The catalytic mechanism of cytochrome P450 BM3 involves a 6 A movement of the bound substrate on reduction.
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Bacillus megaterium↗
Biomedical subjects
G C Roberts
Publications and source records attributed to G C Roberts.
Molecular analysis of the regulation of nisin immunity.
The genetic determinants controlling immunity to nisin are coordinately regulated, along with biosynthesis genes, in response to an environmental signal, nisin or a nisin analogue. The nisR gene product, the putative response regulator of nisin biosynthesis, was found to be a vital component of this induction mechanism. This protein forms part of a two-component regulatory system which controls the expression of genes involved in nisin immunity and biosynthesis. Analysis of the structural requirements of the external signal, using nisin fragments and engineered nisin variants, indicated that the 12 amino-terminal residues of the molecule are a minimum requirement for induction, with an intact ring A being an essential component. Changes throughout the molecule also affected its induction capacity. The production of certain variant nisins by engineered lactococcal strains is reduced in parallel with the strains' immunity to nisin. This can be attributed to inefficient induction by the variant molecule. Treating growing cultures with nisin restored full immunity and maximized the yields of nisin variants by the producer strains.
Chromosome Mapping↗
Effect of goal orientation on achievement beliefs, cognition and strategies in team sport.
Nicholls' motivation conceptual framework pertaining to achievement goals was used to study the relationship between two implicit goal orientations (task and ego) and achievement cognitions and beliefs about the competitive team sport experience. The study examined the relationship between the goal orientations and purposes of team sport, motivational climate, satisfaction, sources of satisfaction, achievement strategies, and perception of ability in team sport. The subjects were 148 students experienced in team sport at a Norwegian university. The scales were translated specifically for the study and factor analyses used to determine the factor structure of the scales. Seventeen factors along the 6 dimensions emerged, and canonical analysis determined that predominantly task-oriented subjects focused on health-related activities within purposes, preferred mastery climates and focused on mastery-oriented criteria to determine satisfaction and other achievement-related beliefs. In contrast, predominantly ego-oriented subjects focused on status enhancement for purpose, preferred performance-oriented climates and focused on ego-oriented criteria to determine satisfaction and other achievement beliefs.
Achievement↗
Structure-activity relationships in the peptide antibiotic nisin: role of dehydroalanine 5.
A mutant of the peptide antibiotic nisin in which the dehydroalanine residue at position 5 has been replaced by an alanine has been produced and structurally characterized. It is shown to have activity very similar to that of wild-type nisin in inhibiting growth of Lactococcus lactis and Micrococcus luteus but is very much less active than nisin as an inhibitor of the outgrowth of spores of Bacillus subtilis. These observations, which parallel those of W. Liu and J. N. Hansen (Appl. Environ. Microbiol. 59:648-651, 1993) on the corresponding mutant of the related antibiotic subtilin, are discussed in terms of the mechanism(s) of action of these antibiotics.
Alanine↗
Goal orientations and perceived purposes of training among elite athletes.
The goal perspective theories of motivated behavior are primarily concerned with the social, psychological, and behavioral antecedents and consequences of two goal perspectives of task orientation and ego orientation. According to Nicholls, goal perspectives may also influence the way individuals perceive the purpose of their involvement in achievement contexts. This study examined among elite athletes the relation of goal orientations to perceived purposes of their involvement in training. Canonical correlation analysis showed that ego-oriented athletes strongly endorsed status reasons for participation in training, whereas those task-involved reported a high preference for social purposes. The results suggest that the personal utility value of practicing in sport as perceived by elite athletes to some extent is different dependent upon their motivational goal perspectives.
Achievement↗
Effect of replacement of ferriprotoporphyrin IX in the haem domain of cytochrome P-450 BM-3 on substrate binding and catalytic activity.
Bacillus megaterium cytochrome P-450 BM-3 (coded by gene CYP102) is a catalytically self-sufficient mono-oxygenase, with both cytochrome P-450 and NADPH:cytochrome P-450 reductase domains, that catalyses the hydroxylation of fatty acids. The natural ferriprotoporphyrin IX has been removed from the haem domain of cytochrome P-450 BM-3 by treatment with acidified acetone, and it has been shown that, under carefully controlled conditions, haem can be added back to the resultant apoprotein to obtain a fully reconstituted haem domain with spectroscopic, substrate-binding and catalytic properties indistinguishable from those of the native domain. Replacement of the natural haem with ferriprotoporphyrin IX dimethyl ester yields a protein which has a higher affinity for the substrate dodecanoic acid and (in the presence of the reductase domain) the same catalytic rate as the native haem domain. Replacement with ferrimesoporphyrin IX yields a protein with the same affinity for substrate, but a reduced catalytic turnover. These results suggest that the haem moiety has a role in the creation of the binding pocket for substrate, and that modification of the electron density on the haem iron effects the catalytic rate.
Bacillus megaterium↗
NMR studies of substrate binding to cytochrome P450 BM3: comparisons to cytochrome P450 cam.
The binding of the substrates sodium laurate and sodium 12-bromolaurate to the heme-containing domain of Bacillus megaterium cytochrome P450 BM3 (CYP102) has been studied by measurement of the relaxation effects of the unpaired electrons of the heme iron on the protons of water and of the bound substrates. Substrate binding leads to a conversion of the heme iron from a low-spin to a high-spin state, as shown by changes in the optical spectrum. The relaxation measurements show that this is accompanied by expulsion of water from the sixth coordination position of the iron, the distance between the iron and the water protons increasing from 2.6 to 5.2 A. Corresponding relaxation measurements on the substrate protons lead to the determination of a number of distances between the iron and protons of the bound substrate and, hence, to information on the position and orientation of the substrate in the binding site. Laurate and 12-bromolaurate are found to bind in a very similar way, in an extended conformation with the carboxylate probably close to Arg47 and the other end of the chain 7.6-7.8 A from the heme iron. It is shown that laurate and pyridine can bind simultaneously to the P450 domain and that the iron-laurate distances in this ternary complex are not significantly different from those in the binary complex. These observations are compared with those on the substrate complex of cytochrome P450 cam, and their implications for structural changes involved in the catalytic cycle are discussed.
Bacillus megaterium↗
Site-specific 15N-labelling of oligonucleotides for NMR: the trp operator and its interaction with the trp repressor.
A convenient and economical method is described for the site-specific 15N-labelling of the 4-amino group of individual cytidine residues in oligonucleotides. This is applied to a 20 base-pair oligonucleotide corresponding to the trp operator; this oligonucleotide and its complex with the E. coli trp repressor are studied by NMR, using 1H-15N HMQC and 15N-edited 1H-1H NOESY experiments.
Bacterial Proteins↗
Nuclear magnetic resonance assignments and secondary structure of bovine S100 beta protein.
S100 beta is a neurite extension factor and has been implicated in Alzheimer's disease and Down's syndrome. It belongs to a group of low molecular weight calcium-binding proteins containing the helix-loop-helix calcium binding motif. The structure of only one S100 protein, calbindin D9k, which has the lowest sequence similarity to the other members of the S100 group has been determined. We report the NMR assignments and secondary structure of calcium-free S100 beta. The secondary structure is similar to that of calbindin D9k, determined using NMR, except that there is clear evidence for an additional well ordered 5-residue alpha-helix in S100 beta.
Amino Acid Sequence↗
Determination of the structure of an N-substituted protoporphyrin isolated from the livers of griseofulvin-fed mice.
Feeding mice with griseofulvin, a widely used anti-fungal agent which induces protoporphyria as a side-effect, leads to the formation in the liver of two green pigments which have been shown to be porphyrin adducts. In this work, the major porphyrin adduct isolated from the livers of griseofulvin-fed mice has been characterized structurally using one- and two-dimensional NMR spectroscopy. The adduct was shown to be an N-alkylated protoporphyrin IX in which the whole of griseofulvin (less a hydrogen atom) is attached to a pyrrole ring nitrogen of the porphyrin. It was shown that the drug-to-porphyrin linkage is an an -O-CH2-Npyrrole = linkage, to either the 4- or 6-position of ring a of griseofulvin. In an attempt to identify which pyrrole nitrogen is involved in this linkage, the 1H spectra of the free base and zinc complex of the adduct were compared with the corresponding spectra of the four regioisomers of N-methylprotoporphyrin. These comparisons indicated that the adduct isolated from the livers of griseofulvin-fed mice is either the NC or the ND regioisomer, although a clear distinction between these two could not be made on the available evidence. The mechanism of formation of the adduct and its relation to griseofulvin-induced protoporphyria are discussed.
Administration, Oral↗
Role of the active-site carboxylate in dihydrofolate reductase: kinetic and spectroscopic studies of the aspartate 26-->asparagine mutant of the Lactobacillus casei enzyme.
A mutant of Lactobacillus casei dihydrofolate reductase, D26N, in which the active site aspartic acid residue has been replaced by asparagine by oligonucleotide-directed mutagenesis has been studied by NMR and optical spectroscopy and its kinetic behavior characterized in detail. On the basis of comparisons of a large number of chemical shifts and NOEs, it is clear that there are only very slight structural differences between the methotrexate complexes of the wild-type and mutant enzymes and that these are restricted to the immediate environment of the substitution. The data suggest a slight difference in orientation of the pteridine ring in the binding site in the mutant enzyme. Both NMR and UV spectroscopy show that methotrexate is protonated on N1 when bound to the wild-type enzyme but not when bound to the mutant. Binding constant measurements by fluorescence quenching and steady-state kinetic measurements of dihydrofolate (FH2) and folate reduction show that the substitution has little or no effect on substrate, coenzyme, and inhibitor binding (< 7-fold increase in Kd) and only a modest effect on kcat (up to a factor of 9 for FH2 and 25 for folate) and kcat/KM (up to a factor of 13 for FH2 and 14 for folate). Measurements of deuterium isotope effects and direct measurements of hydride ion transfer and product release by stopped-flow methods revealed that for the mutant enzyme hydride ion transfer is rate-limiting across the pH range 5-8. This allowed a direct comparison of the rate of hydride ion transfer in the wild-type and mutant enzymes; the asparagine substitution was found to decrease this rate by 62-fold at pH 5.5 and 9-fold at pH 7.5. This effect is much smaller than that seen for the corresponding mutant of Escherichia coli dihydrofolate reductase [Howell, E. E., Villafranca, J. E., Warren, M. S., Oatley, S. J., & Kraut, J. (1986) Science 231, 1123-1128], estimated as a 1000-fold decrease in the rate of hydride ion transfer. The change in pH dependence of kcat resulting from the substitution is consistent with, but does not prove, the idea that the group of pK 6.0 which must be protonated for hydride ion transfer to occur is Asp26. For folate reduction, the pH dependence of kcat is determined by two pKs, one of which, pK 5, disappears in the mutant enzyme, suggesting that it may correspond to ionization of Asp26.(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acid Sequence↗
Changes in the structure of bovine phospholipase A2 upon micelle binding.
Phospholipase A2 (PLA2) is a calcium-dependent enzyme which hydrolyses the 2-acyl ester bond of phospholipids. The extracellular PLA2s are activated by as much as 10000-fold on binding to micelles or vesicles of substrate, possibly due to a conformational change induced in the enzyme. We have studied the complex of bovine pancreatic PLA2 with micelles of SDS by ultracentrifugation, equilibrium dialysis, microcalorimetry, fluorescence and n.m.r. spectroscopy. Ultracentrifugation and equilibrium dialysis measurements showed that on average 1.28 (+/- 0.17) PLA2 molecules and 26.4 (+/- 3.1) SDS molecules are involved in the complex and that there is a rapid equilibrium between micellar species containing one or more enzyme monomers. The estimated heat of formation of the complex, measured calorimetrically as the heat released when PLA2 was injected into excess 10 mM SDS, was 162.3 +/- 1.5) kJ/mol [38.8 (+/- 0.35) kcal/mol] of PLA2 added. The fluorescence of the single tryptophan at position 3 in the N-terminal helix of the protein increases when PLA2 binds to SDS micelles, indicating that this part of the protein is in a more hydrophobic environment in the complex. The structural changes in PLA2 on addition of [2H25]SDS were monitored using n.m.r. spectroscopy. The overall structure of the protein is unchanged, but changes in nuclear Overhauser effects (NOEs) were observed for residues in the N-terminal helix, at the active site region and in a lysine-rich region near the C-terminus. The NOE changes at the N-terminus indicate that this portion of the protein molecule adopts a more ordered, helical conformation when bound to a micelle. We suggest that these conformational changes could be the mechanism by which the enzyme becomes activated in the presence of aggregated substrate.
Amino Acid Sequence↗
Sequencing of oligonucleotides using high performance liquid chromatography and electrospray mass spectrometry.
We report the sequence analysis of an oligodeoxyribonucleotide by electrospray mass spectrometry (ES-MS). For full sequence analysis of the oligomer, d(CGTAGCTACG), an enzyme digestion step followed by off-line high performance liquid chromatography was used. For ES-MS analysis, introducing the sample in an imidazole buffer with 70% acetonitrile increased the electrospray ion currents and helped reduce sodium-adduct ion peaks.
Chromatography, High Pressure Liquid↗
High-level expression and isotopic labeling of Lactobacillus casei dihydrofolate reductase for nuclear magnetic resonance spectroscopy.
Two efficient systems have been used for high-level expression of Lactobacillus casei dihydrofolate reductase in Escherichia coli, including the production of protein generally and specifically labeled with 13C and 15N. A system based on T7 RNA polymerase led to the production of dihydrofolate reductase at a level of 37% of the total soluble protein of the host strain: 50 mg of pure enzyme was obtained from a 1 liter of culture (or 14 mg/g wet weight of cells). In this system, a small amount of the enzyme (less than 5%) was identified as a catalytically active 21-kDa fusion protein. Introduction of a second in-frame (ochre) stop codon did not eliminate the production of this fusion protein. The same expression system was also used to prepare dihydrofolate reductase generally labeled with 15N and to prepare single and double mutants of the enzyme. In order to have an expression system which can be used with a range of auxotrophic strains of E. coli, a system based on the tac promoter was used. This led to the production of dihydrofolate reductase at a level of 29% of total soluble protein; a yield of 40 mg enzyme per liter of culture (or 11 mg/g wet weight of cells). This system was successfully used to produce mutants of the enzyme as well as the enzyme selectively labeled with [gamma-13C]aspartic acid.
Base Sequence↗
Structural studies on FMN domain of cytochrome P450 reductase.
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Electron Transport↗
The interactions of Escherichia coli trp repressor with tryptophan and with an operator oligonucleotide. NMR studies using selectively 15N-labelled protein.
The effects of the binding of the corepressor L-tryptophan and an operator oligonucleotide to Escherichia coli trp repressor have been studied, using selective 15N labelling to permit observation of the backbone amide resonances of 50 of the 107 residues of the protein monomer. Repressor molecules selectively labelled in turn with [15N]alanine, [15N]glutamate, [15N]isoleucine, [15N]leucine and [15N]methionine were prepared by isolating them from prototrophic E. coli cells grown in media containing a mixture of unlabelled and the appropriate 15N-enriched amino acids. Analysis of the heteronuclear correlation spectra of the labelled repressors shows the value of selective labelling in resolving the crosspeaks of, for example, the 19 leucine and 12 glutamate residues. All 50 residues studied show measurable changes in amide 1H and/or 15N chemical shift on the binding of tryptophan and/or the operator oligonucleotide, showing clearly that ligand binding has effects which are transmitted throughout almost the whole protein. Large chemical shift changes on ligand binding are seen in residues in the tryptophan binding site and in the 'helix-turn-helix' DNA-binding domain, but also in residues in helices C and F remote from the ligand binding sites. On operator binding there is selective broadening of the signals of residues in the N-terminal region of the protein and in the DNA-binding domain, perhaps reflecting a conformational equilibrium.
Bacterial Proteins↗
Smooth muscle-specific switching of alpha-tropomyosin mutually exclusive exon selection by specific inhibition of the strong default exon.
Exons 2 and 3 of alpha-tropomyosin are spliced in a strict mutually exclusive manner. Exon 3 is a default choice, being selected in almost all cell types where the gene is expressed. The default selection arises from a competition between the two exons, in which the stronger branch point/pyrimidine tract elements of exon 3 win. Exon 2 is selected predominantly or exclusively only in smooth muscle cells. We show here that the basis for the smooth muscle-specific switching of exon selection is inhibition of exon 3. Exon 3 is still skipped with smooth muscle specificity, even in the absence of exon 2. We have defined two conserved sequence elements, one in each of the introns flanking exon 3, that are essential for this regulation. Mutation of either element severely impairs regulated suppression of exon 3. No other exon or intron sequences appear to be necessary for regulation. We have also demonstrated skipping of exon 3 that is dependent upon both regulatory elements in an in vitro splicing assay. We further show that both splice sites of exon 3 must be inhibited in a concerted fashion to switch to selection of exon 2. This may relate to the requirement for negative elements on both sides of the exon.
Alternative Splicing↗
Direct n.m.r. evidence for substrate-induced conformational changes in a beta-lactamase.
Cefoxitin and other beta-lactam antibiotics with a methoxy group on the alpha-face behave as very poor substrates of the Bacillus licheniformis beta-lactamase. The kinetic properties of the enzyme-cefoxitin system made it theoretically suitable for a detailed structural study of the acyl-enzyme. Unfortunately, soaking the crystals in cefoxitin solution did not allow detection of a crystalline acyl-enzyme complex. In contrast, direct observation by n.m.r. of the stable acyl-enzyme formed with cefoxitin and moxalactam indicated clear modifications of the enzyme structure, which were reflected in the aromatic and high-field methyl regions of the spectrum. The return to the initial free enzyme spectrum was concomitant with the hydrolysis of the acyl-enzyme, the process being slow enough to allow multidimensional n.m.r. experiments.
Anti-Bacterial Agents↗