Asian Mouse Mutagenesis Resource Association (AMMRA): mouse genetics and laboratory animal resources in the Asia Pacific.

The Asian Mouse Mutagenesis Resource Association (AMMRA) is a non-profit organization consisting of major resource and research institutions with rodent expertise from within the Asia Pacific region. For more than a decade, aiming to support biomedical research and stimulate international collaboration, AMMRA has always been a friendly and passionate ally of Asian and Australian member institutions devoted to sharing knowledge, exchanging resources, and promoting biomedical research. AMMRA is also missioned to global connection by working closely with the consortiums such as the International Mouse Phenotyping Consortium and the International Mouse Strain Resource. This review discusses the emergence of AMMRA and outlines its many roles and responsibilities in promoting, assisting, enriching research, and ultimately enhancing global life science research quality.

Hyperthermophilic Carbamate Kinase Stability and Anabolic In Vitro Activity at Alkaline pH.

Carbamate kinases catalyze the conversion of carbamate to carbamoyl phosphate, which is readily transformed into other compounds. Carbamate forms spontaneously from ammonia and carbon dioxide in aqueous solutions, so the kinases have potential for sequestrative utilization of the latter compounds. Here, we compare seven carbamate kinases from mesophilic, thermophilic, and hyperthermophilic sources. In addition to the known enzymes from Enterococcus faecalis and Pyrococcus furiosus, the previously unreported enzymes from the hyperthermophiles Thermococcus sibiricus and Thermococcus barophilus, the thermophiles Fervidobacterium nodosum and Thermosipho melanesiensis, and the mesophile Clostridium tetani were all expressed recombinantly, each in high yield. Only the clostridial enzyme did not show catalysis. In direct assays of carbamate kinase activity, the three hyperthermophilic enzymes display higher specific activities at elevated temperatures, greater stability, and remarkable substrate turnover at alkaline pH (9.9 to 11.4). Thermococcus barophilus and Thermococcus sibiricus carbamate kinases were found to be the most active when the enzymes were tested at 80°C, and maintained activity over broad temperature and pH ranges. These robust thermococcal enzymes therefore represent ideal candidates for biotechnological applications involving aqueous ammonia solutions, since nonbuffered 0.0001 to 1.0 M solutions have pH values of approximately 9.8 to 11.8. As proof of concept, here we also show that carbamoyl phosphate produced by the Thermococcus barophilus kinase is efficiently converted in situ to carbamoyl aspartate by aspartate transcarbamoylase from the same source organism. Using acetyl phosphate to simultaneously recycle the kinase cofactor ATP, at pH 9.9 carbamoyl aspartate is produced in high yield and directly from solutions of ammonia, carbon dioxide, and aspartate.IMPORTANCE Much of the nitrogen in animal wastes and used in fertilizers is commonly lost as ammonia in water runoff, from which it must be removed to prevent downstream pollution and evolution of nitrogenous greenhouse gases. Since carbamate kinases transform ammonia and carbon dioxide to carbamoyl phosphate via carbamate, and carbamoyl phosphate may be converted into other valuable compounds, the kinases provide a route for useful sequestration of ammonia, as well as of carbon dioxide, another greenhouse gas. At the same time, recycling the ammonia in chemical synthesis reduces the need for its energy-intensive production. However, robust catalysts are required for such biotransformations. Here we show that carbamate kinases from hyperthermophilic archaea display remarkable stability and high catalytic activity across broad ranges of pH and temperature, making them promising candidates for biotechnological applications. We also show that carbamoyl phosphate produced by the kinases may be efficiently used to produce carbamoyl aspartate.

Detection of Biosynthetic Precursors, Discovery of Glycosylated Forms, and Homeostasis of Calcitonin in Human Cancer Cells.

The peptide hormone calcitonin is intimately connected with human cancer development and proliferation. Its biosynthesis is reasoned to proceed via glycine-, α-hydroxyglycine-, glycyllysine-, and glycyllysyllysine-extended precursors; however, as a result of the limitations of current analytical methods, until now, there has been no procedure capable of detecting these individual species in cell or tissue samples. Therefore, their presence and dynamics in cancer had not been established. Here, we report the first methodology for the separation, detection, and quantification of calcitonin and each of its precursors in human cancer cells. We also report the discovery and characterization of O-glycosylated calcitonin and its analogous biosynthetic precursors. Through direct and simultaneous analysis of the glycosylated and nonglycosylated species, we interrogate the hormone biosynthesis. This shows that the cellular calcitonin level is maintained to mitigate effects of biosynthetic enzyme inhibitors that substantially change the proportions of calcitonin-related species released into the culture medium.

Clostridium carboxidivorans strain P7T recombinant formate dehydrogenase catalyzes reduction of CO(2) to formate.

Recombinant formate dehydrogenase from the acetogen Clostridium carboxidivorans strain P7(T), expressed in Escherichia coli, shows particular activity towards NADH-dependent carbon dioxide reduction to formate due to the relative binding affinities of the substrates and products. The enzyme retains activity over 2 days at 4°C under oxic conditions.

In situ deprotection and incorporation of unnatural amino acids during cell-free protein synthesis.

The S30 extract from E. coli BL21 Star (DE3) used for cell-free protein synthesis removes a wide range of α-amino acid protecting groups by cleaving α-carboxyl hydrazides; methyl, benzyl, tert-butyl, and adamantyl esters; tert-butyl and adamantyl carboxamides; α-amino form-, acet-, trifluoroacet-, and benzamides; and side-chain hydrazides and esters. The free amino acids are produced and incorporated into a protein under standard conditions. This approach allows the deprotection of amino acids to be carried out in situ to avoid separate processing steps. The advantages of this approach are demonstrated by the efficient incorporation of the chemically intractable (S)-4-fluoroleucine, (S)-4,5-dehydroleucine, and (2S,3R)-4-chlorovaline into a protein through the direct use of their respective precursors, namely, (S)-4-fluoroleucine hydrazide, (S)-4,5-dehydroleucine hydrazide, and (2S,3R)-4-chlorovaline methyl ester. These results also show that the fluoro- and dehydroleucine and the chlorovaline are incorporated into a protein by the normal biosynthetic machinery as substitutes for leucine and isoleucine, respectively.

Substrate-induced conformational change and isomerase activity of dienelactone hydrolase and its site-specific mutants.

Studies of the interactions of dienelactone hydrolase (DLH) and its mutants with both E and Z dienelactone substrates show that the enzyme exhibits two different conformational responses specific for hydrolysis of each of its substrate isomers. DLH facilitates hydrolysis of the Z dienelactone through an unusual charge-relay system that is initiated by interaction between the substrate carboxylate and an enzyme arginine residue that activates an otherwise non-nucleophilic cysteine. The E dienelactone does not display this substrate-arginine binding interaction, but instead induces an alternate conformational response that promotes hydrolysis. Furthermore, the substitution of cysteine 123 for serine (C123S) in DLH, instead of inactivating the enzyme as is typical for this active-site mutation, changes the catalysis from substrate hydrolysis to isomerisation. This is due to the deacylation of the acyl-enzyme intermediates being much slower, thereby increasing their lifetimes and allowing for their interconversion through isomerisation, followed by relactonisation.

ATP Recycling with Cell Lysate for Enzyme-Catalyzed Chemical Synthesis, Protein Expression and PCR.

E. coli lysate efficiently catalyzes acetyl phosphate-driven ATP regeneration in several important biotechnological applications. The utility of this ATP recycling strategy in enzyme-catalyzed chemical synthesis is illustrated through the conversion of uridine to UMP by the lysate from recombinant overexpression of uridine kinase with the E. coli. The UMP is further transformed into UTP through sequential phosphorylations by kinases naturally present in the lysate, in high yield. Cytidine and 5-fluorouridine also give the corresponding NMPs and NTPs with this system. Cell-free protein expression with a processed extract of lysate also proceeds readily when, instead of adding the required NTPs, all four are produced in situ from the NMPs, using acetyl phosphate and relying on endogenous kinase activity. Similarly, dNMPs can be used to produce the dNTPs necessary for DNA synthesis in PCR. These cheap alternative protocols showcase the potential of acetyl phosphate and ATP recycling with readily available cell lysate.

Incorporation of chlorinated analogues of aliphatic amino acids during cell-free protein synthesis.

3-Chloro-Abu and 4-chloro-Nva are biosynthetically incorporated into E. coli peptidyl-Pro cis-trans isomerase B, as substitutes for Val and Leu, respectively. The extent of incorporation is up to ~90%, and substituted protein is catalytically active. By contrast, 4-chloro-Val is not an effective replacement for Ile.