Alternative NAD⁺-dependent formate dehydrogenases in the facultative methylotrophMycobacterium vaccae10

Abstract

Mycobacterium vaccae 10 growing in methanol medium synthesizes two inducible alternative NAD+‐dependent formate dehydrogenases (FDH). In the presence of molybdenum, the dominating form of the enzyme is FDHI with Mr 440 kDa and Km 0.32 mM for sodium formate. FDHI reduced ferricyanide as well as NAD+, and it was reversibly inactivated by formate. NAD+ stabilized FDHI against this inactivation. Under conditions of artificial molybdenum deficiency (tungsten in the medium), the second enzyme (FDHII) appeared with Mr about 93 kDa and Km 8.3 mM for sodium formate, and no FDHI activity was detected. FDHII did not reduce ferricyanide and was not inactivated by formate. The activity of FDHI was restored in tungsten‐grown cells by pulse addition of molybdenum under conditions of blocked protein synthesis, suggesting the pre‐existence of inactive apo‐FDHI.

Transition metal requirement to express high level NAD+-dependent formate dehydrogenase from a serine-type methylotrophic bacterium

“…The apparent Michaelis constant for formate in crude extracts of Methylobacterium sp. RXM was similar to K m values from other methylotrophic organisms [5,8,24]. More significantly, Vma x values in WO42--and MoO42--grown cells were shown to be several times higher when compared with the Vma x for cells grown in nonmetal medium.…”

“…However, these FDHs differed from the FDH described for Methylobacterium sp. RXM at least in two characteristics: in Mycobacterium raccae 10, the cell yields were not shown to be distinct (the final biomass was similar for media containing or not containing metal), and the NAD+/K ferricyanide ratio linked to FDH activity was found to be dependent either on the presence or type of metal anion added [24]. Conversely, in Methylobacterium sp.…”

“…Recently, Karzanov et al [24] reported the presence of two alternative FDHs in the facultative methylotroph Mycobacterium raccae 10. However, these FDHs differed from the FDH described for Methylobacterium sp.…”

NAD+-dependent formate dehydrogenase

“…FDHs as a rule are inducible enzymes synthesized in large quantities (up to 10-15 % of the net protein content) when organisms are grown on C1 compounds [23,45]. Some organisms, e.g.…”

“…Some organisms, e.g. the methylotrophic bacteria Mycobacterium vaccae 10 [45] and Pseudomonas sp. 101 [46], contain more than one FDH, which differ in cellular location, molecular mass and specificity towards the electron acceptor.…”