Shuichiro Goda

A second novel dye-linked L-proline dehydrogenase complex is present in the hyperthermophilic archaeon Pyrococcus horikoshii OT-3

Two distinguishable activity bands for dye‐linked l‐proline dehydrogenase (PDH1 and PDH2) were detected when crude extract of the hyperthermophilic archaeon Pyrococcus horikoshii OT‐3 was run on a polyacrylamide gel. After purification, PDH1 was found to be composed of two different subunits with molecular masses of 56 and 43u2003kDa, whereas PDH2 was composed of four different subunits with molecular masses of 52, 46, 20 and 8u2003kDa. The native molecular masses of PDH1 and PDH2 were 440 and 101u2003kDa, respectively, indicating that PDH1 has an α4β4 structure, while PDH2 has an αβγδ structure. PDH2 was found to be similar to the dye‐linked l‐proline dehydrogenase complex from Thermococcus profundus, but PDH1 is a different type of enzyme. After production of the enzyme in Escherichia coli, high‐performance liquid chromatography showed the PDH1 complex to contain the flavins FMN and FAD as well as ATP. Gene expression and biochemical analyses of each subunit revealed that the β subunit bound FAD and exhibited proline dehydrogenase activity, while the α subunit bound ATP, but unlike the corresponding subunit in the T.u2003profundus enzyme, it exhibited neither proline dehydrogenase nor NADH dehydrogenase activity. FMN was not bound to either subunit, suggesting it is situated at the interface between the α and β subunits. A comparison of the amino‐acid sequences showed that the ADP‐binding motif in the α subunit of PDH1 clearly differs from that in the α subunit of PDH2. It thus appears that a second novel dye‐linked l‐proline dehydrogenase complex is produced in P.u2003horikoshii.

The Sulfolobus tokodaii gene ST1704 codes highly thermostable glucose dehydrogenase

Abstract NAD(P)-dependent glucose-1-dehydrogenase (GDH) has been used for glucose determination and NAD(P)H production in bioreactors. Thermostable glucose dehydrogenase exhibits potential advantage for its application in biological processes. The function of the putative GDH gene (ST1704, 360-encoding amino acids) annotated from the total genome analysis of a thermoacidophilic archeaon Sulfolobus tokodaii strain 7 was investigated to develop more effective application of GDH. The gene encoding S. tokodaii GDH was cloned and the activity was expressed in Escherichia coli , which did not originally possess GDH. This shows that the gene (ST1704) codes the sequence of GDH. The enzyme was effectively purified from the recombinant E. coli with three steps containing a heat treatment and two successive chromatographies. The native enzyme (molecular mass: 160xa0kDa) is composed of a tetrameric structure with a type of subunit (41xa0kDa). The enzyme utilized both NAD and NADP as the coenzyme. The maximum activity for glucose oxidation in the presence of NAD was observed around pH 9 and 75xa0°C in the presence of 20xa0mM Mg 2+ . The enzyme showed broad substrate specificity: several monosaccarides such as 6-deoxy- d -glucose, 2-amino-2-deoxy- d -glucose and d -xylose were oxidized as well as d -glucose as the electron donor. d -Mannose, d -ribose and glucose-6-phosphate were inert as the donor. The enzyme showed high thermostability: remarkable loss of activity was not observed up to 80xa0°C by incubation for 15xa0min at pH 8.0. In addition, the enzyme was stable in a wide pH range of 5.0–10.5 by incubation at 37xa0°C. From the steady-state kinetic analysis, the enzyme reaction of d -glucose oxidation proceeds via a sequential ordered Bi–Bi mechanism: NAD and d -glucose bind to the enzyme in this order and then d -glucono-1,5-lactone and NADH are released from the enzyme in this order. The amino acid sequence alignment showed that S. tokodaii GDH exhibited high homology with the Sulfolobus solfataricus hypothetical glucose dehydrogenase and a Thermoplasma acidophilum one.

L-Threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3: gene cloning and enzymatic characterization

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°C for 20xa0min. The enzyme (molecular mass: 192xa0kDa) is composed of a tetrameric structure with a type of subunit (41xa0kDa). 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 Zn2+, Mn2+ and Co2+ for the activity, and contained one zinc ion/subunit. The Km values for L-threonine and NAD at 50°C were 0.20xa0mM and 0.024xa0mM, 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.

A nicotinamide mononucleotide adenylyltransferase with unique adenylyl group donor specificity from a hyperthermophilic archaeon, Pyrococcus horikoshii OT-3

Abstract A gene encoding a nicotinamide mononucleotide (NMN) adenylyltransferase (NMNAT, EC 2.7.7.1) homologue was identified via genome sequencing in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3. The gene encoded a protein of 186 amino acids with a molecular weight of 21,391. The deduced amino acid sequence of the gene showed 59% identities to the NMNAT from Methanococcus jannaschii . The gene was overexpressed in Escherichia coli , and the produced enzyme was purified to homogeneity. Characterization of the enzyme revealed that it is an extremely thermostable NMNAT; the activity was not lost after incubation at 80xa0°C for 30xa0min. The native molecular mass was estimated to be 77xa0kDa. The K m values for ATP and NMN were calculated to be 0.056 and 0.061xa0mM, respectively. The optimum temperature of the reaction was estimated to be around 90xa0°C. The adenylyl group donor specificity was examined by high-performance liquid chromatography (HPLC). At 70xa0°C, ATP was a prominent donor. However, above 80xa0°C, a relatively small, but significant, NMNAT activity was detected when ATP was replaced by ADP or AMP in the reaction mixture. To date, an NMNAT that utilizes ADP or AMP as an adenylyl group donor has not been found. The present study provides interesting information in which a di- or mono-phosphate nucleotide can be utilized by adenylyltransferase at high temperature.