Commercial enzymatic processes require robust catalysts able to withstand elevated temperatures and long incubations, conditions under which most native enzymes perform poorly. Incremental increases in thermostability can be achieved by repeated rounds of mutagenesis and screening, but general strategies are needed for designing highly thermostable enzymes a priori. Here we show that enzymes can be created that can withstand temperatures ~ 30 °C higher and incubations ≥ 100 times longer than extant forms in a single step using ancestral reconstruction. We exemplify the approach with the first ancestral resurrections of two unrelated enzyme families: cytochrome P450 monooxygenases, that stereo-and regioselectively functionalize un-activated C-H bonds in pharmaceutical, flavour, fragrance and other fine chemical syntheses; and ketol acid reductoisomerases, used to make butanol-based biofuels. This shows thermostability can be designed into proteins using sequence data alone, potentially enhancing the economic feasibility of any process or product requiring a highly stable protein.

Prior evidence supporting the direct observation of phosphorane intermediates in enzymatic phosphoryl transfer reactions was based on the interpretation of electron density corresponding to trigonal species bridging the donor and acceptor atoms. Close examination of the crystalline state of β-phosphoglucomutase, the archetypal phosphorane intermediate-containing enzyme, reveals that the trigonal species is not PO − 3 , but is MgF − 3 (trifluoromagnesate). Although MgF − 3 complexes are transition state analogues rather than phosphoryl group transfer reaction intermediates, the presence of fluorine nuclei in near-transition state conformations offers new opportunities to explore the nature of the interactions, in particular the independent measures of local electrostatic and hydrogen-bonding distributions using 19 F NMR. Measurements on three β-PGM-MgF − 3 -sugar phosphate complexes show a remarkable relationship between NMR chemical shifts, primary isotope shifts, NOEs, cross hydrogen bond F⋯H-N scalar couplings, and the atomic positions determined from the highresolution crystal structure of the β-PGM-MgF − 3 -G6P complex. The measurements provide independent validation of the structural and isoelectronic MgF − 3 model of near-transition state conformations.19F NMR | phosphoryl transfer enzyme | transition state analogue | trifluoromagnesate T he mono-and diesters of phosphoric acid have commanding and ubiquitous roles in all species of life. As structural components they show remarkable stability to spontaneous hydrolysis under near physiological conditions (25°C), with half-lives for P-O bond cleavage in phosphate diesters estimated at ca. 10 7 years and for monoesters ca. 10 12 years (1, 2). Yet, they are susceptible to enzyme-catalyzed hydrolysis and phosphoryl group transfer reactions either between two oxygens, or between oxygen and nitrogen or sulfur, with turnover numbers adequate to support a vast array of biological processes, e.g. Serratia nuclease k cat ca. 2; 500 s −1 (3), E. coli alkaline phosphatase k cat ≥ 45 s −1 (4), and human protein tyrosine phosphatase β k cat ca.

Maturation-promoting factor (MPF) is known to be a key regulator of both mitotic and meiotic cell cycles. MPF is a complex of a B cyclin and the cyclin-dependent kinase cdkl (p34cdc2). Oocyte maturation and its arrest at metaphase of meiosis II (MII) are regulated by changes in MPF activity. In this study, experiments were conducted to examine the dynamics of MPF activity and its constituent proteins during in vitro maturation of bovine oocytes. Bovine oocytes displayed relatively low levels of MPF (histone H1 kinase) activity at the germinal vesicle stage during the first 8 h of maturation. MPF activity increased gradually thereafter, and its first peak of activity occurred at 12-14 h of maturation (presumptive metaphase I), which was followed by an abrupt reduction in activity at 16-18 h, during presumptive anaphase and telophase. MPF activity then increased, reaching a plateau at 20-24 h of maturation (MII stage). This high level of MPF activity was maintained for several hours but decreased gradually after 30 h of maturation and became barely detectable by 48 h of in vitro maturation (IVM) culture. At each time point, there was a significant variation among individual oocytes in histone H1 kinase activity, which was probably due to asynchronous maturation. Abundance of cdk1 increased gradually during the first 8 h and then remained relatively constant except for an apparent reduction at 18-22 h of IVM. The level of cyclin B2 increased quickly during the initial 2 h of culture, and this high level was maintained until 16 h, after which a significant reduction was observed between 18 and 22 h of IVM. The de novo synthesis of cyclin B2, however, exhibited a biphasic oscillation during maturation, with peaks before the onset of MI and of MII. These results have defined the profiles of MPF activity and its individual components during bovine oocyte maturation in vitro. We conclude that active MPF regulates bovine oocyte maturation and that de novo synthesis of cyclin B2 occurs during the process of maturation.

The missing link: Reiterative ligations based on non‐cysteine and cysteine‐based acyl acceptors have been used to prepare synthetic polypeptides and proteins with multiple sites of glycosylation. Highly complex positionally defined glycopolypeptides corresponding to single glycoforms can be formed from simple components.

A pathway has been devised, wherein a phenolic ester of a C-terminal peptide is ligated with an N-terminal peptide through two consecutive acyl migrations. In the first transacylation, the C-terminus is transferred from a phenol to a newly liberated ortho-thiol function. Subsequently, the acyl group is transported to a proximal benzylamine through a six-membered transition state.