David P. Clark

Structure-based mutagenesis approaches toward expanding the substrate specificity of D-2-deoxyribose-5-phosphate aldolase.

2-Deoxyribose-5-phosphate aldolase (DERA, EC 4.1.2.4) catalyzes the reversible aldol reaction between acetaldehyde and D-glyceraldehyde-3-phosphate to generate D-2-deoxyribose-5-phosphate. It is unique among the aldolases as it catalyzes the reversible asymmetric aldol addition reaction of two aldehydes. In order to expand the substrate scope and stereoselectivity of DERA, structure-based substrate design as well as site-specific mutation has been investigated. Using the 1.05 A crystal structure of DERA in complex with its natural substrate as a guide, five site-directed mutants were designed in order to improve its activity with the unnatural nonphosphorylated substrate, D-2-deoxyribose. Of these, the S238D variant exhibited a 2.5-fold improvement over the wild-type enzyme in the retroaldol reaction of 2-deoxyribose. Interestingly, this S238D mutant enzyme was shown to accept 3-azidopropinaldehyde as a substrate in a sequential asymmetric aldol reaction to form a deoxy-azidoethyl pyranose, which is a precursor to the corresponding lactone and the cholesterol-lowering agent Lipitor. This azidoaldehyde is not a substrate for the wild-type enzyme. Another structure-based design of new nonphosphorylated substrates was focused on the aldol reaction with inversion in enantioselectivity using the wild type or the S238D variant as the catalyst and 2-methyl-substituted aldehydes as substrates. An example was demonstrated in the asymmetric synthesis of a deoxypyranose as a new effective synthon for the total synthesis of epothilones. In addition, to facilitate the discovery of new enzymatic reactions, the engineered E. coli strain SELECT (Deltaace, adhC, DE3) was developed to be used in the future for selection of DERA variants with novel nonphosphorylated acceptor specificity.

The use of suicide substrates to select mutants of Escherichia coli lacking enzymes of alcohol fermentation

SummaryMutants of Escherichia coli resistant to chloroethanol or to chloroacetaldehyde were selected. Such mutants were found to lack the fermentative coenzyme A (CoA) linked acetaldehyde dehydrogenase activity. Most also lacked the associated fermentative enzyme alcohol dehydrogenase. Both types of mutants, those lacking acetaldehyde dehydrogenase alone or lacking both enzymes, mapped close to the regulatory adhC gene at 27 min on the E. coli genetic map. The previously described acd mutants which lack acetaldehyde dehydrogenase and which map at 63 min were shown to be pleiotropic, affecting respiration and growth on a variety of substrates. It therefore seems likely that the structural genes for both the acetaldehyde and alcohol dehydrogenases lie in the adhCE operon. This interpretation was confirmed by the isolation of temperature sensitive chloracetaldehyde-resistant mutants, some of which produced thermolabile acetaldehyde dehydrogenase and alcohol dehydrogenase and were also found to map at the adh locus. Reversion analysis indicated that mutants lacking one or both enzymes carried single mutations. The gene order in the adh region was determined by three point crosses to be trp - zch:: Tn10 - adh - galU- bglY - tyrT - chlC.

[57] Bacterial mutants for the study of lipid metabolism

Publisher Summary This chapter discusses the bacterial mutants for the study of lipid metabolism. The major advantage to using the bacterium Escherichia coli for biochemical investigations is the possibility of genetic manipulation. Genetic methods may be used to eliminate specific enzymes from the cell, allowing unambiguous allocation of their in vivo role. The fatty acid composition of E. coli may be altered by feeding a variety of fatty acids to mutants defective in fatty acid biosynthesis. The fab D and fab E mutants are defective in the synthesis of all fatty acids, and were isolated as temperature-sensitive mutants. Supplementation of fab D or fab E mutants with saturated fatty acids plus unsaturated fatty acids (UFA) is only partly successful in maintaining growth at the restrictive temperature. The fab A and fab B mutants are specifically defective in the synthesis of UFA and may be supplemented with a variety of fatty acids, including cis - and trans -UFA, cyclopropane, branched, and halogenated fatty acids. A third mutation with no phenotype is cfa . These mutants have a defective cyclopropane fatty acid synthase and are deficient in the conversion of unsaturated fatty acids to their cyclopropane derivatives. This conversion occurs after the fatty acyl residues are attached to the phospholipids and happens only as cells enter the stationary phase.

One-step gene amplification by Mu-mediated transposition of E. coli genes to a multicopy plasmid.

Abstract A general in vivo method to amplify the number of copies of a specific gene in one step is described. The method is directly applicable to any selectable gene of Escherichia coli and is based on the Mu-mediated transposition of segments of host chromosomes into the conjugative, multicopy plasmid R6K. Using this method we have cloned the β-hydroxydecanoyl thioester dehydrase structural gene, fabA , into the R6K plasmid. Strains carrying the resultant plasmid produced 13 to 21 times more dehydrase than control strains.

Permeability and Susceptibility of Escherichia coli to β- Lactam Compounds

In general, the susceptibility of envelope mutants of Escherichia coli to β-lactam compounds correlates well with the permeability of these antibiotics through the outer membrane. However, several mutants were isolated which, although apparently freely permeable to penicillins, showed only marginal increases in susceptibility to these agents.

Genetic Deregulation of Ethanol-Related Genes

Wild-type Escherichia coli strains are unable to use ethanol as a carbon source. However, they do produce ethanol as a fermentation product under certain conditions of anaerobic growth. In the absence of nitrate, at acidic pH, and especially in the presence of high phosphate concentrations, E. coli ferments glucose mainly to lactic acid. However, at alkaline pH and in the absence of phosphate an equimolar mixture of acetate plus ethanol is the major product (11).