1. L-Threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3: gene cloning and enzymatic characterization
Yasuhiro Shimizu, Haruhiko Sakuraba, Ryushi Kawakami, Shuichiro Goda, Yutaka Kawarabayasi, Toshihisa Ohshima Extremophiles. 2005 Aug;9(4):317-24. doi: 10.1007/s00792-005-0447-2. Epub 2005 May 18.
A gene encoding the L-threonine dehydrogenase homologue has been identified in a hyperthermophlic archaeon Pyrococcus horikoshii OT3 via genome sequencing. The gene was cloned and expressed in Escherichia coli. The purified enzyme from the recombinant E. coli was extremely thermostable; the activity was not lost after incubation at 100 degrees C for 20 min. The enzyme (molecular mass: 192 kDa) is composed of a tetrameric structure with a type of subunit (41 kDa). The enzyme is specific for NAD and utilizes L-threonine, L-serine and DL-threo-3-phenylserine as the substrate. The enzyme required divalent cations such as Zn(2+), Mn(2+) and Co(2+) for the activity, and contained one zinc ion/subunit. The K(m) values for L-threonine and NAD at 50 degrees C were 0.20 mM and 0.024 mM, respectively. Kinetic analyses indicated that the L-threonine oxidation reaction proceeds via a random mechanism with regard to the binding of L-threonine and NAD. The enzyme showed pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of NADH. This is the first description of the characteristics of an L-threonine dehydrogenase from the archaea domain.
2. Overexpression of serine hydroxymethyltransferase from halotolerant cyanobacterium in Escherichia coli results in increased accumulation of choline precursors and enhanced salinity tolerance
Rungaroon Waditee-Sirisattha, Daungjai Sittipol, Yoshito Tanaka, Teruhiro Takabe FEMS Microbiol Lett. 2012 Aug;333(1):46-53. doi: 10.1111/j.1574-6968.2012.02597.x. Epub 2012 Jun 11.
Serine hydroxymethyltransferase (SHMT) is a key enzyme in cellular one-carbon pathway and has been studied in many living organisms from bacteria to higher plants and mammals. However, biochemical and molecular characterization of SHMT from photoautotrophic microorganisms remains a challenge. Here, we isolated the SHMT gene from a halotolerant cyanobacterium Aphanothece halophytica (ApSHMT) and expressed it in Escherichia coli. Purified recombinant ApSHMT protein exhibited catalytic reactions for dl-threo-3-phenylserine as well as for l-serine. Catalytic reaction for l-serine was strongly inhibited by NaCl, but not to that level with glycine betaine. Overexpression of ApSHMT in E. coli resulted in the increased accumulation of glycine and serine. Choline and glycine betaine levels were also significantly increased. Under high salinity, the growth rate of ApSHMT-expressing cells was faster compared to its respective control. High salinity also strongly induced the transcript level of ApSHMT in A. halophytica. Our results indicate the importance of a novel pathway; salt-induced ApSHMT increased the level of glycine betaine via serine and choline and conferred the tolerance to salinity stress.
