Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 ϫ g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmicphase cells. In contrast, sterol ⌬ 24 -methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.

Reduction of enoyl -acyl-carrier-protein (ACP) substrates by enoyl-ACP reductase is a key regulatory step in fatty acid elongation of Escherichia coli. Two enoyl-ACP reductase activities have been described in E. coli, one specific for NADH, the other for NADPH as cofactor. Because of their distinct enzymatic properties, these activities were ascribed to two different proteins. The NADH-dependent enoyl-ACP reductase of E. coli has previously been identified as the FabI protein, which is the target of a group of antibacterial compounds, the diazaborines. We now demonstrate that both enoyl-ACP reductase activities reside in FabI. In crude cell extracts of FabI-overproducing strains, both NADH-dependent and NADPHdependent enoyl-ACP reductase activities are increased. Mutations in the fabl gene that lead either to temperature-sensitive growth or diazaborine resistance result in the reduction of both activities. When FabI is purified in pH 6.5 buffers, the protein exhibits NADH-dependent and NADPH-dependent reductase activities. Both enzymatic activities are inhibited by diazaborine. The NADPH-dependent enoyl-ACP reductase activity, however, turned out to be approximately eight times more resistant to diazaborine. The difference in sensitivity indicates that binding of either NADPH or NADH to FabI results in distinct changes in the configuration of the protein or, alternatively, it is different due to the different charge of the cofactors. These effects might be responsible for the differences in the enzymatic properties. Both reductase activities of the FabI protein are inhibited by physiologically relevant concentrations of palmitoyl-CoA, which might be important in regulating endogenous fatty acid biosynthesis in E. coli in the presence of exogenous fatty acids.

No abstract

Conjugation and bacteriophage P1 transduction experiments in Escherichia coli showed that resistance to the antibacterial compound diazaborine is caused by an allelic form of the envM gene. The envM gene from Salmonella typhimurium was cloned and sequenced. It codes for a 27,765-dalton protein. The plasmids carrying this DNA complemented a conditionally lethal envM mutant of E. coli. Recombinant plasmids containing gene envM from a diazaborine-resistant S. typhimurium strain conferred the drug resistance phenotype to susceptible E. coli cells. A guanine-to-adenine exchange in the envM gene changing a Gly codon to a Ser codon was shown to be responsible for the resistance character. Upstream of envM a small gene coding for a 10,445-dalton protein was identified. Incubating a temperature-sensitive E. coli envM mutant at the nonpermissive temperature caused effects on the cells similar to those caused by treatment with diazaborine, i.e., inhibition of fatty acid, phospholipid, and lipopolysaccharide biosynthesis, induction of a 28,000-dalton inner membrane protein, and change in the ratio of the porins OmpC and OmpF.

1,2-Dihydro-1-hydroxy-2-(organosulfonyl)areno[d] [1,2,3]diazaborines 2 (arene = benzene, naphthalene, thiophene, furan, pyrrole) were synthesized by reaction of (organosulfonyl)hydrazones of arene aldehydes or ketones with tribromoborane in the presence of ferric chloride. The activities of 2 against bacteria in vitro and in vivo (Escherichia coli) were determined and structure-activity relationships are discussed. Included in this study are 2,3-dihydro-1-hydroxy-2-(p-tolylsulfonyl)-1H-2,1-benzazaborole+ ++ (3) and 1-hydroxy-1,2,3,4-tetrahydro-2-(p-tolylsulfonyl)-2,1-benzazabor ine (4) as well as the carbacyclic benzodiazaborine analogue 4-hydroxy-3-(p-tolylsulfonyl)isoquinoline (7). The nature of the active species is briefly discussed.