Stable mutant strains

One type of the constituent metallocenters in the MoFe protein has the properties of a somewhat independent structural entity. This component, referred to as the FeMo cofactor (FeMo-co), was first identified by Shah and Brill (1977) as the stable metallocluster extracted from acid-denatured MoFe protein. The FeMo-co was able to fully activate a defective protein in the extracts of mutant strain UW45, a protein which subsequently was shown to contain the P clusters but not the EPR-active center. The isolated cofactor accounted for the total S = t system observed by EPR and Mdssbauer spectroscopies of the holo-MoFe protein (Rawlings et al., 1978). Elemental analysis indicated a composition of Mo Fee-8 Se-g for the cofactor, which, if there are two FeMo-co s per a2 2> accounts for all the molybdenum and approximately half the iron in active enzyme (Nelson etai, 1983). Although FeMo-co has been extensively studied [reviewed in Burgess (1990)] the structure remains enigmatic. To date, all attempts to crystallize the cofactor have failed. This is possibly due to the instability and resultant heterogeneity of the cofactor when removed from the protein. Also, there is a paucity of appropriate models for spectral comparison (see Coucouvanis, 1991, for a recent discussion). Final resolution of this elusive structure may require its determination as a component of the holoprotein. 

Dibenzofuran is highly stable to oxidizing agents. 2-Dibenzofuranol is oxidized to 2-(2-hydroxyphenyl)-l,4-benzoquinone by sodium periodate in aqueous acetic acid, whereas reaction of 1-dibenzofuranol or 4-dibenzo-furanols with Fremy s salt produces the dibenzofuran-l,4-quinones. The oxidation of dibenzofuran with microorganisms has been studied. A mutant strain of a Beijerinckia bacterium oxidizes dibenzofuran to a mixture of cis-2,3-dihydroxy-2,3-dihydrodibenzofuran and cis-l,2-dihy-droxy-l,2-dihydrodibenzofuran. The former compound was found to be too... 

A report (1991) may lead to the ultimate solution of the acid instability problem the production of 6-deoxyerythromycin A by the use of a mutant strain of Saccharopolyspora erythrea. This was accomplished by a targeted disruption of the gene ery F, believed to code for the P450 hydroxylase enzyme, which normally produces the C-6 OH function. This new mutant antibiotic was found to be acid stable and as effective as EM-A in mice (orally) against several pathogenic bacteria. 

The microlocalisation technique with the stable isotope Li uses a beam of neutrons in an atomic reactor. The Li nucleus absorbs a neutron and immediately undergoes fission to produce an a-particle and a H atom, which create tracks in a suitable detector placed in contact with Li-containing tissue. The tissue distribution in the rat, brain lithium distribution in the mouse " and the rat, ° distribution in the mouse embryo, kinetics in the mouse brain, and distribution in mutant strains of mice with dysmyelination have been studied. 

In spite of the large number of high-activity mutant strains isolated, none of them preserved its original activity after further selection. Since nitrosoalkylamides (NAA) were used to derive the parental strain M82 (Grusina et al., 1974), it is possible that the genome of this strain is already saturated with NAA-inducible point mutations, so that the treatment with NMB, another NAA mutagen, could not elicit stable mutants in this strain. 

Arzberger CF, Peterson WH, Fred EB (1920) Certain factors that influence acetone production by Bacillus acetoethylicum. J Biol Chem 44 465—479 Azeddoug H, Hubert J, Reysset G (1992) Stable inheritance of shuttle vectors based on plasmid pIM13 in a mutant strain of Clostridium acetobutylicum. J Gen Microbiol 138 1371-1378... 

Squalene synthase (EC 2.5.1.21) known as a membrane-bound enzyme condenses two molecules of famesyl diphosphate (FPP) in a stable intermediate, the presqualene diphosphate (PS) which is then reduced by NAD(P)H to form squalene (S). In spite of various hypotheses concerning the binding sites and reaction centers, the catalytic machinery for the two reactions and the mechanism(s) responsible for the sterol mediated regulation of squalene synthase (SQS), have not been elucidated. Sterol auxotrophic mutant strains (1) can be used as tools for such studies. Therefore, before going to purify SQS from plant sources (2), we have undertaken the purification and comparative characterization of squalene synthase (SQS) from three yeast strains the wild-type FLIOO, the mutant strain erglOB and the recombinant strain FK5188 pMF13. 

The usual method of study is to suggest a possible precursor and to feed it to the biosynthesizing system. The precursor has to be labelled in some way to trace it through the sequence of reactions, and that is usually by some isotopic element. It may be a radio-active isotope, such as H, " 0, or that can be followed by its radiation or it can be a stable heavy isotope, such as H, C, N, or 0, that can be traced by mass spectrometry or nuclear magnetic resonance (NMR) spectroscopy (Table 5.1). Another possible way is to use mutant strains of an organism that lack the enzymes to complete a particular synthesis, or to add a specific enzyme inhibitor, so that intermediates accumulate and can be identified. A mutant strain of yeast was important in discovering mevalonic acid and its place in terpene biosynthesis (Chapter 6) and a number of mutants of the bacterium Escherichia coli helped to understand the shikimic acid pathway (Chapter 8). 

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