Nicotinamide Adenine Dinucleotide Phosphate-Dependent Formate Dehydrogenase from Clostridium thermoaceticum: Purification and Properties

Abstract

The nicotinamide adenine dinucleotide phosphate (NADP)-dependent formate dehydrogenase in Clostridium thermoaceticum used, in addition to its natural electron acceptor, methyl and benzyl viologen. The enzyme was purified to a specific activity of 34 (micromoles per minute per milligram of protein) with NADP as electron acceptor. Disc gel electrophoresis of the purified enzyme yielded two major and two minor protein bands, and during centrifugation in sucrose gradients two components of apparent molecular weights of 270,000 and 320,000 were obtained, both having formate dehydrogenase activity. The enzyme preparation catalyzed the reduction of riboflavine 5'-phosphate flavine adenine dinucleotide and methyl viologen by using reduced NADP as a source of electrons. It also had reduced NADP oxidase activity. The enzyme was strongly inhibited by cyanide and ethylenediaminetetraacetic acid. It was also inhibited by hypophosphite, an inhibition that was reversed by formate. Sulfite inhibited the activity with NADP but not with methyl viologen as acceptor. The apparent Km at 55 C and pH 7.5 for formate was 2.27 x 10-4 M with NADP and 0.83 x 10-I with methyl viologen as acceptor. The apparent Km for NADP was 1.09 x 10-' M and for methyl viologen was 2.35 x 10-3M. NADP showed substrate inhibition at 5 x 10-3 M and higher concentrations. With NADP as electron acceptor, the enzyme had a broad pH optimum between 7 and 9.5. The apparent temperature optimum was 85 C. In the absence of substrates, the enzyme was stable at 70 C but was rapidly inactivated at temperatures above 73 C. The enzyme was very sensitive to oxygen but was stabilized by thiol-iron complexes and formate. Oi the homoacetate fermentation of glucose or fructose by Clostridium thermoaceticum and Clostridium formicoaceticum, CO2 is used as an

Tungsten, the Surprisingly Positively Acting Heavy Metal Element for Prokaryotes

“…46 Its NADP-dependent activity could just be increased by purification to 0.018 U/mg of protein [one unit (U) is defined as 1 μmol/min], 47 but the starting activity was already increased to 0.086 U/mg of protein using a high concentration of molybdate (0.5 mM) and selenite (1 μM) in the growth medium. 48 Purification starts from 0.455 U/mg of protein if tungstate (36 μM) is present during growth. 49 Using the β-emitter 185 W tungstate as a label and anaerobic conditions throughout the purification procedure, the FDH activity and radioactivity finally coelute in one single band at a fixed ratio.…”

Oxalate metabolism by the acetogenic bacteriumMoorella thermoacetica

“…Oxalate-or formate-dependent reduction of NAD þ was also not observed. In this regard, M. thermoacetica is known to contain a NADP þ -dependent formate dehydrogenase [28]. Furthermore, the activity ratio for oxalate-and formate-dependent reduction of NADP þ was nearly 10-fold less than the activity ratio observed with benzyl viologen, suggesting that formate dehydrogenase and the oxalate-catabolizing enzyme system have differential electron acceptor speci¢cities.…”

Tungsten in biological systems

“…The fact that some of them are produced in well studied microorganisms but only under unusual growth conditions [27][28][29][30], while some others are found in 'exotic' species such as hyperthermophilic archaea [31][32][33][34], has severely hindered progress in this area. Similarly, many of the known tungstoenzymes are very oxygen-sensitive [1][2][3]8,[27][28][29][32][33][34][35], which can make even their detection problematic. One of the objectives of this review is to demonstrate that the full spectrum of tungstoenzymes has yet to be fully recognized or appreciated.…”

“…In natural environments, the high stability of the oxo compounds of W(VI) compared with W(IV), and of sulfur derivatives of Mo(IV), such as MoS 2, compared with Mo(VI), such as molybdates, is due mainly to the reaction shown in Eq. (2) (where AH = -31 kcal/mol; [46])• H2MoO 4 + WS 2 ---> MoS 2 + H2WO 4 (2) Most natural environments also contain Ca, Fe and Mn, and the reaction shown in Eq. (2) is reinforced by the stability of the following associations: CaWO [46,52].…”

“…The notion that tungsten (chemical symbol: W) has a functional role in biological systems was substantiated only very recently. The first indications were reported in the early 1970s when the growth of certain acetogenic clostridia was shown to be stimulated by the addition of tungstate to their growth media [1][2][3][4][5][6][7]. It was a decade later, however, before the first naturally occurring W-containing protein was purified to homogeneity [8].…”