Gabriele Diekert

Biosynthetic evidence for a nickel tetrapyrrole structure of factor F430 from Methanobacterium thermoautotrophicum

Methanogenic bacteria have been shown to require nickel for growth [I]. From these organisms a yellow nickel containing compound can be isolated [2-61, that has been designated factor FJso [7]. Its mass/m01 nickel was determined to be 1500 and egso to be near 23 000 cm-‘. 1 (mol Ni)-’ [5,6]. The structure and function of this compound is unknown. Incorporation studies with [ 14C]succinate as labelled precursor indicate that factor F,,,, may be a tetrapyrrole compound. 8 mol succinate/mol nickel were found to be incorporated into the factor, which is the amount predicted for a tetrapyrrole [4]. Tetrapyrrole biosynthesis proceeds via 6-aminolevulinic acid @-ALA) and porphobilinogen as intermediates. Here we show with Methanobacterium thermoautotrophicum that factor F,,, becomes labelled when the organism is grown in the presence of F-[4-14C]ALA. Dependent on the S-ALA concentration in the growth medium up to 8 mol s-ALA/m01 nickel were incorporated. Only tetrapyrroles are known to be synthesized from 8 mol &-ALA; therefore the incorporation data are taken as evidence that factor F J3,, has a nickel tetrapyrrole structure.

Biological role of nickel

Abstract Several enzymes and one cofactor have recently been shown to contain nickel. For example, urease of jack beans has been found to be a nickel protein and factor F 430 from methanogenic bacteria to be a nickel tetrapyrrole.

O-demethylase from Acetobacterium dehalogenans--substrate specificity and function of the participating proteins.

The ether-cleaving O-demethylase isolated from syringate-grown cells of Acetobacterium dehalogenans (formerly named strain MC) consists of four proteins, components A, B, C and D. The enzyme system converts only phenyl methyl ethers with a hydroxyl group in the ortho position to the methoxyl moiety. The presence of a carboxyl group in the aromatic compound was not required for O-demethylase reaction. Component B mediated the conversion of vanillate to 3,4-dihydroxybenzoate in the presence of the Ti(III)-reduced corrinoid-containing component A. After addition of component D and tetrahydrofolate, methyl tetrahydrofolate was formed from vanillate in stoichiometric amounts. Titanium(III) citrate as a reductant could be replaced by H2, methyl viologen or ferredoxin, partially purified hydrogenase, purified component C obtained from A. dehalogenans, and ATP. From these findings, it was deduced that component B serves as vanillate:corrinoid protein methyltransferase (methyltransferase I) mediating the methyl transfer from vanillate to the reduced corrinoid protein component A. Component D functions as methylcorrinoid protein:tetrahydrofolate transferase (methyltransferase II). The role of component C is probably that of an activating protein reversing accidental oxidation of the protein-bound cob(I)alamin to cob(II)alamin in the presence of ATP and reducing equivalents supplied by the enzymatic oxidation of hydrogen.

Purification of the nickel protein carbon monoxide dehydrogenase of Clostridium thermoaceticum

The carbon monoxide dehydrogenase was purified from Clostridium thermoaceticum to apparent homogeneity. The 120‐fold purified enzyme with app. M r 250000 had a nickel content of 10 ± 2 μmol Ni/protein.

Incorporation of methionine-derived methyl groups into factor F430 by Methanobacterium thermoautotrophicum

Factor F4a0 is a low M, yellow compound with an absorption maximum at 430 nm present in all methanogenic bacteria [ 1,2]. It is probably the prosthetic group of methyl coenzyme M reductase [3], which catalyzes the reduction of methyl CoM to methane. In [4-61 factor F,,, was found to contain nickel, which explained why methanogenic bacteria are dependent on this transition element for growth [7]. The mass/m01 nickel was determined to be 1500 and ~~a,, to be near 23 000 cm-‘. 1. (mol Ni))’ [4-61. Labelling studies with [r4C]succinate [8] and 6-[14C]aminolevulinic acid (S-ALA) [9] indicate that factor F,,, has a nickel tetrapyrrole structure; 8 mol6 -ALA/ mol Ni are incorporated into the factor. The arrangement of the tetrapyrrole is probably macrocyclic (as in the porphyrins) rather than linear (as in the bile pigments) because nickel does not dissociate from the factor, neither in strong acids (6 N HCl) nor under alkaline conditions (pH 13) [8]. The detailed structure of factor F4a0 is not known. Chlorophylls, hemes, sirohemes and vitamin Bra are the macrocyclic tetrapyrroles of biological importance known to date. The 4 tetrapyrroles are either derived from protoporphyrin IX (chlorophylls, hemes and cytochromes) or from sirohydrochlorin (siroheme and vitamin Brz) (fig.1). Both protoporphyrin IX and sirohydrochlorin are synthesized from uroporphyrinogen III; the former by 6 consecutive decarboxylations, including oxidative decarboxylations; the latter by 2 reductive methylations with S-adenosyl methionine. Uroporphyrinogen III is formed from 8 &-ALA.

Retentive Memory of Bacteria: Long-Term Regulation of Dehalorespiration in Sulfurospirillum multivorans

The gram-negative, strictly anaerobic epsilonproteobacterium Sulfurospirillum multivorans is able to gain energy from dehalorespiration with tetrachloroethene (perchloroethylene [PCE]) as a terminal electron acceptor. The organism can also utilize fumarate as an electron acceptor. Prolonged subcultivation of S. multivorans in the absence of PCE with pyruvate as an electron donor and fumarate as an electron acceptor resulted in a decrease of PCE dehalogenase (PceA) activity. Concomitantly, the pceA transcript level equally decreased as shown by reverse transcriptase PCR. After 35 subcultivations (approximately 105 generations), a pceA transcript was not detectable and the PceA protein and activity were completely absent. In such long-term subcultivated S. multivorans cells, the biosynthesis of catalytically active PceA was restored to the initial level within about 50 h (approximately three generations) by the addition of PCE or trichloroethene. Single colonies obtained from PceA-depleted cultures were able to induce PCE dechlorination, indicating that long-term subcultured cells still contained the functional pceA gene. The results point to a novel type of long-term regulation of PCE dehalogenase gene expression in S. multivorans.

Energetics of Acetogenesis from C1 Units

Homoacetogenic bacteria (also called “acetogenic bacteria”) are strictly anaerobic eubacteria, which catalyze the synthesis of acetate from C1 units in their catabolism. The homoacetogens have proved to be a very heterogenous group of microorganisms: they are gram positive as well as gram negative, spore- or non-spore forming, thermophilic and mesophilic, motile and nonmotile, and so on. Usually they are able to grow on a variety of substrates, some of which are quite unusual for anaerobic microorganisms. Because most of the homoacetogens are able to grow at the expense of H2 plus CO2 as the sole energy source, the reduction of 2 CO2 to acetate must be coupled to the synthesis of ATP. This chapter will deal mainly with the utilization of C1 substrates and the energy conservation coupled to the synthesis of acetate from these substrates. For general overviews on homoacetogenic bacteria, the reader is referred to Chapters 1 and 7, as well as other recent reviews (Diekert, 1992; Schink and Bomar, 1992; Drake, 1992).

Drei neue Nickelenzyme aus anaeroben Bakterien

Until recently nickel was not considered to be an element of biological importance. Nutritional studies have shown, however, that many eucaryotic and procaryotic organisms are dependent on the transition metal for growth. Four enzymes are presently known to contain nickel: urease from plants and from bacteria; methyl CoM reductase from methanogenic bacteria; all “uptake” hydrogenases investigated so far; and carbon monoxide dehydrogenase from anaerobic bacteria. The prosthetic group of the methyl CoM reductase has been identified as a nickel tetrapyrrole, the structure of which has been elucidated.

The Ether-Cleaving Methyltransferase System of the Strict Anaerobe Acetobacterium dehalogenans: Analysis and Expression of the Encoding Genes

Anaerobic O-demethylases are inducible multicomponent enzymes which mediate the cleavage of the ether bond of phenyl methyl ethers and the transfer of the methyl group to tetrahydrofolate. The genes of all components (methyltransferases I and II, CP, and activating enzyme [AE]) of the vanillate- and veratrol-O-demethylases of Acetobacterium dehalogenans were sequenced and analyzed. In A. dehalogenans, the genes for methyltransferase I, CP, and methyltransferase II of both O-demethylases are clustered. The single-copy gene for AE is not included in the O-demethylase gene clusters. It was found that AE grouped with COG3894 proteins, the function of which was unknown so far. Genes encoding COG3894 proteins with 20 to 41% amino acid sequence identity with AE are present in numerous genomes of anaerobic microorganisms. Inspection of the domain structure and genetic context of these orthologs predicts that these are also reductive activases for corrinoid enzymes (RACEs), such as carbon monoxide dehydrogenase/acetyl coenzyme A synthases or anaerobic methyltransferases. The genes encoding the O-demethylase components were heterologously expressed with a C-terminal Strep-tag in Escherichia coli, and the recombinant proteins methyltransferase I, CP, and AE were characterized. Gel shift experiments showed that the AE comigrated with the CP. The formation of other protein complexes with the O-demethylase components was not observed under the conditions used. The results point to a strong interaction of the AE with the CP. This is the first report on the functional heterologous expression of acetogenic phenyl methyl ether-cleaving O-demethylases.