Crystal structures of archaeal 2-oxoacid:ferredoxin oxidoreductases from Sulfolobus tokodaii

Abstract

As the first three-dimensional structure of the two-subunit type 2-oxoacid:ferredoxin oxidoreductases (OFOR) from archaea, we solved the crystal structures of STK_23000/STK_22980 (StOFOR1) and STK_24350/STK_24330 (StOFOR2) from Sulfolobus tokodaii. They showed similar overall structures, consisting of two a- and b-subunit heterodimers containing thiamin pyrophosphate (TPP) cofactor and [4Fe-4S] cluster, but lack an intramolecular ferredoxin domain. Unlike other OFORs, StOFORs can utilize both pyruvate and 2-oxoglutarate, playing a key role in the central metabolism. In the structure of StOFOR2 in unreacted pyruvate complex form, carboxylate group of pyruvate is recognized by Arg344 and Thr257 from the a-subunit, which are conserved in pyruvate:ferredoxin oxidoreductase from Desulfovbrio africanus (DaPFOR). In the structure of StOFOR1 co-crystallized with 2-oxobutyrate, electron density corresponding to a 1-hydroxypropyl group (post-decarboxylation state) was observed at the thiazole ring of TPP. The binding pockets of the StOFORs surrounding the methyl or propyl group of the ligands are wider than that of DaPFOR. Mutational analyses indicated that several residues were responsible for the broad 2-oxoacid specificity of StOFORs. We also constructed a possible complex structural model by placing a Zn2+-containing dicluster ferredoxin of S. tokodaii into the large pocket of StOFOR2, providing insight into the electron transfer between the two redox proteins.

New light on ancient enzymes – in vitro CO2 Fixation by Pyruvate Synthase of Desulfovibrio africanus and Sulfolobus acidocaldarius

“…For the native PFORdaf, some kinetic data have been published previously including an apparent K m value for pyruvate of 5.5 m m and we did not re‐determine these parameters in the present study. For PFORsac we found a lower apparent K m for pyruvate of 0.21 mM, in the same order of magnitude as recently reported for the respective enzyme of S. tokodaii ( K m = 0.32 m m , V max = 7.5 U·mg −1 ) .…”

A Reverse TCA Cycle 2-Oxoacid:Ferredoxin Oxidoreductase that Makes C-C Bonds from CO2

“…Prior to our work, structures of OFORs have been limited to three different types, two PFORs (MtPFOR 7 and the cognate enzyme from Desulfovibiro africanus [DaPFOR] [20][21][22] ), an oxaloacetate oxidoreductase (OOR) from M. thermoacetica (MtOOR), 23,24 and two OFORs from Sulfolobus tokodaii of cryptic function (StOFORs). 25 Even with these structures in hand, many open questions about OFOR chemistry remain, including the issues of electron transfer described above. For example, how OFORs differentiate 2-oxoacids and/or their acyl-CoAs to proceed in either CO 2 reduction or 2-oxoacid oxidation is still unknown.…”

“…The structures that are available report on enzymes that bind small substrates, 2-oxoacids with six non-H atoms, instead of larger substrates, i.e., 2-oxoglutarate, so we do not yet understand how larger substrates are accommodated. Even though StOFOR is reactive toward 2-oxoglutarate, 25 it is not substrate specific, so identification of a physiological substrate and specific function has been difficult; mutational studies shift reactivity in ways that the structures cannot account for. Consequently, a structure of a substrate-specific OGOR with a full description of structure-function relationships will greatly expand our understanding of OFOR chemistry.…”

“…In StOFORs, a lysine (Lys48b) was also proposed to play a similar role ( Figure 3E). 25 The Environment of the [4Fe-4S] Cluster Is Different for Enzymes that Lack an Fd Domain The [4Fe-4S] cluster in MmOGOR is located 10.6 Å away from the TPP active site (from the edge of the cluster to the C2 position of TPP), which is also the position equivalent to the proximal cluster in PFOR and OOR. The proximity between cofactors indicates the [4Fe-4S] cluster is capable of facile electron-transfer and thereby plays an essential role in the two distinct one-electron processes of OFOR reactions ( Figure 1D).…”

Oxidative Stickland reactions in an obligate aerobic organism – amino acid catabolism in the Crenarchaeon Sulfolobus solfataricus

“…Their limited presence in (facultative) anaerobes is probably due to the oxygen sensitivity of this type of oxidoreductases . In contrast, however, the 2‐oxoacid:ferredoxin oxidoreductase from Sulfolobus tokodaii has been shown to be oxygen resistant . The activity of oxidoreductases in S. solfataricus demonstrated by the presence of organic acids in this work suggests that the oxygen resistance of this group of enzymes is common to the genus Sulfolobus .…”