We developed a method for rapidly generating thermostable enzyme variants. Our strategy is to introduce the gene coding for a given enzyme from a mesophilic organism into a thermophile, Bacillus stearothermophilus. Variants that retain the enzymatic activity at the higher growth temperatures of the thermophile are then selected. This strategy was applied to kanamycin nucleotidyltransferase, which confers resistance to the antibiotic kanamycin. B. stearothermophilus carrying the wild-type enzyme is resistant to the antibiotic at 47C but not at 55C and above. Variants that were kanamycin resistant at 63°C were obtained by selection of spontaneous mutants, by passage ofa shuttle plasmid through theEscherichia colimutD5 mutator strain and introduction into B. stearothermophilus by transformation, and by growing the thermophile in a chemostat. The kanamycin nucleotidyltransferases purified from these variants were all more resistant to irreversible thermal inactivation than is the wild-type enzyme, and all have the same single amino acid replacement, aspartate to tyrosine at position 80. Mutants that are even more heat stable were derived from the frt variant by selecting for kanamycin resistance at 70C, and these carry the additional change of threonine to lysine at position 130. This strategy is applicable to other enzymatic activities that are selectable in thermophiles or that can be screened for by plate assays.The ability of some microorganisms to grow at extreme temperatures (1) implies that their enzymes are stable and active at these temperatures. This is largely borne out when enzymes from thermophilic sources are studied in vitro; such enzymes are indeed more thermostable than the equivalents isolated from phylogenetically related mesophilic organisms (2). Correlations between an increase in the proportion of hydrophobic residues and the degree of thermostability have been observed (3-5). Internal electrostatic interactions (6) and disulfide linkages (7) have also been proposed as features that stabilize proteins. However, the role of individual amino acid residues in enhancing the resistance to thermal denaturation of an enzyme from a thermophile is not known.We wish to understand the contributions of individual amino acids to the overall stability of a protein's structure. Comparisons between enzymes from mesophiles and thermophiles are complicated because these proteins, although homologous, usually differ in more than one residue. More precise inferences can be based on comparisons of temperature-sensitive mutations which encode, as a result of single amino acid changes, proteins that retain activity but are less resistant to heat denaturation than the wild-type counterpart. Thus, for example, many temperature-sensitive mutations of phage T4 lysozyme have been studied with the aim of correlating the changes in stability with changes in the protein structure (8). However, an x-ray crystallographic study of one of these mutant enzymes (9) revealed that a variant in which a histidine residue repla...

Kanamycin nucleotidyltransferase, as originally isolated from Staphylococcus aureus, inactivates the antibiotic kanamycin by catalyzing the transfer of a nucleotidyl group from nucleoside triphosphates such as ATP to the 4'-hydroxyl group of the aminoglycoside. The molecular structure of the enzyme described here was determined by X-ray crystallographic analysis to a resolution of 3.0 A. Crystals employed in the investigation belonged to the space group P4(3)2(1)2 with unit cell dimensions of a = b = 78.9 A and c = 219.2 A. An electron density map phased with seven heavy-atom derivatives revealed that the molecules packed in the crystalline lattice as dimers exhibiting local 2-fold rotation axes. Subsequent symmetry averaging and solvent flattening improved the quality of the electron density such that it was possible to completely trace the 253 amino acid polypeptide chain. Each monomer is divided into two distinct structural domains: the N-terminal motif composed of residues Met 1-Glu 127 and the C-terminal half delineated by residues Ala 128-Phe 253. The N-terminal region is characterized by a five-stranded mixed beta-pleated sheet whereas the C-terminal domain contains five alpha-helices, four of which form an up-and-down alpha-helical bundle very similar to that observed in cytochrome c'. The two subunits wrap about one another to form an ellipsoid with a pronounced cleft that could easily accommodate the various aminoglycosides known to bind to the enzyme.

The dha regulon in KkebsieUa pneumoniae enables the organism to grow anaerobically on glycerol and produce 1,3-propanediol (1,3-PD). Escherichia coli, which does not have a dha system, is unable to grow anaerobically on glycerol without an exogenous electron acceptor and does not produce 1,3-PD. A genomic library of K. pneumoniae ATCC 25955 constructed in E. coli AG1 was enriched for the ability to grow anaerobically on glycerol and dihydroxyacetone and was screened for the production of 1,3-PD. The cosmid pTC1 (42.5 kb total with an 18.2-kb major insert) was isolated from a 1,3-PD-producing strain of E. coli and found to possess enzymatic activities associated with four genes of the dha regulon: glycerol dehydratase (dhaB), 1,3-PD oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK). All four activities were inducible by the presence of glycerol. When E. coli AG1/pTC1 was grown on complex medium plus glycerol, the yield of 1,3-PD from glycerol was 0.46 mol/mol. The major fermentation by-products were formate, acetate, and D-lactate. 1,3-PD is an intermediate in organic synthesis and polymer production. The 1,3-PD fermentation provides a useful model system for studying the interaction of a biochemical pathway in a foreign host and for developing strategies for metabolic pathway engineering. Metabolic pathway engineering (MPE, also metabolic engineering), the modification, design, and construction of biochemical pathways, is an emerging discipline of potential importance to the chemical, biochemical, food, and environmental industries. MacQuitty (19) has called MPE the fourth wave of biotechnology following classical fermentation, recombinant DNA technology, and protein engineering. Recent progress in MPE has been reviewed by Bailey (2). We have selected the conversion of glycerol to 1,3propanediol (1,3-PD) as a model system for the study of MPE. Our reasons are as follows. (i) The pathway is relatively simple, consisting of only two enzymes, a dehydratase (glycerol dehydratase [EC 4.2.1.30] or diol dehydratase [EC 4.2.1.28] and 1,3-PD oxidoreductase [EC 1.1.1.202]); (ii) the pathway possesses features of a more complex metabolic network (e.g., the dehydratase is a multicomponent enzyme and requires coenzyme B12, and

The pheromone a factor, secreted by Saccharomyces cerevisiae cells of the a mating type, serves to synchronize the opposite mating type (a cells) at GI as a prelude to fusion of the two cell types. We found that, in vitro, a factor inhibited the' membrane-bound adenylate cyclase of these cells in a dose-dependent manner. Moreover, one class (steS) of a cell mutants that grow normally at either 230 or 340C but that are unable to respond to a factor or to mate at the higher temperature possessed an adenylate cyclase activity that was not inhibited by a factor at 340C but was fully sensitive to inhibition at 230C. Furthermore, addition of cyclic AMP to a cell culture medium shortened the period of pheromone-induced GI arrest. We conclude that inhibition of adenylate cyclase activity by a factor may constitute, at least in part, the biochemical mode of action of the pheromone in vivo.Haploid cells of opposite mating type of Saccharomyces cerevisiae can fuse pairwise to form diploid cells. This process is triggered by the action of diffusible oligopeptide pheromones (reviewed in ref. 1). a Factor, the pheromone secreted by a haploids, elicits in a haploids the following developmentally specific events, in order of their appearance: an increase in adhesiveness (2); arrest of growth at the G1 stage of the cell cycle with concomitant cessation of nuclear DNA replication (3); and anisotropic cell wall synthesis (4) resulting eventually in the formation of morphologically abnormal cells if mating is precluded by the absence of a cells (5). Exposure of a cells to a factor thus synchronizes the culture as unbudded mononucleate cells primed for mating with their a counterparts.Although much is known about the a factor molecule, including confirmation of its primary structure (6) by solid-phase peptide synthesis (7), little work has been done to elucidate the biochemical mechanism of its action. By analogy with mammalian peptide hormones and neurotransmitters that act via cyclic AMP as an intracellular "second messenger" (8, 9), we decided to investigate the effect of cyclic AMP on the response of a cells to a factor in vio and the effect of a factor on yeast adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in vitro. We show that the a pheromone inhibits cyclic AMP production by the enzyme and that this may constitute at least part of its mode of action at the molecular level.MATERIALS AND METHODS Chemicals. All biochemicals were of the highest grade commercially available. Radiochemicals were purchased from New England Nuclear. All other chemicals were reagent grade. Purification of a factor and chemical synthesis of the entire molecule and partial segments have been described (7,10). Commercially prepared synthetic a factor (Peninsula Labs, San Carlos, CA) was also used, after extensive purification (7).Organisms and Growth Conditions. The standard laboratory yeast strains, X2180-1A (a), X2180-1B (a), and P2180A (a/at), and conditions for their cultivation have been described (11). The collection ...

A feruloyl esterase (FAE) gene was isolated from a rumen microbial metagenome, cloned into E. coli, and expressed in active form. The enzyme (RuFae2) was identified as a type C feruloyl esterase. The RuFae2 alone released ferulic acid from rice bran, wheat bran, wheat-insoluble arabinoxylan, corn fiber, switchgrass, and corn bran in the order of decreasing activity. Using a saturating amount of RuFae2 for 100 mg substrate, a maximum of 18.7 and 80.0 μg FA was released from 100 mg corn fiber and wheat-insoluble arabinoxylan, respectively. Addition of GH10 endoxylanase (EX) synergistically increased the release of FA with the highest level of 6.7-fold for wheat bran. The synergistic effect of adding GH11 EX was significantly smaller with all the substrates tested. The difference in the effect of the two EXs was further analyzed by comparing the rate in the release of FA with increasing EX concentration using wheat-insoluble arabinoxylan as the substrate.