly warmed to 77 K. We also describe preliminary EPR photolysis studies that have yielded new intermediates.

Aromatic hydroxylation catalyzed by toluene 4-monooxygenase in organic solvent/aqueous buffer mixtures.

Toluene 4-monooxygenase is a four-protein component diiron enzyme complex. The enzyme catalyzes the hydroxylation of toluene to give p-cresol with approximately 96% regioselectivity. The performance of the enzyme in two-phase reaction systems consisting of toluene, hexane, or perfluorohexane and an aqueous buffer was tested. In each of the cosolvent systems, containing up to 93% (v/v) of solvent, the enzyme was active and exhibited regioselectivity indistinguishable from the aqueous reaction. Using the perfluorohexane/buffer system, a number of polycyclic aromatic hydrocarbons were oxidized that were not readily oxidized in aqueous buffer. An instability of the hydroxylase component and a substantial uncoupling of NADH utilization and product formation were observed in reactions that were continued for longer than approximately 3 min. More stable enzyme complexes will be needed for broad applicability of this hydroxylating system in nonaqueous media.

Solution structure of the toluene 4-monooxygenase effector protein (T4moD).

Toluene 4-monooxygenase (T4MO) from Pseudomonas mendocina catalyzes the NADH- and O(2)-dependent hydroxylation of toluene to form p-cresol. The complex consists of an NADH oxidoreductase (T4moF), a Rieske ferredoxin (T4moC), a diiron hydroxylase [T4moH, with (alphabetagamma)(2) quaternary structure], and a catalytic effector protein (T4moD). The solution structure of the 102-amino acid T4moD effector protein has been determined from 2D and 3D (1)H, (13)C, and (15)N NMR spectroscopic data. The structural model was refined through simulated annealing by molecular dynamics in torsion angle space (DYANA software) with input from 1467 experimental constraints, comprising 1259 distance constraints obtained from NOEs, 128 dihedral angle constraints from J-couplings, and 80 hydrogen bond constraints. Of 60 conformers that met the acceptance criteria, the 20 that best satisfied the input constraints were selected to represent the solution structure. With exclusion of the ill-defined N- and C-terminal segments (Ser1-Asn11 and Asp99-Met102), the atomic root-mean-square deviation for the 20 conformers with respect to the mean coordinates was 0.71 A for the backbone and 1.24 A for all non-hydrogen atoms. The secondary structure of T4moD consists of three alpha-helices and seven beta-strands arranged in an N-terminal betaalphabetabeta and a C-terminal betaalphaalphabetabetabeta domain topology. Although the published NMR structures of the methane monooxygenase effector proteins from Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath) have a similar secondary structure topology, their three-dimensional structures differ from that of T4moD. The major differences in the structures of the three effector proteins are in the relative orientations of the two beta-sheets and the interactions between the alpha-helices in the two domains. The structure of T4moD is closer to that of the methane monooxygenase effector protein from M. capsulatus (Bath) than that from M. trichosporium OB3b. The specificity of T4moD as an effector protein was investigated by replacing it in reconstituted T4MO complexes with effector proteins from monooxygenases from other bacterial species: Pseudomonas pickettii PKO1 (TbuV, toluene 3-monooxygenase); Pseudomonas species JS150 (TbmC, toluene 2-monooxygenase); and Burkeholderia cepacia G4 (S1, toluene 2-monooxygenase). The results showed that the closely related TbuV effector protein (55% sequence identity) provided partial activation of the complex, whereas the more distantly related TbmC (34% sequence identity) and S1 (29% sequence identity) did not. The (1)H NMR chemical shifts of the side-chain amide protons of Asn34, a conserved, structurally relevant amino acid, were found to be similar in spectra of effector proteins T4moD and TbuV but not in the spectrum of TbmC. This suggests that the region around Asn34 may be involved in structural aspects contributing to functional specificity.

Transformation of 2,4,6-trinitrotoluene by purified xenobiotic reductase B from Pseudomonas fluorescens I-C.

The enzymatic transformation of 2,4,6-trinitrotoluene (TNT) by purified XenB, an NADPH-dependent flavoprotein oxidoreductase from Pseudomonas fluorescens I-C, was evaluated by using natural abundance and [U-(14)C]TNT preparations. XenB catalyzed the reduction of TNT either by hydride addition to the aromatic ring or by nitro group reduction, with the accumulation of various tautomers of the protonated dihydride-Meisenheimer complex of TNT, 2-hydroxylamino-4,6-dinitrotoluene, and 4-hydroxylamino-2, 6-dinitrotoluene. Subsequent reactions of these metabolites were nonenzymatic and resulted in predominant formation of at least three dimers with an anionic m/z of 376 as determined by negative-mode electrospray ionization mass spectrometry and the release of approximately 0.5 mol of nitrite per mol of TNT consumed. The extents of the initial enzymatic reactions were similar in the presence and in the absence of O(2), but the dimerization reaction and the release of nitrite were favored under aerobic conditions or under anaerobic conditions in the presence of NADP(+). Reactions of chemically and enzymatically synthesized and high-pressure liquid chromatography-purified TNT metabolites showed that both a hydroxylamino-dinitrotoluene isomer and a tautomer of the protonated dihydride-Meisenheimer complex of TNT were required precursors for the dimerization and nitrite release reactions. The m/z 376 dimers also reacted with either dansyl chloride or N-1-naphthylethylenediamine HCl, providing evidence for an aryl amine functional group. In combination, the experimental results are consistent with assigning the chemical structures of the m/z 376 species to various isomers of amino-dimethyl-tetranitrobiphenyl. A mechanism for the formation of these proposed TNT metabolites is presented, and the potential enzymatic and environmental significance of their formation is discussed.

Chemical and posttranslational modification of Escherichia coli acyl carrier protein for preparation of dansyl-acyl carrier proteins.

Escherichia coli acyl carrier protein (ACP) contains a single tyrosine residue at position 71. The combined o-nitration of apo-ACP Y71 by tetranitromethane and reduction to 3-aminotyrosyl-apo-ACP were performed to introduce a specific site for attachment of a dansyl fluorescent label. Conditions for purification and characterization of dansylaminotyrosyl-apo-ACP are reported. Dansylaminotyrosyl-apo-ACP was enzymatically phosphopantetheinylated and acylated in vitro with an overall approximately 30% yield of purified stearoyl-dansylaminotyrosyl-ACP starting from unmodified apo-ACP. The steady-state kinetic parameters k(cat) = 22 min(-1) and K(M) = 2.7 microM were determined for reaction of stearoyl-dansylaminotyrosyl-ACP with stearoyl-ACP Delta(9)-desaturase. These results show that dansylaminotyrosyl-ACP will function well for studying binding interactions with the Delta(9)-desaturase and suggest similar possibilities for other ACP-dependent enzymes. The efficient in vivo phosphopantetheinylation of E. coli apo-ACP by coexpression with holo-ACP synthase in E. coli BL21(DE3) using fructose as the carbon source is also reported.

Optimized expression and purification of toluene 4-monooxygenase hydroxylase.

Toluene 4-monooxygenase is a four-protein complex that catalyzes the O(2)- and NADH-dependent oxidation of toluene to p-cresol. The influence of various expression systems on the host cell growth characteristics, purified protein yields, and specific activity of the hydroxylase (T4moH) component of the complex was evaluated by considering the cell mass obtained per liter of fermentation culture medium, the purified protein obtained per gram of cell mass, and the specific activity of purified T4moH. The specific activity of purified T4moH was determined to be 1200-1250 nmol of p-cresol formed per minute per milligram of T4moH in air-saturated 50 mM phosphate buffer, pH 7.5, at 25 degrees C in the presence of optimal concentrations of the other protein components of the complex, saturating toluene (5.8 mM at 25 degrees C), and saturating NADH (1 mM). This value was obtained for T4moH purified from several different expression systems and apparently represents the maximal specific activity of the enzyme complex for toluene hydroxylation. By manipulation of vectors and gene inserts to eliminate adventitious catalytic turnover of NADH, up to 60-fold increase in the volumetric yield of T4moH activity was obtained from recombinant fermentations in Escherichia coli BL21(DE3).

Differential regulation of the stearoyl-CoA desaturase genes by thiazolidinediones in 3T3-L1 adipocytes.

Two stearoyl-CoA desaturase (SCD) isoforms can be expressed during the differentiation of 3T3-L1 preadipocytes into adipocytes. Here we report on the effects of the peroxisome proliferator-activated receptor gamma ligand troglitazone (TRO) on scd1 and scd2 mRNA levels as determined by Northern blotting, on SCD protein expression as determined by Western blotting, and on total lipid composition as determined by GC during differentiation. In preadipocytes, scd1 mRNA and SCD protein were not detected, whereas scd2 mRNA was detected. These cells have high levels of palmitate (16:0), stearate (18:0), and monounsaturated oleate (Delta(9)-18:1) and low levels of monounsaturated palmitoleate (Delta(9)-16:1). In MDI (methylisobutylxanthine, dexamethasone, and insulin)-treated cells, scd1 mRNA and SCD protein were increased approximately 100-fold relative to preadipocyte levels, the scd2 mRNA level was increased 2-fold, Delta(9)-16:1 was increased approximately 20-fold, and 18:0 was decreased approximately 3-fold. In TRO-treated cells, the scd1 mRNA level was lower than that observed in preadipocytes, while the scd2 mRNA level was similar. TRO also decreased scd1 mRNA in primary adipocytes. The TRO-treated cells contained a Delta(9)-18:1 level typical of MDI-treated cells whereas, conversely, these cells also contained a low Delta(9)-16:1 level typical of preadipocytes. The implications of these correlations for the regulatory and enzymatic mechanism(s) used to establish and maintain lipid composition are discussed.

Resonance Raman studies of the stoichiometric catalytic turnover of a substrate-stearoyl-acyl carrier protein delta(9) desaturase complex.

Resonance Raman spectroscopy has been used to study the effects of substrate binding (stearoyl-acyl carrier protein, 18:0-ACP) on the diferric centers of Ricinus communis 18:0-ACP Delta(9) desaturase. These studies show that complex formation produces changes in the frequencies of nu(s)(Fe-O-Fe) and nu(as)(Fe-O-Fe) consistent with a decrease in the Fe-O-Fe angle from approximately 123 degrees in the oxo-bridged diferric centers of the as-isolated enzyme to approximately 120 degrees in oxo-bridged diferric centers of the complex. Analysis of the shifts in nu(s)(Fe-O-Fe) and nu(as)(Fe-O-Fe) as a function of 18:0-ACP concentration also suggests that 4e(-)-reduced Delta9D containing two diferrous centers has a higher affinity for 18:0-ACP than resting Delta9D containing two diferric centers. Catalytic turnover of a stoichiometric complex of 18:0-ACP and Delta9D was used to investigate whether an O-atom from O(2) would be incorporated into a bridging position of the resultant mu-oxo-bridged diferric centers during the desaturation reaction. Upon formation of approximately 70% yield of 18:1-ACP product in the presence of (18)O(2), no incorporation of an (18)O atom into the mu-oxo bridge position was detected. The result with 18:0-ACP Delta(9) desaturase differs from that obtained during the tyrosyl radical formation reaction of the diiron enzyme ribonucleotide reductase R2 component, which proceeds with incorporation of an O-atom from O(2) into the mu-oxo bridge of the resting diferric site. The possible implications of these results for the O-O bond cleavage reaction and the nature of intermediates formed during Delta9D catalysis are discussed.

Toluene monooxygenase-catalyzed epoxidation of alkenes.

Several toluene monooxygenase-producing organisms were tested for their ability to oxidize linear alkenes and chloroalkenes three to eight carbons long. Each of the wild-type organisms degraded all of the alkenes that were tested. Epoxides were produced during the oxidation of butene, butadiene, and pentene but not hexene or octadiene. A strain of Escherichia coli expressing the cloned toluene-4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 was able to oxidize butene, butadiene, pentene, and hexene but not octadiene, producing epoxides from all of the substrates that were oxidized. A T4MO-deficient variant of P. mendocina KR1 oxidized alkenes that were five to eight carbons long, but no epoxides were detected, suggesting the presence of multiple alkene-degrading enzymes in this organism. The alkene oxidation rates varied widely (ranging from 0. 01 to 0.33 micromol of substrate/min/mg of cell protein) and were specific for each organism-substrate pair. The enantiomeric purity of the epoxide products also varied widely, ranging from 54 to >90% of a single epoxide enantiomer. In the absence of more preferred substrates, such as toluene or alkenes, the epoxides underwent further toluene monooxygenase-catalyzed transformations, forming products that were not identified.

Desaturation of trans-octadecenoyl-acyl carrier protein by stearoyl-acyl carrier protein delta9 desaturase.

Positional isomers of mono-unsaturated 18:1-ACP have been used as substrates for stearoyl-acyl carrier protein delta9 desaturase to test whether a C-H bond abstraction from either the C-9 or C-10 position could lead to rearranged products diagnostic for the production of an allylic radical intermediate. The reconstituted enzyme complex was able to desaturate trans-delta11-18:1-ACP and trans-delta7-18:1-ACP, but not trans-delta9-18:1-ACP, or any of the corresponding cis-isomers. Enzymatic desaturation of trans-delta11-18:1-ACP gave a single product, cis-delta9,trans-delta11-18:2-ACP, as characterized by gas chromatography-electron ionization mass spectrometry of the molecular ions, the fragmentation products of pyrrolidide and 4,4-dimethyloxazoline derivatives, and by comparison of chromatographic retention times with authentic standards. Reaction of trans-delta7-18:1-ACP gave two enzymic products, trans-delta7,cis-delta9-18:2 (approximately 80%) and trans-delta7,cis-delta11-18:2 (approximately 20%). The major product was likely formed in a reaction identical to that of 18:0-ACP desaturation, while the minor product was likely formed by alternative placement of the C-10 and C-11 positions of the substrate analog in a cis configuration relative to the diiron oxidant. Since none of the products observed are indicative of rearrangements originating with an allylic radical, a discussion of the origins and possible implications of these results is presented.

Threonine 201 in the diiron enzyme toluene 4-monooxygenase is not required for catalysis.

The diiron enzyme toluene 4-monooxygenase from Pseudomonas mendocina KR1 catalyzes the NADH- and O(2)-dependent hydroxylation of toluene. A combination of sequence alignments and spectroscopic studies indicate that T4MO has an active site structure closely related to the crystallographically characterized methane monooxygenase hydroxylase. In the methane monooxygenase hydroxylase, active site residue T213 has been proposed to participate in O(2) activation by analogy to certain proposals made for cytochrome P450. In this work, mutagenesis of the comparable residue in the toluene 4-monooxygenase hydroxylase, T201, has been used to investigate the role of an active site hydroxyl group in catalysis. Five isoforms (T201S, T201A, T201G, T201F, and T201K) that retain catalytic activity based on an in vivo indigo formation assay were identified, and detailed characterizations of the purified T201S, T201A, and T201G variants are reported. These isoforms have k(cat) values of 1.2, 1.0, and 0.6 s(-)(1), respectively, and k(cat)/K(M) values that vary by only approximately 4-fold relative to that of the native isoform. Moreover, these isoforms exhibit 80-90% coupling efficiency, which also compares favorably to the >94% coupling efficiency determined for the native isoform. For the T201S, T201A, and T201G isoforms, the regiospecificity of toluene hydroxylation was nearly identical to that of the natural isoform, with p-cresol representing 90-95% of the total product distribution. In contrast, the T201F isoform caused a substantial shift in the product distribution, and gave o- and p-cresol in a 1:1 ratio. In addition, the amount of benzyl alcohol was increased approximately 10-fold with the T201F isoform. For reaction with p-xylene, previous studies have shown that the native isoform reacted to give 4-methybenzyl alcohol and 2, 5-dimethylphenol in a 4:1 ratio [Pikus, J. D., Studts, J. M., McClay, K., Steffan, R. J., and Fox, B. G. (1997) Biochemistry 36, 9283-9289]. For comparison, the T201S, T201A, and T201F isoforms gave a slightly relaxed 3:1 ratio of these products, while the T201G isoform gave a dramatically relaxed 1:1 ratio. On the basis of these studies, we conclude that the hydroxyl group of T201 is not essential to maintaining the turnover rate or the coupling of the toluene 4-monooxygenase complex. However, changing the volume occupied by the side chain at the position of T201 can lead to alterations in the regiospecificity of the hydroxylation, presumably by producing different orientations for substrate binding during catalysis.

Cloning and sequence analysis of two Pseudomonas flavoprotein xenobiotic reductases.

The genes encoding flavin mononucleotide-containing oxidoreductases, designated xenobiotic reductases, from Pseudomonas putida II-B and P. fluorescens I-C that removed nitrite from nitroglycerin (NG) by cleavage of the nitroester bond were cloned, sequenced, and characterized. The P. putida gene, xenA, encodes a 39,702-Da monomeric, NAD(P)H-dependent flavoprotein that removes either the terminal or central nitro groups from NG and that reduces 2-cyclohexen-1-one but did not readily reduce 2,4,6-trinitrotoluene (TNT). The P. fluorescens gene, xenB, encodes a 37,441-Da monomeric, NAD(P)H-dependent flavoprotein that exhibits fivefold regioselectivity for removal of the central nitro group from NG and that transforms TNT but did not readily react with 2-cyclohexen-1-one. Heterologous expression of xenA and xenB was demonstrated in Escherichia coli DH5alpha. The transcription initiation sites of both xenA and xenB were identified by primer extension analysis. BLAST analyses conducted with the P. putida xenA and the P. fluorescens xenB sequences demonstrated that these genes are similar to several other bacterial genes that encode broad-specificity flavoprotein reductases. The prokaryotic flavoprotein reductases described herein likely shared a common ancestor with old yellow enzyme of yeast, a broad-specificity enzyme which may serve a detoxification role in antioxidant defense systems.

Role of hydrophobic partitioning in substrate selectivity and turnover of the ricinus communis stearoyl acyl carrier protein delta(9) desaturase.

Stearoyl acyl carrier protein Delta(9) desaturase (Delta9D) uses a diiron center to catalyze the NADPH- and O(2)-dependent desaturation of stearoyl acyl carrier protein (ACP) to form oleoyl-ACP. The reaction of recombinant Ricinus communis Delta9D with natural and nonnatural chain length acyl-ACPs was used to examine the coupling of the reconstituted enzyme complex, the specificity for position of double-bond insertion, the kinetic parameters for the desaturation reaction, and the selectivity for acyl chain length. The coupling of NADPH and O(2) consumption and olefin production was found to be maximal for 18:0-ACP, and the loss of coupling observed for the more slowly desaturated acyl-ACPs was attributed to autoxidation of the electron-transfer chain. Analysis of steady-state kinetic parameters for desaturation of acyl-ACPs having various acyl chain lengths revealed that the K(M) values were similar ( approximately 2.5-fold difference) for 15:0-18:0-ACP, while the k(cat) values increased by approximately 26-fold for the same range of acyl chain lengths. A linear increase in log (k(cat)/K(M)) was observed upon lengthening of the acyl chain from 15:0- to 18:0-ACP, while no further increase was observed for 19:0-ACP. The similarity of the k(cat)/K(M) values for 18:0- and 19:0-ACPs and the retained preference for double-bond insertion at the Delta(9) position with 19:0-ACP (>98% desaturation at the Delta(9) position) suggest that the active-site channel past the diiron center can accommodate at least one more methylene group than is found in the natural substrate. The DeltaDeltaG(binding) estimated from the change in k(cat)/K(M) for increasing substrate acyl-chain length was -3 kJ/mol per methylene group, similar to the value of -3.5 kJ/mol estimated for the hydrophobic partition of long-chain fatty acids (C-7 to C-21) from water to heptane [Smith, R. , and Tanford, C. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 289-293]. Since the K(M) values are overall similar for all acyl-ACPs tested, the progressive increase in hydrophobic binding energy available from increased chain length is apparently utilized to enhance catalytic steps, which thus provides the underlying physical mechanism for acyl chain selectivity observed with Delta9D.

Mössbauer studies of the formation and reactivity of a quasi-stable peroxo intermediate of stearoyl-acyl carrier protein Delta 9-desaturase.

Stearoyl-ACP Delta(9)-desaturase (Delta 9D) is a diiron enzyme that catalyzes 18:0-ACP desaturation. Each subunit of homodimeric resting Delta 9D contains a diferric cluster, while chemical reduction by 4e(-) produces a diferrous cluster in each subunit. Reaction of 4e(-)-reduced Delta 9D with 18:0-ACP and O(2) yields a blue chromophore (lambda(max) approximately 700 nm) that exhibits a vibrational spectrum indicative of a micro-1,2-peroxo complex; this species has been designated peroxo Delta9D. In contrast to other enzymic peroxodiiron intermediates, peroxo Delta 9D is long-lived (t(1/2) approximately 30 min at 25 degrees C) and decays via an oxidase reaction without formation of either H(2)O(2) or product (18:1-ACP). In this work, optical, transient kinetic, and Mössbauer techniques have been used to further investigate the origin and nature of this unusual peroxodiiron complex. Rapid mixing of 4e(-) Delta 9D with O(2)-equilibrated 18:0-ACP produced peroxo Delta 9D as revealed by a temperature-dependent, pseudo-first-order absorption increase at 700 nm (k = 46 s(-)(1) at 6 degrees C). The Mössbauer spectrum of peroxo Delta 9D, accounting for 96% of the total iron, consists of two quadrupole doublets present in equal proportions: delta(1) = 0.68(1) mm/s, and Delta E(Q)(1) = 1.90(2) mm/s; delta(2) = 0.64(1) mm/s, and Delta E(Q)(2) = 1.06(2) mm/s. Decay of the 700 nm optical band (k = 0.004 min(-)(1) at 6 degrees C) correlates with the complete conversion of peroxo Delta 9D into a complex called peroxo-cycled Delta 9D, which exhibits two new doublets present in equal proportions: delta(1) = 0.57(2) mm/s, and Delta E(Q)(1) = 1. 91(3) mm/s; delta(2) = 0.52(2) mm/s, and Delta E(Q)(2) = 1.41(3) mm/s. Thus, peroxo Delta 9D contains two asymmetric diferric clusters and reacts to yield peroxo-cycled Delta 9D, also containing two asymmetric diferric clusters that most probably represent a substrate complex state. The clusters of both peroxo Delta 9D and peroxo-cycled Delta 9D have a diamagnetic ground state. Because peroxo Delta 9D and peroxo-cycled Delta 9D are observed only in the presence of 18:0-ACP, substrate binding appears to have introduced asymmetry into the Delta 9D diiron clusters. In situ photolysis of peroxo Delta 9D at 4.2 K in the Mössbauer cryostat caused the release of O(2) and the reappearance of a diferrous Delta 9D.18:0-ACP complex with slightly changed parameters, suggesting a constrained cluster configuration was produced by the photolysis event. Annealing the photolyzed sample for 30 min at 77 K quantitatively restored the Mössbauer spectrum of peroxo Delta 9D, showing that the released O(2) was effectively sequestered within the active site.

Application of fed-batch fermentation to the preparation of isotopically labeled or selenomethionyl-labeled proteins.

An increasing demand for isotopically labeled samples for spectroscopic and crystallographic studies has led to a corresponding need for effective and efficient methods for producing these samples. The present work is based on the strategy of using an isotopically labeled compound as the growth-limiting nutrient during protein expression in Escherichia coli (DE3) strains. By using dissolved O2 and agitation rate data, the cell growth, feeding of the isotopic label, induction of protein expression, and the harvest of cells can be coordinated in a feedback controlled fermenter in a simple, easily defined manner. This approach is demonstrated for the nutrient-limited production of [U-15N]- and [U-13C, U-15N]-labeled toluene 4-monooxygenase effector protein in E. coli BL21(DE3) with isotopic abundance identical to that of the labeled precursors. For selective labeling, demonstrated with selenomethionine using methionine auxotroph E. coli B834(DE3), approximately 80-85% incorporation was obtained from methionine-dependent growth of the auxotroph followed by selenomethionine feeding and protein induction upon methionine depletion. This selective labeling is accomplished in a single culture, does not require washing or resuspension, minimizes costly incorporation of label into host cell mass prior to induction, and can be easily adapted to selective labeling with other amino acids. Moreover, cell mass yield from these experiments can be readily optimized to provide the desired level of protein for a given investigation from a single growth and purification. This combination provides an efficient, controllable option for isotopic labeling experiments.

Spinach holo-acyl carrier protein: overproduction and phosphopantetheinylation in Escherichia coli BL21(DE3), in vitro acylation, and enzymatic desaturation of histidine-tagged isoform I.

Spinach ACP isoform I was overexpressed in Escherichia coli BL21(DE3) using a gene synthesized from codons associated with high-level expression in E. coli. The synthetic gene has extensive changes in codon usage (23 of 77 total codons) relative to that of the originally synthesized plant gene (P. D. Beremand et al., 1987, Arch. Biochem. Biophys. 256, 90-100). After expression of the new synthetic gene, purified ACP and ACP-His6 were obtained in yields of up to 70 mg L-1 of culture medium, compared to approximately 1-6 mg L-1 of purified ACP obtained from the gene composed of predicted spinach codons. In either shaken flask or fermentation culture, approximately 15% conversion to holo-ACP or holo-ACP-His6 was obtained regardless of the level of protein expression. However, coexpression of ACP-His6 with E. coli holo-ACP synthase in E. coli BL21(DE3) during pH- and dissolved O2-controlled fermentation routinely yielded greater than 95% conversion to holo-ACP-His6. Electrospray ionization mass spectrometric analysis of the purified recombinant ACPs revealed that the amino terminal Met was efficiently removed, but only if the bacterial cell lysates were prepared in the absence of EDTA. This observation is consistent with the inhibition of endogenous Met-aminopeptidase by removal of catalytically essential Co(II) and introduces the importance of considering the catalytic properties of host enzymes providing ad hoc posttranslational modification of recombinant proteins. Stearoyl-ACP-His6 was shown to be indistinguishable from stearoyl-ACP as a substrate for enzymatic acylation and desaturation. In combination, these studies provide a coordinated scheme to produce and characterize quantities of acyl-ACPs sufficient to support expanded biophysical and structural studies.