Biomedical subjects

Brian G Fox

Publications and source records attributed to Brian G Fox.

44 records · Page 3Linked to original sources

PubMed2003

Insight into the mechanism of aromatic hydroxylation by toluene 4-monooxygenase by use of specifically deuterated toluene and p-xylene.

The present studies address the mechanism of aromatic hydroxylation used by the natural and G103L isoforms of the diiron enzyme toluene 4-monooxygenase. These isoforms have comparable catalytic parameters but distinct regiospecificities for toluene hydroxylation. Hydroxylation of ring-deuterated p-xylene by the natural isoform revealed a substantial inverse isotope effect of 0.735, indicating a change in hybridization from sp(2) to sp(3) for hydroxylation at a carbon atom bearing the deuteron. During the hydroxylation of 4-(2)H(1)- and 3,5-(2)H(2)-toluene, similar magnitudes of intramolecular isotope effects and patterns of deuterium retention were observed from both isoforms studied, indicating that the active-site mutation affected substrate orientation but did not influence the mechanism of hydroxylation. The results with deuterated toluenes show inverse intramolecular isotope effects for hydroxylation at the position of deuteration, normal secondary isotope effects for hydroxylation adjacent to the position of deuteration, near-quantitative deuterium retention in m-cresol obtained from 4-(2)H(1)-toluene, and partial loss of deuterium from all phenolic products obtained from 3,5-(2)H(2)-toluene. This combination of results suggests that an active site-directed opening of position-specific transient epoxide intermediates may contribute to the chemical mechanism and the high degree of regiospecificity observed for aromatic hydroxylation in this evolutionarily specialized diiron enzyme.

Binding Sites↗

PubMed2003

Crystallization and preliminary analysis of native and N-terminal truncated isoforms of toluene-4-monooxygenase catalytic effector protein.

Single crystals have been obtained of the toluene 4-monooxygenase catalytic effector protein, the SeMet-enriched protein and a truncated isoform missing ten amino acids from the N-terminus. Complete X-ray diffraction data sets have been collected and analyzed to 2.0, 3.0 and 1.96 A resolution for the native, SeMet and truncated isoform crystals, respectively. The native and SeMet proteins crystallized in space group P6(1)22 (unit-cell parameters a = b = 86.41 +/- 0.15, c = 143.90 +/- 0.27 A), whereas the truncated isoform crystallized in space group P2(1)3 (a = b = c = 86.70 +/- 0.47 A). Matthews coefficient calculations suggest either two or three molecules per asymmetric unit in the P6(1)22 space group and two molecules per asymmetric unit in the P2(1)3 space group. Experimental phases from MAD analysis of the SeMet isoform and molecular replacement of the truncated isoform confirm the presence of two molecules per asymmetric unit in each case. These crystallographic results are the first available for the evolutionarily related but functionally diversified catalytic effector proteins from the multicomponent diiron monooxygenase family.

Crystallization↗

PubMed2003

Effects of sterculic acid on stearoyl-CoA desaturase in differentiating 3T3-L1 adipocytes.

The effects of sterculic acid on cell size, adiposity, and fatty acid composition of differentiating 3T3-L1 adipocytes are correlated with stearoyl-CoA desaturase (SCD) expression (mRNA and protein levels) and enzyme activity. Fluorescence-activated cell scanning (FACS) analysis showed that adipocytes differentiated with methylisobutylxanthine, dexamethasone, and insulin (MDI) plus 100 microM sterculic acid comprised a population of predominantly large cells with reduced adiposity compared to MDI-treated cells. Although both groups had similar amounts of total fat, their fatty acid profiles were strikingly different: MDI-treated cells had high levels of the unsaturated palmitoleic (Delta(9)-16:1) and oleic (Delta(9)-18:1) acids, whereas the cells cultured with MDI plus sterculic acid accumulated palmitic (16:0) and stearic (18:0) acids together with a marked reduction in Delta(9)-16:1. Although the cells treated with MDI plus sterculic acid had similar levels of scd1 and scd2 mRNAs and antibody-detectable SCD protein as the MDI-treated cells, the SCD enzyme activity was inhibited more than 90%. The accumulation of 16:0 and 18:0, together with normal levels of fatty acid synthase (FAS) and aP2 mRNAs, shows that de novo synthesis and elongation of fatty acids, as well as cell differentiation, were not affected by sterculic acid. Because of the increase in cell size in the sterculic acid-treated cells, the insulin-stimulated 2-deoxyglucose (2-DOG) uptake was determined. Compared to MDI-treated cells, the 2-DOG uptake in the cells treated with sterculic acid was not affected. These results indicate that sterculic acid directly inhibits SCD activity, possibly by a turnover-dependent reaction, without affecting the processes required for adipocyte differentiation, scd gene expression or SCD protein translation.

3T3 Cells↗

PubMed2002

Fluorescence anisotropy studies of enzyme-substrate complex formation in stearoyl-ACP desaturase.

Stearoyl-acyl carrier protein Delta(9)-desaturase (delta9D) catalyzes regio- and stereospecific insertion of cis double bonds into acyl chains attached to acyl carrier protein. Steady-state and stopped-flow fluorescence anisotropy measurements using acylated forms of dansyl- and fluoresceinyl-ACPs revealed equilibrium dissociation constants and dissociation rate constants for 16:0-, 17:0-, and 18:0-ACPs with resting and chemically 4e(-) reduced delta9D. Binding of 1 nM 18:0-fluoresceinyl-ACP to one subunit of the dimeric resting delta9D was observed with K(D1) = 13 +/- 3 nM. No significant difference in the K(D1) value was observed for 4e(-) delta9D. An approximately 4-fold increase in K(D1) per methylene group was observed upon shortening the acyl chain from 18:0 to 17:0 and then 16:0. In different experiments performed with 850 nM 18:0-dansyl-ACP, binding to the second subunit of resting delta9D was estimated to have K(D2) approximately 350 +/- 40 nM. The K(D2) values exhibited a similar dependence on acyl chain length as observed for the K(D1) values. The k(off) values measured by stopped-flow anisotropy measurements for reversal of the enzyme-substrate complex were also acyl-chain length dependent and increased 130-fold for 16:0-ACP (130 s(-)(1)) relative to 18:0-ACP (1 s(-)(1)). Increases in acyl chain length are thus associated with the presently reported increases in the K(D) and k(off) values. These results indicate that acyl chain length selectivity derives in major part from partition of the enzyme-substrate complex between substrate release and subsequent steps in catalysis.

Acyl Carrier Protein↗

PubMed2002

N-isotope effects on the Raman spectra of Fe(2)S(2) ferredoxin and Rieske ferredoxin: evidence for structural rigidity of metal sites.

The diiron ferredoxins have a common diamond-core structure with two bridging sulfides, but differ in the nature of their terminal ligands: either four cysteine thiolates in the Fe(2)S(2) ferredoxins or two cysteine thiolates and two histidine imidazoles in the Rieske ferredoxins. Contributions of the bridging (b) and terminal (t) ligands to the resonance Raman spectra of the Fe(2)S(2) ferredoxins have been distinguished previously by isotopic substitution of the bridging sulfides. We now find that uniform (15)N-labeling of Anabaena Fe(2)S(2) ferredoxin results in shifts of -1 cm(-1) in the Fe-S(t) stretching modes at 282, 340, and 357 cm(-1). The (15)N dependence is ascribed to kinematic coupling of the Fe-S(Cys) stretch with deformations of the cysteine backbone, including the amide nitrogen. No (15)N dependence occurs for the nu(Fe-S(b)) modes at 395 and 426 cm(-1). Similar effects are observed for the Rieske center in T4MOC ferredoxin from the toluene-4-monooxygenase system of Pseudomonas mendocina. Upon selective (15)N-labeling of the alpha-amino group of cysteine, the vibrational modes at 321, 332, 350, and 362 cm(-1) all undergo shifts of -1 to -2 cm(-1), thereby identifying them as combinations of nu(Fe-S(t)) and delta(Cys). These same four modes undergo similar isotope shifts when T4MOC ferredoxin is selectively labeled with (15)N-histidine ((15)N in either the alpha1,delta1 or delta1,epsilon2 positions). Thus, the Fe-S(Cys) stretch must also be undergoing kinematic coupling with vibrations of the Fe-His moiety. The extensive kinematic coupling of iron ligand vibrations observed in both the Fe(2)S(2) and Rieske ferredoxins presumably arises from the rigidity of the protein framework and is reminiscent of the behavior of cupredoxins. In both cases, the structural rigidity is likely to play a role in minimizing the reorganization energy for electron transfer.

Anabaena↗

PubMed2002

Desaturation, chain scission, and register-shift of oxygen-substituted fatty acids during reaction with stearoyl-ACP desaturase.

Stearoyl acyl carrier protein Delta(9) desaturase catalyzes the NADPH- and O(2)-dependent insertion of a cis double bond between the C-9 and C-10 positions of the acyl chain in the kinetically preferred natural substrate 18:0-ACP. In this work, substrate analogues with an oxygen atom singly replacing the methylene groups at the 8, 9, 10, and 11 positions of the stearoyl chain were synthesized, converted to acyloxy-ACPs, and used as probes of desaturase reactivity. Evidence for desaturation, acyloxy chain scission, and register-shift in binding prior to chain scission was obtained. Reactions with acyloxy-ACPs having either O-8 or O-11 substitutions gave a single desaturation product consistent with the insertion of a cis double bond between C-9 and C-10. The k(cat)/K(M) values for the O-8- and O-11-substituted acyloxy-ACPs were comparable to that of the natural substrate, indicating that the presence of an ether group adjacent to the site of reactivity did not significantly interfere either with the desaturation reaction or with the binding of substrate in the proper register for desaturation between C-9 and C-10. For reactions with the O-9 and O-10 acyloxy-ACPs, the k(cat) values were decreased to approximately 3% of that observed for 18:0-ACP, and upon reaction, the acyloxy chain was broken to yield an omega-hydroxy fatty alkanoyl-ACP and a volatile long-chain aldehyde. For the O-9 substitution, 8-hydroxyoctanoate and 1-nonanal were obtained, corresponding to the anticipated binding register and subsequent reaction between the O-9 and C-10 positions. In contrast, the O-10 substitution yielded 9-hydroxynonanoyl-ACP and 1-octanal, corresponding to an obligate "register-shift" of acyloxy chain binding prior to reaction between the O-10 and C-11 positions. Register-shift is thus defined as a mechanistically relevant misalignment of acyl chain binding that results in reaction at positions other than between C-9 and C-10. The inability of the O-10 acyloxy probe to undergo reaction between the C-9 and O-10 positions provides evidence that the Delta9D-catalyzed desaturation of stearoyl-ACP may initiate at C-10. Possible mechanisms of the acyl chain scission and implications of these results for the desaturation mechanism are considered.

Catalysis↗

PubMed2002

Molecular differences caused by differentiation of 3T3-L1 preadipocytes in the presence of either dehydroepiandrosterone (DHEA) or 7-oxo-DHEA.

The effects of dehydroepiandrosterone (DHEA) and 7-oxo-DHEA on the cell size, adiposity, and fatty acid composition of differentiating 3T3-L1 preadipocyte cells are correlated with stearoyl-CoA desaturase (SCD) expression (mRNA and protein levels) and enzyme activity. Fluorescence-activated cell sorting shows that preadipocyte cells treated with methylisobutylxanthine, dexamethasone, and insulin (MDI) plus DHEA comprise a population distribution of predominantly large cells with reduced adiposity. In contrast, cells treated with MDI plus 7-oxo-DHEA comprise a population distribution of almost equal proportions of small and large cells that have an adiposity equivalent to cells differentiated with MDI alone. The cells treated with MDI plus DHEA have significantly reduced levels of total fatty acid, mainly due to a dramatic reduction in the level of palmitoleic (Delta(9)-16:1) acid. The cells treated with MDI plus 7-oxo-DHEA have a significantly increased level of total fat, primarily due to increased levels of Delta(9)-16:1 and palmitic (16:0) acids. At the molecular level, the DHEA-treated cells contain lowered amounts of SCD1 mRNA and antibody-detectable desaturase protein, while 7-oxo-DHEA-treated cells contained elevated levels of SCD1 mRNA and protein. Inhibition of differentiation in DHEA-treated cells was also suggested by a reduction in the mRNA level of the adipogenic gene aP2. At the level of microsomal enzymatic activity, SCD activity was decreased in DHEA-treated cells while the SCD activity was increased in 7-oxo-DHEA-treated cells. The changes in mRNA levels and enzyme activity were concentration-dependent and appeared as early as day 3 of the differentiation protocol. The results show that DHEA and 7-oxo-DHEA have distinct modes of action with respect to the complex transcriptional cascade required for differentiation. Furthermore, differences in the insulin-stimulated uptake of 2-deoxyglucose and in the activity of carnitine palmitoyl transferase observed from either DHEA- or 7-oxo-DHEA-treated cells support the ability of DHEA to produce a thermogenic effect in differentiating preadipocytes, while 7-oxo-DHEA promotes differentiation without other changes typical of thermogenesis.

3T3 Cells↗

PubMed2002

Combined participation of hydroxylase active site residues and effector protein binding in a para to ortho modulation of toluene 4-monooxygenase regiospecificity.

Toluene 4-monooxygenase (T4MO) is a diiron hydroxylase that exhibits high regiospecificity for para hydroxylation. This fidelity provides the basis for an assessment of the interplay between active site residues and protein complex formation in producing an essential biological outcome. The function of the T4MO catalytic complex (hydroxylase, T4moH, and effector protein T4moD) is evaluated with respect to effector protein concentration, the presence of T4MO electron-transfer components (Rieske ferredoxin, T4moC, and NADH oxidoreductase), and use of mutated T4moH isoforms with different hydroxylation regiospecificities. Steady-state kinetic analyses indicate that T4moC and T4moD form complexes of similar affinity with T4moH. At low T4moD concentrations, the steady-state hydroxylation rate is linearly dependent on T4moD-T4moH complex formation, whereas regiospecificity and the coupling efficiency between NADH consumption and hydroxylation are associated with intrinsic properties of the T4moD-T4moH complex. The optimized complex gives both efficient coupling and high regiospecificity with p-cresol representing >96% of total products from toluene. Similar coupling and regiospecificity for para hydroxylation are obtained with T3buV (an effector protein from a toluene 3-monooxygenase), demonstrating that effector protein binding does not uniquely determine or alter the regiospecificity of toluene hydroxylation. The omission of T4moD causes an approximately 20-fold decrease in hydroxylation rate, nearly complete uncoupling, and a decrease in regiospecificity so that p-cresol represents approximately 60% of total products. Similar shifts in regiospecificity are observed in oxidations of alternative substrates in the absence or upon the partial removal of either T4moD or T3buV from toluene oxidations. The mutated T4moH isoforms studied have apparent V(max)/K(M) specificities differing by approximately 2-4-fold and coupling efficiencies ranging from 88% to 95%, indicating comparable catalytic function, but also exhibit unique regiospecificity patterns for all substrates tested, suggesting unique substrate binding preferences within the active site. The G103L isoform has enhanced selectivity for ortho hydroxylation with all substrates tested except nitrobenzene, which gives only m-nitrophenol. The regiospecificity of the G103L isoform is comparable to that observed from naturally occurring variants of the toluene/benzene/o-xylene monooxygenase subfamily. Evolutionary and mechanistic implications of these findings are considered.

Binding Sites↗