A Wasserfallen's research while affiliated with Universidade NOVA de Lisboa and other places

The A-type flavoproteins (ATF) are modular proteins involved in multi-component electron transfer pathways, having oxygen reductase activity. They are complex flavoproteins containing two distinct structural domains, one having an FMN in a flavodoxin-like fold and the other a binuclear iron centre within a metallo-beta-lactamase-like fold. Here, we report the purification and characterisation of a recombinant ATF from the cyanobacterium Synechoystis sp. PCC 6803, which has the unique feature of comprising an additional third domain with similarities towards flavin:NAD(P)H reductases. The latter was expressed independently as a truncated protein form and found to be capable of receiving electrons from NADH as well as to indiscriminately bind either one FAD or one FMN with equivalent affinities. Further kinetic studies have shown that the intact ATF is an NADH:oxygen oxidoreductase, with the catalytic ability to fully reduce oxygen to water. Thus, this constitutes an example on how structural modules found within partner proteins from an electron transfer pathway can be combined in a single polypeptide chain achieving identical catalytic activities.

The methanogenic archaeon Methanobacterium thermoautotrophicum Marburg is infected by the double-stranded DNA phage ΨM2. The complete phage genome sequence of 26 111 bp was established. Thirty-one open reading frames (orfs), all of them organized in the same direction of transcription, were identified. On the basis of comparison of the deduced amino acid sequences to known proteins and by searching for conserved motifs, putative functions were assigned to the products of six orfs. These included three proteins involved in packaging DNA into the capsid, two putative phage structural proteins and a protein related to the Int family of site-specific recombinases. Analysis of the N-terminal amino acid sequences of three phage-encoded proteins led to the identification of two genes encoding structural proteins and of peiP, the structural gene of pseudomurein endoisopeptidase. This enzyme is involved in the lysis of host cells, and it appears to belong to a novel enzyme family. peiP was overexpressed in Escherichia coli, and its product was shown to catalyse the in vitro lysis of M. thermoautotrophicum cells. Comparison of the phage ΨM2 DNA sequence with parts of the sequence of the wild-type phage ΨM1 suggests that ΨM2 is a deletion derivative, which formed by homologous recombination between two copies of a direct repeat.

Sequence comparison of pseudomurein endoisopeptidases PeiW encoded by the defective prophage PsiM100 of Methanothermobacter wolfeii, and PeiP encoded by phage PsiM2 of Methanothermobacter marburgensis, revealed that the two enzymes share only limited similarity. Their amino acid sequences comprise an N-terminal domain characterized by the presence of direct repeats and a C-terminal domain with a catalytic triad C-H-D as in thiol proteases and animal transglutaminases. Both PeiW and PeiP catalyze the in vitro lysis of M. marburgensis cells under reducing conditions and exhibit characteristics of metal-activated peptidases. Optimal temperature and pH were determined to be 63 degrees C and 6.4 for His-tagged PeiP and 71 degrees C and 6.4 for His-tagged PeiW, respectively. Database search results suggest that PeiW and PeiP are the first two experimentally identified members of a novel family of proteases in a superfamily of archaeal, bacterial, and eukaryotic protein homologs of animal transglutaminases.

A family of flavoproteins, called A-type flavoproteins, is described. It consists of 14 protein sequences of 385−597 amino acids in length, 7 from methanogens (domain : Archaea), 5 from phototrophic prokaryotes, one from Escherichia coli, and a partial sequence from the sulfate reducer Desulfovibrio gigas (domain : Bacteria). No similar sequence could be found in the domain Eucarya. All sequences show significant similarity over a 385−400 amino acid portion overlapping a recognizable flavodoxin signature starting at positions 245−285 of the common core sequence. Cofactor analysis and, to some extent, analysis of the primary structure of six A-type flavoproteins, three of which are structurally characterized here, support the existence of four sub-families : (a) simple flavoproteins binding only FMN; (b) diflavin flavoproteins binding FMN and FAD; (c) a flavorubredoxin binding FMN and iron; (d) a hemoflavoprotein. The possible involvement of A-type flavoproteins in the metabolism of oxygen, as suggested for D. gigas hemoflavoprotein [Gomes, C. M., Silva, G., Oliveira, S., LeGall, J., Liu, M.-Y., Xavier, A. V., Rodrigues-Pousada, C. & Teixeira, M. (1997) J. Biol. Chem.272, 22 502−22 508], is discussed.

Methanothermobacter wolfeii (formerlyMethanobacterium wolfei), a thermophilic methanoarchaeon whose cultures lyse upon hydrogen starvation, carries a defective prophage called ΨM100 on its chromosome. The nucleotide sequence of ΨM100 and its flanking regions was established and compared to that of the previously sequenced phage ΨM2 of Methanothermobacter marburgensis (formerly Methanobacterium thermoautotrophicum Marburg). The ΨM100 genome extends over 28,798 bp, and its borders are defined by flanking 21-bp direct repeats of a pure-AT sequence, which very likely forms the core of the putative attachment site where the crossing over occurred during integration. A large fragment of 2,793 bp, IFa, apparently inserted into ΨM100 but is absent in the genome of ΨM2. The remaining part of the ΨM100 genome showed 70.8% nucleotide sequence identity to the whole genome of ΨM2. Thirty-four open reading frames (ORFs) on the forward strand and one ORF on the reverse strand were identified in the ΨM100 genome. Comparison of ΨM100-encoded ORFs to those encoded by phage ΨM2 and to other known protein sequences permitted the assignment of putative functions to some ORFs. The ORF28 protein of ΨM100 was identified as the previously known autolytic enzyme pseudomurein endoisopeptidase PeiW produced by M. wolfeii.

Members of the Archaea domain are extremely diverse in their adaptation to extreme environments, yet also widespread in "normal" habitats. Altogether, among the best characterized archaeal representatives all mechanisms of gene transfer such as transduction, conjugation, and transformation have been discovered, as briefly reviewed here. For some halophiles and mesophilic methanogens, usable genetic tools were developed for in vivo studies. However, on an individual basis no single organism has evolved into the "E. coli of Archaea" as far as genetics is concerned. Currently, and unfortunately, most of the genome sequences available are those of microorganisms which are either not amenable to gene transfer or not among the most promising candidates for genetic studies.

The objective of the present study was to investigate the effects of seven different pure fatty acids on rumen fermentation using the rumen simulation technique (RUSITEC). The fatty acids were supplied to a complete ruminant diet at a proportion of 50 g x kg(-1) dietary dry matter and compared with an unsupplemented control. Methane release and methanogenic counts were suppressed by the fatty acids C12 : 0, C14 : 0 and C18 : 2 whereas C8 : 0, C10 : 0, C16 : 0 and C18 : 0 showed no corresponding effects. Apart from C12 : 0 and C18 : 2, C8 : 0 and C10 : 0 also adversely affected ciliate protozoa suggesting independence from the methane-suppressing effect of medium-chain fatty acids (MCFA). Although MCFA but not C18 : 2 reduced ruminal fibre degradation, the influence on other fermentation traits remained low. In conclusion, the supply of certain fatty acids to ruminant diets seems to have the potential to reduce methane release.

Comparison of the updated complete nucleotide sequences of the two related plasmids pME2001 and pME2200 from the thermophilic archaeon Methanothermobacter marburgensis (formerly Methanobacterium thermoautotrophicum) strains Marburg and ZH3, respectively, revealed an almost identical common backbone structure and five plasmid-specific inserted fragments (IFs), four of which are flanked by perfect or nearly perfect direct repeats 25-52 bp in length. A 4354-bp minimal replicon was derived from the alignment of the two plasmids, which encodes one putative antisense RNA related to replication control and five open reading frames (ORFs) organized in two operons. The first operon consists of four ORFs, the third of which, i.e. ORF3, contains a helix-turn-helix motif and a purine NTP-binding motif often found in proteins involved in DNA metabolic processes. The database search results suggest that ORF3 might function as a replication initiator protein. The large putative Rep protein encoded by pME2001 was overexpressed in Escherichia coli as an N-terminal His-tagged version using pET28a and a compatible helper plasmid that coexpresses minor tRNAs, argU and ileX to compensate for codon usage difference. ORFs 1, 2, and 3 are organized in a sequence reminiscent of that described in E. coli plasmids of the R1 family, cop-tap-rep. ORF6 encoded by IF1, one of the pME2200-specific elements, showed significant similarity to ORF6 encoded by archaeal phage psiM2 of M. marburgensis strain Marburg and may confer the apparent immunity of its host strain ZH3 to infection by phage psiM2. Our data indicate that M. marburgensis plasmids may evolve by a series of gene duplication and excision events.

A novel two-component enzyme system from Escherichia coli involving a flavorubredoxin (FlRd) and its reductase was studied in terms of spectroscopic, redox, and biochemical properties of its constituents. FlRd contains one FMN and one rubredoxin (Rd) center per monomer. To assess the role of the Rd domain, FlRd and a truncated form lacking the Rd domain (FlRdDeltaRd), were characterized. FlRd contains 2.9+/-0.5 iron atoms/subunit, whereas FlRdDeltaRd contains 2.1+/-0.6 iron atoms/subunit. While for FlRd one iron atom corresponds to the Rd center, the other two irons, also present in FlRdDeltaRd, are most probably due to a di-iron site. Redox titrations of FlRd using EPR and visible spectroscopies allowed us to determine that the Rd site has a reduction potential of -140+/-15 mV, whereas the FMN undergoes reduction via a red-semiquinone, at -140+/-15 mV (Fl(ox)/Fl(sq)) and -180+/-15 mV (Fl(sq)/Fl(red)), at pH 7.6. The Rd site has the lowest potential ever reported for a Rd center, which may be correlated with specific amino acid substitutions close to both cysteine clusters. The gene adjacent to that encoding FlRd was found to code for an FAD-containing protein, (flavo)rubredoxin reductase (FlRd-reductase), which is capable of mediating electron transfer from NADH to Desulfovibrio gigas Rd as well as to E. coli FlRd. Furthermore, electron donation was found to proceed through the Rd domain of FlRd as the Rd-truncated protein does not react with FlRd-reductase. In vitro, this pathway links NADH oxidation with dioxygen reduction. The possible function of this chain is discussed considering the presence of FlRd homologues in all known genomes of anaerobes and facultative aerobes.

A novel two-component enzyme system from Escherichia coli involving a flavorubredoxin (FlRd) and its reductase was studied in terms of spectroscopic, redox, and biochemical properties of its constituents. FlRd contains one FMN and one rubredoxin (Rd) center per monomer. To assess the role of the Rd domain, FlRd and a truncated form lacking the Rd domain (FlRdDeltaRd), were characterized. FlRd contains 2.9+/-0.5 iron atoms/subunit, whereas FlRdDeltaRd contains 2.1+/-0.6 iron atoms/subunit. While for FlRd one iron atom corresponds to the Rd center, the other two irons, also present in FlRdDeltaRd, are most probably due to a di-iron site. Redox titrations of FlRd using EPR and visible spectroscopies allowed us to determine that the Rd site has a reduction potential of -140+/-15 mV, whereas the FMN undergoes reduction via a red-semiquinone, at -140+/-15 mV (Fl(ox)/Fl(sq)) and -180+/-15 mV (Fl(sq)/Fl(red)), at pH 7.6. The Rd site has the lowest potential ever reported for a Rd center, which may be correlated with specific amino acid substitutions close to both cysteine clusters. The gene adjacent to that encoding FlRd was found to code for an FAD-containing protein, (flavo)rubredoxin reductase (FlRd-reductase), which is capable of mediating electron transfer from NADH to Desulfovibrio gigas Rd as well as to E. coli FlRd. Furthermore, electron donation was found to proceed through the Rd domain of FlRd as the Rd-truncated protein does not react with FlRd-reductase. In vitro, this pathway links NADH oxidation with dioxygen reduction. The possible function of this chain is discussed considering the presence of FlRd homologues in all known genomes of anaerobes and facultative aerobes.