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A structural phylogeny for understanding 2-oxoacid oxidoreductase function.

ABSTRACT: 2-Oxoacid:ferredoxin oxidoreductases (OFORs) are essential enzymes in microbial one-carbon metabolism. They use thiamine pyrophosphate to reversibly cleave carbon-carbon bonds, generating low potential (?-500mV) electrons. Crystallographic analysis of a recently discovered OFOR, an oxalate oxidoreductase (OOR), has provided a second view of OFOR architecture and active site composition. Using these recent structural data along with the previously determined structures of pyruvate:ferredoxin oxidoreductase, structure-function relationships in this superfamily have been expanded and re-evaluated. Additionally, structural motifs have been defined that better serve to distinguish one OFOR subfamily from another and potentially uncover novel OFORs.

SUBMITTER: Gibson MI

PROVIDER: S-EPMC5381805 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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A structural phylogeny for understanding 2-oxoacid oxidoreductase function.

Gibson Marcus I MI Chen Percival Yang-Ting PY Drennan Catherine L CL

Current opinion in structural biology 20160614

2-Oxoacid:ferredoxin oxidoreductases (OFORs) are essential enzymes in microbial one-carbon metabolism. They use thiamine pyrophosphate to reversibly cleave carbon-carbon bonds, generating low potential (∼-500mV) electrons. Crystallographic analysis of a recently discovered OFOR, an oxalate oxidoreductase (OOR), has provided a second view of OFOR architecture and active site composition. Using these recent structural data along with the previously determined structures of pyruvate:ferredoxin oxid ...[more]

PMID: 27315560

Similar Datasets

The Structure of an Oxalate Oxidoreductase Provides Insight into Microbial 2-Oxoacid Metabolism.

Project description:Thiamine pyrophosphate (TPP), a derivative of vitamin B1, is a versatile and ubiquitous cofactor. When coupled with [4Fe-4S] clusters in microbial 2-oxoacid:ferredoxin oxidoreductases (OFORs), TPP is involved in catalyzing low-potential redox reactions that are important for the synthesis of key metabolites and the reduction of N2, H(+), and CO2. We have determined the high-resolution (2.27 Å) crystal structure of the TPP-dependent oxalate oxidoreductase (OOR), an enzyme that allows microbes to grow on oxalate, a widely occurring dicarboxylic acid that is found in soil and freshwater and is responsible for kidney stone disease in humans. OOR catalyzes the anaerobic oxidation of oxalate, harvesting the low-potential electrons for use in anaerobic reduction and fixation of CO2. We compare the OOR structure to that of the only other structurally characterized OFOR family member, pyruvate:ferredoxin oxidoreductase. This side-by-side structural analysis highlights the key similarities and differences that are relevant for the chemistry of this entire class of TPP-utilizing enzymes.

| S-EPMC4498597 | biostudies-literature

Identification and characterization of oxalate oxidoreductase, a novel thiamine pyrophosphate-dependent 2-oxoacid oxidoreductase that enables anaerobic growth on oxalate.

Project description:Moorella thermoacetica is an anaerobic acetogen, a class of bacteria that is found in the soil, the animal gastrointestinal tract, and the rumen. This organism engages the Wood-Ljungdahl pathway of anaerobic CO(2) fixation for heterotrophic or autotrophic growth. This paper describes a novel enzyme, oxalate oxidoreductase (OOR), that enables M. thermoacetica to grow on oxalate, which is produced in soil and is a common component of kidney stones. Exposure to oxalate leads to the induction of three proteins that are subunits of OOR, which oxidizes oxalate coupled to the production of two electrons and CO(2) or bicarbonate. Like other members of the 2-oxoacid:ferredoxin oxidoreductase family, OOR contains thiamine pyrophosphate and three [Fe(4)S(4)] clusters. However, unlike previously characterized members of this family, OOR does not use coenzyme A as a substrate. Oxalate is oxidized with a k(cat) of 0.09 s(-1) and a K(m) of 58 μM at pH 8. OOR also oxidizes a few other 2-oxoacids (which do not induce OOR) also without any requirement for CoA. The enzyme transfers its reducing equivalents to a broad range of electron acceptors, including ferredoxin and the nickel-dependent carbon monoxide dehydrogenase. In conjunction with the well characterized Wood-Ljungdahl pathway, OOR should be sufficient for oxalate metabolism by M. thermoacetica, and it constitutes a novel pathway for oxalate metabolism.

| S-EPMC3003350 | biostudies-literature

Toward an Understanding of the Structural and Mechanistic Aspects of Protein-Protein Interactions in 2-Oxoacid Dehydrogenase Complexes.

Project description:The 2-oxoglutarate dehydrogenase complex (OGDHc) is a key enzyme in the tricarboxylic acid (TCA) cycle and represents one of the major regulators of mitochondrial metabolism through NADH and reactive oxygen species levels. The OGDHc impacts cell metabolic and cell signaling pathways through the coupling of 2-oxoglutarate metabolism to gene transcription related to tumor cell proliferation and aging. DHTKD1 is a gene encoding 2-oxoadipate dehydrogenase (E1a), which functions in the L-lysine degradation pathway. The potentially damaging variants in DHTKD1 have been associated to the (neuro) pathogenesis of several diseases. Evidence was obtained for the formation of a hybrid complex between the OGDHc and E1a, suggesting a potential cross talk between the two metabolic pathways and raising fundamental questions about their assembly. Here we reviewed the recent findings and advances in understanding of protein-protein interactions in OGDHc and 2-oxoadipate dehydrogenase complex (OADHc), an understanding that will create a scaffold to help design approaches to mitigate the effects of diseases associated with dysfunction of the TCA cycle or lysine degradation. A combination of biochemical, biophysical and structural approaches such as chemical cross-linking MS and cryo-EM appears particularly promising to provide vital information for the assembly of 2-oxoacid dehydrogenase complexes, their function and regulation.

| S-EPMC8146983 | biostudies-literature

A reverse TCA cycle 2-oxoacid:ferredoxin oxidoreductase that makes C-C bonds from CO<sub>2</sub>.

Project description:2-oxoglutarate:ferredoxin oxidoreductase (OGOR) is a thiamine pyrophosphate (TPP) and [4Fe-4S] cluster-dependent enzyme from the reductive tricarboxylic acid (rTCA) cycle that fixes CO2 to succinyl-CoA, forming 2-oxoglutarate and CoA. Here we report an OGOR from the rTCA cycle of Magnetococcus marinus MC-1, along with all three potential ferredoxin (Fd) redox partners. We demonstrate MmOGOR operates bidirectionally (both CO2-fixing and 2-oxoglutarate oxidizing), and that only one Fd (MmFd1) supports efficient catalysis. Our 1.94-Å and 2.80-Å resolution crystal structures of native and substrate-bound forms of MmOGOR reveal the determinants of substrate specificity and CoA-binding in an OGOR, and illuminate the [4Fe-4S] cluster environment, portraying the electronic conduit allowing MmFd1 to be wired to the bound-TPP. Structural and biochemical data further identify Glu45α as a mobile residue that impacts catalytic bias toward CO2-fixation although it makes no direct contact with TPP-bound intermediates, indicating that reaction directionality can be tuned by second layer interactions. (149 of 150 words limit).

| S-EPMC6508887 | biostudies-literature

Light-driven carbon-carbon bond formation via CO<sub>2</sub> reduction catalyzed by complexes of CdS nanorods and a 2-oxoacid oxidoreductase.

Project description:Redox enzymes are capable of catalyzing a vast array of useful reactions, but they require redox partners that donate or accept electrons. Semiconductor nanocrystals provide a mechanism to convert absorbed photon energy into redox equivalents for enzyme catalysis. Here, we describe a system for photochemical carbon-carbon bond formation to make 2-oxoglutarate by coupling CO2 with a succinyl group. Photoexcited electrons from cadmium sulfide nanorods (CdS NRs) transfer to 2-oxoglutarate:ferredoxin oxidoreductase from Magnetococcus marinus MC-1 (MmOGOR), which catalyzes a carbon-carbon bond formation reaction. We thereby decouple MmOGOR from its native role in the reductive tricarboxylic acid cycle and drive it directly with light. We examine the dependence of 2-oxoglutarate formation on a variety of factors and, using ultrafast transient absorption spectroscopy, elucidate the critical role of electron transfer (ET) from CdS NRs to MmOGOR. We find that the efficiency of this ET depends strongly on whether the succinyl CoA (SCoA) cosubstrate is bound at the MmOGOR active site. We hypothesize that the conformational changes due to SCoA binding impact the CdS NR-MmOGOR interaction in a manner that decreases ET efficiency compared to the enzyme with no cosubstrate bound. Our work reveals structural considerations for the nano-bio interfaces involved in light-driven enzyme catalysis and points to the competing factors of enzyme catalysis and ET efficiency that may arise when complex enzyme reactions are driven by artificial light absorbers.

| S-EPMC6955356 | biostudies-literature

An unprecedented function for a tungsten-containing oxidoreductase.

Project description:Five tungstopterin-containing oxidoreductases were characterized from the hyperthermophile Pyrococcus furiosus. Each enzyme catalyzes the reversible conversion of one or more aldehydes to the corresponding carboxylic acid, but they have different specificities. The physiological functions of only two of these enzymes are known: one, termed GAPOR, is a glycolytic enzyme that oxidizes glyceraldehyde-3-phosphate, while the other, termed AOR, oxidizes multiple aldehydes generated during peptide fermentation. Two of the enzymes have known structures (AOR and FOR). Herein, we focus on WOR5, the fifth tungstopterin enzyme to be discovered in P. furiosus. Expression of WOR5 was previously shown to be increased during cold shock (growth at 72 ℃), although the physiological substrate is not known. To gain insight into WOR5 function, we sought to determine both its structure and identify its intracellular substrate. Crystallization experiments were performed with a concentrated cytoplasmic extract of P. furiosus grown at 72 ℃ and the structure of WOR5 was deduced from the crystals that were obtained. In contrast to a previous report, WOR5 is heterodimeric containing an additional polyferredoxin-like subunit with four [4Fe-4S] clusters. The active site structure of WOR5 is substantially different from that of AOR and FOR and the significant electron density observed adjacent to the tungsten cofactor of WOR5 was modeled as an aliphatic sulfonate. Biochemical assays and product analysis confirmed that WOR5 is an aliphatic sulfonate ferredoxin oxidoreductase (ASOR). A catalytic mechanism for ASOR is proposed based on the structural information and the potential role of ASOR in the cold-shock response is discussed.

| S-EPMC10293930 | biostudies-literature

Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency.

Project description:NADPH-cytochrome P450 oxidoreductase (CYPOR) is essential for electron donation to microsomal cytochrome P450-mediated monooxygenation in such diverse physiological processes as drug metabolism (approximately 85-90% of therapeutic drugs), steroid biosynthesis, and bioactive metabolite production (vitamin D and retinoic acid metabolites). Expressed by a single gene, CYPOR's role with these multiple redox partners renders it a model for understanding protein-protein interactions at the structural level. Polymorphisms in human CYPOR have been shown to lead to defects in bone development and steroidogenesis, resulting in sexual dimorphisms, the severity of which differs significantly depending on the degree of CYPOR impairment. The atomic structure of human CYPOR is presented, with structures of two naturally occurring missense mutations, V492E and R457H. The overall structures of these CYPOR variants are similar to wild type. However, in both variants, local disruption of H bonding and salt bridging, involving the FAD pyrophosphate moiety, leads to weaker FAD binding, unstable protein, and loss of catalytic activity, which can be rescued by cofactor addition. The modes of polypeptide unfolding in these two variants differ significantly, as revealed by limited trypsin digestion: V492E is less stable but unfolds locally and gradually, whereas R457H is more stable but unfolds globally. FAD addition to either variant prevents trypsin digestion, supporting the role of the cofactor in conferring stability to CYPOR structure. Thus, CYPOR dysfunction in patients harboring these particular mutations may possibly be prevented by riboflavin therapy in utero, if predicted prenatally, or rescued postnatally in less severe cases.

| S-EPMC3158178 | biostudies-literature

Structural and mechanistic insights into unusual thiol disulfide oxidoreductase.

Project description:Cytoplasmic desulfothioredoxin (Dtrx) from the anaerobe Desulfovibrio vulgaris Hildenborough has been identified as a new member of the thiol disulfide oxidoreductase family. The active site of Dtrx contains a particular consensus sequence, CPHC, never seen in the cytoplasmic thioredoxins and generally found in periplasmic oxidases. Unlike canonical thioredoxins (Trx), Dtrx does not present any disulfide reductase activity, but it presents instead an unusual disulfide isomerase activity. We have used NMR spectroscopy to gain insights into the structure and the catalytic mechanism of this unusual Dtrx. The redox potential of Dtrx (-181 mV) is significantly less reducing than that of canonical Trx. A pH dependence study allowed the determination of the pK(a) of all protonable residues, including the cysteine and histidine residues. Thus, the pK(a) values for the thiol group of Cys(31) and Cys(34) are 4.8 and 11.3, respectively. The His(33) pK(a) value, experimentally determined for the first time, differs notably as a function of the redox states, 7.2 for the reduced state and 4.6 for the oxidized state. These data suggest an important role for His(33) in the molecular mechanism of Dtrx catalysis that is confirmed by the properties of mutant DtrxH33G protein. The NMR structure of Dtrx shows a different charge repartition compared with canonical Trx. The results presented are likely indicative of the involvement of this protein in the catalysis of substrates specific of the anaerobe cytoplasm of DvH. The study of Dtrx is an important step toward revealing the molecular details of the thiol-disulfide oxidoreductase catalytic mechanism.

| S-EPMC3265852 | biostudies-literature

Progress in the structural understanding of voltage-gated calcium channel (CaV) function and modulation.

Project description:Voltage-gated calcium channels (CaVs) are large, transmembrane multiprotein complexes that couple membrane depolarization to cellular calcium entry. These channels are central to cardiac action potential propagation, neurotransmitter and hormone release, muscle contraction, and calcium-dependent gene transcription. Over the past six years, the advent of high-resolution structural studies of CaV components from different isoforms and CaV modulators has begun to reveal the architecture that underlies the exceptionally rich feedback modulation that controls CaV action. These descriptions of CaV molecular anatomy have provided new, structure-based insights into the mechanisms by which particular channel elements affect voltage-dependent inactivation (VDI), calcium‑dependent inactivation (CDI), and calcium‑dependent facilitation (CDF). The initial successes have been achieved through structural studies of soluble channel domains and modulator proteins and have proven most powerful when paired with biochemical and functional studies that validate ideas inspired by the structures. Here, we review the progress in this growing area and highlight some key open challenges for future efforts.

| S-EPMC3018750 | biostudies-literature

Structural Analysis Provides Mechanistic Insight into Nicotine Oxidoreductase from Pseudomonas putida.

Project description:The first structure of nicotine oxidoreductase (NicA2) was determined by X-ray crystallography. Pseudomonas putida has evolved nicotine-degrading activity to provide a source of carbon and nitrogen. The structure establishes NicA2 as a member of the monoamine oxidase family. Residues 1-50 are disordered and may play a role in localization. The nicotine-binding site proximal to the isoalloxazine ring of flavin shows an unusual composition of the classical aromatic cage (W427 and N462). The active site architecture is consistent with the proposed binding of the deprotonated form of the substrate and the flavin-dependent oxidation of the pyrrolidone C-N bond followed by nonenzymatic hydrolysis.

| S-EPMC6250430 | biostudies-literature

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