A study of 31 temperature-sensitive mutants of mouse cytomegalovirus has indicated that two mutants (tsm1, tsm31) may be defective in immediate-early/early functions, two (tsm2, tsm3) may be defective in early functions and six (tsm9, tsm18, tsm22, tsm23, tsm28, tsm30) may be defective in early/late functions while the remainder are late function-defective mutants as determined by temperature-s...

Comprehensive and programmable protein mutagenesis is critical for understanding structure-function relationships and improving protein function. There is thus a need for robust and unbiased molecular biological approaches for the construction of the requisite comprehensive protein libraries. Here we demonstrate that plasmid recombineering is a simple and robust in vivo method for the generatio...

The predicted effect of effective population size on the distribution of fitness effects and substitution rate is critically dependent on the relationship between sequence and fitness. This highlights the importance of using models that are informed by the molecular biology, biochemistry, and biophysics of the evolving systems. We describe a computational model based on fundamental aspects of b...

Rieske nonheme iron oxygenases form a large class of aromatic ring-hydroxylating dioxygenases found in microorganisms. These enzymes enable microorganisms to tolerate and even exclusively utilize aromatic compounds for growth, making them good candidates for use in synthesis of chiral intermediates and bioremediation. Studies of the chemical stability and thermostability of these enzymes thus b...

Ak.1 protease, a thermostable subtilisin isolated originally from Bacillus st. Ak.1, was purified to homogeneity from the Escherichia coli clone PB5517. It is active against substrates containing neutral or hydrophobic branched-chain amino acids at the P(1) site, such as valine, alanine or phenylalanine. The K(m) and k(cat) of the enzyme decrease with decreasing temperature, though not to the s...

Eight erythrocyte enzymes were examined for thermostability in an unselected sample of 100 newborn infants. Three thermolabile variants, one each of lactate dehydrogenase, glucosephosphate isomerase, and glucose-6-phosphate dehydrogenase, were identified, none of which was detectable as a variant by standard electrophoretic techniques. All were inherited. This frequency of 3.8 heritable thermos...

The gene encoding a novel penicillin G acylase (PGA), designated pgaW, was cloned from Achromobacter xylosoxidans and overexpressed in Escherichia coli. The pgaW gene contains an open reading frame of 2586 nucleotides. The deduced protein sequence encoded by pgaW has about 50% amino acid identity to several well-characterized PGAs, including those of Providencia rettgeri, Kluyvera cryocrescens,...

We probe the hypothesis of corresponding states, according to which homologues from mesophilic and thermophilic organisms are in corresponding states of similar rigidity and flexibility at their respective optimal temperatures. For this, the local distribution of flexible and rigid regions in 19 pairs of homologous proteins from meso- and thermophilic organisms is analyzed and related to activi...

A leading intellectual challenge in evolutionary genetics is to identify the specific phenotypes that drive adaptation. Enzymes offer a particularly promising opportunity to pursue this question, because many enzymes' contributions to organismal fitness depend on a comparatively small number of experimentally accessible properties. Moreover, on first principles the demands of enzyme thermostabi...

Twelve mutations were constructed to improve the thermostability of glucoamylase from Aspergillus awamori based on the results of molecular dynamics simulations. The thermal unfolding of the catalytic domain followed a putative hierarchical behavior. In addition, the unfolding of the 13 alpha-helices obeyed the random ordered mechanism, in which the alpha-helices 8, 1 and 11 unfolded more rapid...