Pyruvate–Ferredoxin Oxidoreductase

Abstract

Introduction. Giardia intestinalis is a unicellular parasite of worldwide distribution. It causes an intestinal illness known as giardiasis, and it is probably the earliest diverging eukaryotic microorganism. Previously, changes have been reported in the expression of mRNAs at several stages of the life cycle; however specific enzymatic activity changes have not been explored. Objective. The expression of pyruvate ferredoxin oxidoreductase (PFOR) and alcohol dehydrogenase E (ADHE) enzymes was measured in cyst and trophozoite stages, and during the excystation process. Materials and methods. Recombinant proteins were generated for PFOR and ADHE to be used as antigens in the production of polyclonal antibodies for the detection of native proteins by Western Blot. The enzymatic activity of ADHE and glutamate dehydrogenase (GDH) was evaluated by spectrophotometric assays. Results. PFOR (139 kDa) and ADHE (97 kDa) proteins were detected in trophozoites, but not in cysts. During excystation, ADHE protein was detected after the first phase of induction, but the PFOR protein appeared only after the second phase. This indicated that both proteins were synthesized during excystation, although at different times. ADHE enzymatic activity was present only in trophozoites and not in cysts whereas GDH activity was detected in both stages. Conclusion. These results conclusively showed that PFOR and ADHE enzymes were translated during the excystation process and is strong evidence that active protein synthesis was occurring during excystation.

Radical reactions of thiamin pyrophosphate in 2-oxoacid oxidoreductases

“…The quaternary structures separate into four groups: homodimers (α 2 ); dimers of heterotetramers (αβγδ) 2 ; dimers of heterotrimers (αβγ) 2 ; and heterodimers (αβ) 2 . The dimers are proposed to have arisen from various gene fusions from an ancestral heterotetrmeric protein such as those found in archaea [18]. In some cases, e. g., in the H. salinarium enzyme, a fusion of the α and γ subunits can be isolated as a homodimer containing only the binding site of TPP and Cys residues corresponding to a single [4Fe-4S] cluster.…”

Metalloproteins in the Biology of Heterocysts

“…The overall structure of PFOR enzymes is also rather variable. Most bacterial members form homodimers (A 2 ), yet dimers of heterodimers (a 2 b 2 ) are also found in a few cases, whereas PFORs in archaea are heterotetramers (αβγδ), which are considered to resemble the ancestral protein arrangement [450,453]. Despite the differences in subunit composition, they share a similar structure and topology.…”

“…Despite the differences in subunit composition, they share a similar structure and topology. Therefore, subunits A contain domains homologous to the four α, β, γ, and δ subunits of archaeal members, while ab dimers exhibit domains homologues to α, β, and γ subunits, but lack a domain similar to subunits δ [450,453].…”

“…In archaeal heterotetrameric PFORs, the subunit δ harbors the binding motifs to coordinate two Fd-like [4Fe–4S] clusters and the subunit β contains a conserved binding site for a thiamine pyrophosphate cofactor and the Cys residues to coordinate one [4Fe–4S] cluster, while subunits α and γ are involved in catalytic and structural roles [450,453]. As in heterotetrameric PFORs, bacterial homodimeric enzymes dispose the same cofactors in a similar arrangement per monomer.…”

“…As in heterotetrameric PFORs, bacterial homodimeric enzymes dispose the same cofactors in a similar arrangement per monomer. Although the structure of PFORs from heterocyst-forming cyanobacteria has not been resolved, PFORs from this group of cyanobacteria exhibit sequence similarity to that of bacterial homologs and are predicted to contain the same metal clusters and the thiamine pyrophosphate cofactor [447,453]. Thiamine pyrophosphate and one [4Fe–4S] cluster are buried in close proximity within the protein near the substrate binding site [450].…”