Specificity of Human Sulfiredoxin for Reductant and Peroxiredoxin Oligomeric State.

Human peroxiredoxins (Prx) are a family of antioxidant enzymes involved in a myriad of cellular functions and diseases. During the reaction with peroxides (e.g., H2O2), the typical 2-Cys Prxs change oligomeric structure between higher order (do)decamers and disulfide-linked dimers, with the hyperoxidized inactive state (-SO2H) favoring the multimeric structure of the reduced enzyme. Here, we present a study on the structural requirements for the repair of hyperoxidized 2-Cys Prxs by human sulfiredoxin (Srx) and the relative efficacy of physiological reductants hydrogen sulfide (H2S) and glutathione (GSH) in this reaction. The crystal structure of the toroidal Prx1-Srx complex shows an extended active site interface. The loss of this interface within engineered Prx2 and Prx3 dimers yielded variants more resistant to hyperoxidation and repair by Srx. Finally, we reveal for the first time Prx isoform-dependent use of and potential cooperation between GSH and H2S in supporting Srx activity.

Crystal Structure and Substrate Specificity of Human Thioesterase 2: INSIGHTS INTO THE MOLECULAR BASIS FOR THE MODULATION OF FATTY ACID SYNTHASE.

The type I fatty acid synthase (FASN) is responsible for the de novo synthesis of palmitate. Chain length selection and release is performed by the C-terminal thioesterase domain (TE1). FASN expression is up-regulated in cancer, and its activity levels are controlled by gene dosage and transcriptional and post-translational mechanisms. In addition, the chain length of fatty acids produced by FASN is controlled by a type II thioesterase called TE2 (E.C. 3.1.2.14). TE2 has been implicated in breast cancer and generates a broad lipid distribution within milk. The molecular basis for the ability of the TE2 to compete with TE1 for the acyl chain attached to the acyl carrier protein (ACP) domain of FASN is unknown. Herein, we show that human TE1 efficiently hydrolyzes acyl-CoA substrate mimetics. In contrast, TE2 prefers an engineered human acyl-ACP substrate and readily releases short chain fatty acids from full-length FASN during turnover. The 2.8 Å crystal structure of TE2 reveals a novel capping domain insert within the α/ß hydrolase core. This domain is reminiscent of capping domains of type II thioesterases involved in polyketide synthesis. The structure also reveals that the capping domain had collapsed onto the active site containing the Ser-101-His-237-Asp-212 catalytic triad. This observation suggests that the capping domain opens to enable the ACP domain to dock and to place the acyl chain and 4'-phosphopantetheinyl-linker arm correctly for catalysis. Thus, the ability of TE2 to prematurely release fatty acids from FASN parallels the role of editing thioesterases involved in polyketide and non-ribosomal peptide synthase synthases.

Proline dehydrogenase 2 (PRODH2) is a hydroxyproline dehydrogenase (HYPDH) and molecular target for treating primary hyperoxaluria.

The primary hyperoxalurias (PH), types 1-3, are disorders of glyoxylate metabolism that result in increased oxalate production and calcium oxalate stone formation. The breakdown of trans-4-hydroxy-L-proline (Hyp) from endogenous and dietary sources of collagen makes a significant contribution to the cellular glyoxylate pool. Proline dehydrogenase 2 (PRODH2), historically known as hydroxyproline oxidase, is the first step in the hydroxyproline catabolic pathway and represents a drug target to reduce the glyoxylate and oxalate burden of PH patients. This study is the first report of the expression, purification, and biochemical characterization of human PRODH2. Evaluation of a panel of N-terminal and C-terminal truncation variants indicated that residues 157-515 contain the catalytic core with one FAD molecule. The 12-fold higher k(cat)/K(m) value of 0.93 M⁻¹·s⁻¹ for Hyp over Pro demonstrates the preference for Hyp as substrate. Moreover, an anaerobic titration determined a K(d) value of 125 µM for Hyp, a value ~1600-fold lower than the K(m) value. A survey of ubiquinone analogues revealed that menadione, duroquinone, and CoQ1 reacted more efficiently than oxygen as the terminal electron acceptor during catalysis. Taken together, these data and the slow reactivity with sodium sulfite support that PRODH2 functions as a dehydrogenase and most likely utilizes CoQ10 as the terminal electron acceptor in vivo. Thus, we propose that the name of PRODH2 be changed to hydroxyproline dehydrogenase (HYPDH). Three Hyp analogues were also identified to inhibit the activity of HYPDH, representing the first steps toward the development of a novel approach to treat all forms of PH.

Metabolism of [13C5]hydroxyproline in vitro and in vivo: implications for primary hyperoxaluria.

Hydroxyproline (Hyp) metabolism is a key source of glyoxylate production in the body and may be a major contributor to excessive oxalate production in the primary hyperoxalurias where glyoxylate metabolism is impaired. Important gaps in our knowledge include identification of the tissues with the capacity to degrade Hyp and the development of model systems to study this metabolism and how to suppress it. The expression of mRNA for enzymes in the pathway was examined in 15 different human tissues. Expression of the complete pathway was identified in liver, kidney, pancreas, and small intestine. HepG2 cells also expressed these mRNAs and enzymes and were shown to metabolize Hyp in the culture medium to glycolate, glycine, and oxalate. [(18)O]- and [(13)C(5)]Hyp were synthesized and evaluated for their use with in vitro and in vivo models. [(18)O]Hyp was not suitable because of an apparent tautomerism of [(18)O]glyoxylate between enol and hydrated forms, which resulted in a loss of isotope. [(13)C(5)]Hyp, however, was metabolized to [(13)C(2)]glycolate, [(13)C(2)]glycine, and [(13)C(2)]oxalate in vitro in HepG2 cells and in vivo in mice infused with [(13)C(5)]Hyp. These model systems should be valuable tools for exploring various aspects of Hyp metabolism and will be useful in determining whether blocking Hyp catabolism is an effective therapy in the treatment of primary hyperoxaluria.

Structural and biochemical studies of human 4-hydroxy-2-oxoglutarate aldolase: implications for hydroxyproline metabolism in primary hyperoxaluria.

BACKGROUND: 4-hydroxy-2-oxoglutarate (HOG) aldolase is a unique enzyme in the hydroxyproline degradation pathway catalyzing the cleavage of HOG to pyruvate and glyoxylate. Mutations in this enzyme are believed to be associated with the excessive production of oxalate in primary hyperoxaluria type 3 (PH3), although no experimental data is available to support this hypothesis. Moreover, the identity, oligomeric state, enzymatic activity, and crystal structure of human HOGA have not been experimentally determined. METHODOLOGY/PRINCIPAL FINDINGS: In this study human HOGA (hHOGA) was identified by mass spectrometry of the mitochondrial enzyme purified from bovine kidney. hHOGA performs a retro-aldol cleavage reaction reminiscent of the trimeric 2-keto-3-deoxy-6-phosphogluconate aldolases. Sequence comparisons, however, show that HOGA is related to the tetrameric, bacterial dihydrodipicolinate synthases, but the reaction direction is reversed. The 1.97 Å resolution crystal structure of hHOGA bound to pyruvate was determined and enabled the modeling of the HOG-Schiff base intermediate and the identification of active site residues. Kinetic analyses of site-directed mutants support the importance of Lys196 as the nucleophile, Tyr168 and Ser77 as components of a proton relay, and Asn78 and Ser198 as unique residues that facilitate substrate binding. CONCLUSIONS/SIGNIFICANCE: The biochemical and structural data presented support that hHOGA utilizes a type I aldolase reaction mechanism, but employs novel residue interactions for substrate binding. A mapping of the PH3 mutations identifies potential rearrangements in either the active site or the tetrameric assembly that would likely cause a loss in activity. Altogether, these data establish a foundation to assess mutant forms of hHOGA and how their activity could be pharmacologically restored.

Use of dimedone-based chemical probes for sulfenic acid detection evaluation of conditions affecting probe incorporation into redox-sensitive proteins.

Sulfenic acids, formed as transient intermediates during the reaction of cysteine residues with peroxides, play significant roles in enzyme catalysis and regulation, and are also involved in the redox regulation of transcription factors and other signaling proteins. Therefore, interest in the identification of protein sulfenic acids has grown substantially in the past few years. Dimedone, which specifically traps sulfenic acids, has provided the basis for the synthesis of a novel group of compounds that derivatize 1,3-cyclohexadione, a dimedone analogue, with reporter tags such as biotin for affinity capture and fluorescent labels for visual detection. These reagents allow identification of the cysteine sites and proteins that are sensitive to oxidation and permit identification of the cellular conditions under which such oxidations occur. We have shown that these compounds are reactive and specific toward sulfenic acids and that the labeled proteins can be detected at high sensitivity using gel analysis or mass spectrometry. Here, we further characterize these reagents, showing that the DCP-Bio1 incorporation rates into three sulfenic acid containing proteins, papaya papain, Escherichia coli fRMsr, and the Salmonella typhimurium peroxiredoxin AhpC, are significantly different and, in the case of fRMsr, are unaffected by changes in buffer pH from 5.5 and 8.0. We also provide protocols to label protein sulfenic acids in cellular proteins, either by in situ labeling of intact cells or by labeling at the time of lysis. We show that the addition of alkylating reagents and catalase to the lysis buffer is critical in preventing the formation of sulfenic acid subsequent to cell lysis. Data presented herein also indicate that the need to standardize, as much as possible, the protein and reagent concentrations during labeling. Finally, we introduce several new test or control proteins that can be used to evaluate labeling procedures and efficiencies.

Protein engineering of the quaternary sulfiredoxin.peroxiredoxin enzyme.substrate complex reveals the molecular basis for cysteine sulfinic acid phosphorylation.

Oxidative stress can damage the active site cysteine of the antioxidant enzyme peroxiredoxin (Prx) to the sulfinic acid form, Prx-SO(2)(-). This modification leads to inactivation. Sulfiredoxin (Srx) utilizes a unique ATP-Mg(2+)-dependent mechanism to repair the Prx molecule. Using selective protein engineering that involves disulfide bond formation and site-directed mutagenesis, a mimic of the enzyme.substrate complex has been trapped. Here, we present the 2.1 A crystal structure of human Srx in complex with PrxI, ATP, and Mg(2+). The Cys(52) sulfinic acid moiety was substituted by mutating this residue to Asp, leading to a replacement of the sulfur atom with a carbon atom. Because the Srx reaction cannot occur, the structural changes in the Prx active site that lead to the attack on ATP may be visualized. The local unfolding of the helix containing C52D resulted in the packing of Phe(50) in PrxI within a hydrophobic pocket of Srx. Importantly, this structural rearrangement positioned one of the oxygen atoms of Asp(52) within 4.3 A of the gamma-phosphate of ATP bound to Srx. These observations support a mechanism where phosphorylation of Prx-SO(2)(-) is the first chemical step.

Reduction of cysteine sulfinic acid in peroxiredoxin by sulfiredoxin proceeds directly through a sulfinic phosphoryl ester intermediate.

Sulfiredoxin (Srx) catalyzes a novel enzymatic reaction, the reduction of protein cysteine sulfinic acid, Cys-SO(2)(-). This reaction is unique to the typical 2-Cys peroxiredoxins (Prx) and plays a role in peroxide-mediated signaling by regulating the activity of Prxs. Two mechanistic schemes have been proposed that differ regarding the first step of the reaction. This step involves either the direct transfer of the gamma-phosphate of ATP to the Prx molecule or through Srx acting as a phosphorylated intermediary. In an effort to clarify this step of the Srx reaction, we have determined the 1.8A resolution crystal structure of Srx in complex with ATP and Mg(2+). This structure reveals the role of the Mg(2+) ion to position the gamma-phosphate toward solvent, thus preventing an in-line attack by the catalytic residue Cys-99 of Srx. A model of the quaternary complex is consistent with this proposal. Furthermore, phosphorylation studies on several site-directed mutants of Srx and Prx, including the Prx-Asp mimic of the Prx-SO(2)(-) species, support a mechanism where phosphorylation of Prx-SO(2)(-) is the first chemical step.

Structure of the sulphiredoxin-peroxiredoxin complex reveals an essential repair embrace.

Typical 2-Cys peroxiredoxins (Prxs) have an important role in regulating hydrogen peroxide-mediated cell signalling. In this process, Prxs can become inactivated through the hyperoxidation of an active site Cys residue to Cys sulphinic acid. The unique repair of this moiety by sulphiredoxin (Srx) restores peroxidase activity and terminates the signal. The hyperoxidized form of Prx exists as a stable decameric structure with each active site buried. Therefore, it is unclear how Srx can access the sulphinic acid moiety. Here we present the 2.6 A crystal structure of the human Srx-PrxI complex. This complex reveals the complete unfolding of the carboxy terminus of Prx, and its unexpected packing onto the backside of Srx away from the Srx active site. Binding studies and activity analyses of site-directed mutants at this interface show that the interaction is required for repair to occur. Moreover, rearrangements in the Prx active site lead to a juxtaposition of the Prx Gly-Gly-Leu-Gly and Srx ATP-binding motifs, providing a structural basis for the first step of the catalytic mechanism. The results also suggest that the observed interactions may represent a common mode for other proteins to bind to Prxs.

Crystal structure of the thioesterase domain of human fatty acid synthase inhibited by Orlistat.

Human fatty acid synthase (FAS) is uniquely expressed at high levels in many tumor types. Pharmacological inhibition of FAS therefore represents an important therapeutic opportunity. The drug Orlistat, which has been approved by the US Food and Drug Administration, inhibits FAS, induces tumor cell-specific apoptosis and inhibits the growth of prostate tumor xenografts. We determined the 2.3-A-resolution crystal structure of the thioesterase domain of FAS inhibited by Orlistat. Orlistat was captured in the active sites of two thioesterase molecules as a stable acyl-enzyme intermediate and as the hydrolyzed product. The details of these interactions reveal the molecular basis for inhibition and suggest a mechanism for acyl-chain length discrimination during the FAS catalytic cycle. Our findings provide a foundation for the development of new cancer drugs that target FAS.

Free methionine-(R)-sulfoxide reductase from Escherichia coli reveals a new GAF domain function.

The reduction of methionine sulfoxide (MetO) is mediated by methionine sulfoxide reductases (Msr). The MsrA and MsrB families can reduce free MetO and MetO within a peptide or protein context. This process is stereospecific with the S- and R-forms of MetO repaired by MsrA and MsrB, respectively. Cell extracts from an MsrA(-)B(-) knockout of Escherichia coli have several remaining Msr activities. This study has identified an enzyme specific for the free form of Met-(R)-O, fRMsr, through proteomic analysis. The recombinant enzyme exhibits the same substrate specificity and is as active as MsrA family members. E. coli fRMsr is, however, 100- to 1,000-fold more active than non-selenocysteine-containing MsrB enzymes for free Met-(R)-O. The crystal structure of E. coli fRMsr was previously determined, but no known function was assigned. Thus, the function of this protein has now been determined. The structural similarity of the E. coli and yeast proteins suggests that most fRMsrs use three cysteine residues for catalysis and the formation of a disulfide bond to enclose a small active site cavity. This latter feature is most likely a key determinant of substrate specificity. Moreover, E. coli fRMsr is the first GAF domain family member to show enzymatic activity. Other GAF domain proteins substitute the Cys residues and others to specifically bind cyclic nucleotides, chromophores, and many other ligands for signal potentiation. Therefore, Met-(R)-O may represent a signaling molecule in response to oxidative stress and nutrients via the TOR pathway in some organisms.

The thioredoxin domain of Neisseria gonorrhoeae PilB can use electrons from DsbD to reduce downstream methionine sulfoxide reductases.

The PilB protein from Neisseria gonorrhoeae is located in the periplasm and made up of three domains. The N-terminal, thioredoxin-like domain (NT domain) is fused to tandem methionine sulfoxide reductase A and B domains (MsrA/B). We show that the alpha domain of Escherichia coli DsbD is able to reduce the oxidized NT domain, which suggests that DsbD in Neisseria can transfer electrons from the cytoplasmic thioredoxin to the periplasm for the reduction of the MsrA/B domains. An analysis of the available complete genomes provides further evidence for this proposition in other bacteria where DsbD/CcdA, Trx, MsrA, and MsrB gene homologs are all located in a gene cluster with a common transcriptional direction. An examination of wild-type PilB and a panel of Cys to Ser mutants of the full-length protein and the individually expressed domains have also shown that the NT domain more efficiently reduces the MsrA/B domains when in the polyprotein context. Within this frame-work there does not appear to be a preference for the NT domain to reduce the proximal MsrA domain over MsrB domain. Finally, we report the 1.6A crystal structure of the NT domain. This structure confirms the presence of a surface loop that makes it different from other membrane-tethered, Trx-like molecules, including TlpA, CcmG, and ResA. Subtle differences are observed in this loop when compared with the Neisseria meningitidis NT domain structure. The data taken together supports the formation of specific NT domain interactions with the MsrA/B domains and its in vivo recycling partner, DsbD.

Structural basis for the retroreduction of inactivated peroxiredoxins by human sulfiredoxin.

Sufiredoxins (Srx) repair the inactivated forms of typical two-Cys peroxiredoxins (Prx) implicated in hydrogen peroxide-mediated cell signaling. The reduction of the cysteine sulfinic acid moiety within the active site of the Prx by Srx involves novel sulfur chemistry and the use of ATP and Mg(2+). The 1.65 A crystal structure of human Srx (hSrx) exhibits a new protein fold and a unique nucleotide binding motif containing the Gly98-Cys99-His100-Arg101 sequence at the N-terminus of an alpha-helix. HPLC analysis of the reaction products has confirmed that the site of ATP cleavage is between the beta- and gamma-phosphate groups. Cys99 and the gamma-phosphate of ATP, modeled within the active site of the 2.0 A ADP product complex structure, are adjacent to large surface depressions containing additional conserved residues. These features and the necessity for significant remodeling of the Prx structure suggest that the interactions between hSrx and typical two-Cys Prxs are specific. Moreover, the concave shape of the hSrx active site surface appears to be ideally suited to interacting with the convex surface of the toroidal Prx decamer.