The catalytic architecture of leukotriene C4 synthase with two arginine residues

Leukotriene (LT) C(4) and its metabolites, LTD(4) and LTE(4), are involved in the pathobiology of bronchial asthma. LTC(4) synthase is the nuclear membrane-embedded enzyme responsible for LTC(4) biosynthesis, catalyzing the conjugation of two substrates that have considerably different water solubility; that amphipathic LTA(4) as a derivative of arachidonic acid and a water-soluble glutathione (GSH). A previous crystal structure revealed important details of GSH binding and implied a GSH activating function for Arg-104. In addition, Arg-31 was also proposed to participate in the catalysis based on the putative LTA(4) binding model. In this study enzymatic assay with mutant enzymes demonstrates that Arg-104 is required for the binding and activation of GSH and that Arg-31 is needed for catalysis probably by activating the epoxide group of LTA(4).     

Structural and Biochemical Characterization of an Active Arylamine N-Acetyltransferase Possessing a Non-canonical Cys-His-Glu Catalytic Triad

Arylamine N-acetyltransferases (NATs), a class of xenobiotic-metabolizing enzymes, catalyze the acetylation of aromatic amine compounds through a strictly conserved Cys-His-Asp catalytic triad. Each residue is essential for catalysis in both prokaryotic and eukaryotic NATs. Indeed, in (HUMAN)NAT2 variants, mutation of the Asp residue to Asn, Gln, or Glu dramatically impairs enzyme activity. However, a putative atypical NAT harboring a catalytic triad Glu residue was recently identified in Bacillus cereus ((BACCR)NAT3) but has not yet been characterized. We report here the crystal structure and functional characterization of this atypical NAT. The overall fold of (BACCR)NAT3 and the geometry of its Cys-His-Glu catalytic triad are similar to those present in functional NATs. Importantly, the enzyme was found to be active and to acetylate prototypic arylamine NAT substrates. In contrast to (HUMAN) NAT2, the presence of a Glu or Asp in the triad of (BACCR)NAT3 did not significantly affect enzyme structure or function. Computational analysis identified differences in residue packing and steric constraints in the active site of (BACCR)NAT3 that allow it to accommodate a Cys-His-Glu triad. These findings overturn the conventional view, demonstrating that the catalytic triad of this family of acetyltransferases is plastic. Moreover, they highlight the need for further study of the evolutionary history of NATs and the functional significance of the predominant Cys-His-Asp triad in both prokaryotic and eukaryotic forms.     

Structural and mechanistic insights into the interaction of cytochrome P4503A4 with bromoergocryptine, a type I ligand

Cytochrome P4503A4 (CYP3A4), a major human drug-metabolizing enzyme, is responsible for the oxidation and clearance of the majority of administered drugs. One of the CYP3A4 substrates is bromoergocryptine (BEC), a dopamine receptor agonist prescribed for the inhibition of prolactin secretion and treatment of Parkinson disease, type 2 diabetes, and several other pathological conditions. Here we present a 2.15 Å crystal structure of the CYP3A4-BEC complex in which the drug, a type I heme ligand, is bound in a productive mode. The manner of BEC binding is consistent with the in vivo metabolite analysis and identifies the 8′ and 9′ carbons of the proline ring as the primary sites of oxidation. The crystal structure predicts the importance of Arg²¹² and Thr²²⁴ for binding of the tripeptide and lysergic moieties of BEC, respectively, which we confirmed experimentally. Our data support a three-step BEC binding model according to which the drug binds first at a peripheral site without perturbing the heme spectrum and then translocates into the active site cavity, where formation of a hydrogen bond between Thr²²⁴ and the N1 atom of the lysergic moiety is followed by a slower conformational readjustment of the tripeptide group modulated by Arg²¹².     

Molecular and catalytic properties of three rat leukotriene C(4) synthase homologs

The committed step in the biosynthesis of cysteinyl-leukotrienes is catalyzed by leukotriene C(4) synthase as well as microsomal glutathione S-transferase (MGST) type 2 and type 3, which belong to a family of membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG). We cloned and characterized these three enzymes from the rat to allow a side-by-side comparison of structural and catalytic properties. The proteins are 79.6-86.7% identical to the human orthologs. Rat MGST3 fails to convert leukotriene A(4) into leukotriene C(4), which in turn challenges the proposed catalytic role of a conserved Arg and Tyr residue for the leukotriene C(4) synthase reaction. Comparative inhibitor studies of all three enzymes, using MK-886 and cysteinyl-leukotrienes, indicate that their catalytic centers originate from structurally related and overlapping active sites. Hence, it seems feasible to design enzyme inhibitors, which simultaneously target several members of this protein family to yield compounds with increased anti-inflammatory action.     

Crystal structure of heterodimeric hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26 reveals that the small subunit is directly involved in the product chain length regulation

Hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26 (Ml-HexPPs) is a heterooligomeric type trans-prenyltransferase catalyzing consecutive head-to-tail condensations of three molecules of isopentenyl diphosphates (C(5)) on a farnesyl diphosphate (FPP; C(15)) to form an (all-E) hexaprenyl diphosphate (HexPP; C(30)). Ml-HexPPs is known to function as a heterodimer of two different subunits, small and large subunits called HexA and HexB, respectively. Compared with homooligomeric trans-prenyltransferases, the molecular mechanism of heterooligomeric trans-prenyltransferases is not yet clearly understood, particularly with respect to the role of the small subunits lacking the catalytic motifs conserved in most known trans-prenyltransferases. We have determined the crystal structure of Ml-HexPPs both in the substrate-free form and in complex with 7,11-dimethyl-2,6,10-dodecatrien-1-yl diphosphate ammonium salt (3-DesMe-FPP), an analog of FPP. The structure of HexB is composed of mostly antiparallel α-helices joined by connecting loops. Two aspartate-rich motifs (designated the first and second aspartate-rich motifs) and the other characteristic motifs in HexB are located around the diphosphate part of 3-DesMe-FPP. Despite the very low amino acid sequence identity and the distinct polypeptide chain lengths between HexA and HexB, the structure of HexA is quite similar to that of HexB. The aliphatic tail of 3-DesMe-FPP is accommodated in a large hydrophobic cleft starting from HexB and penetrating to the inside of HexA. These structural features suggest that HexB catalyzes the condensation reactions and that HexA is directly involved in the product chain length control in cooperation with HexB.     

Novel folding and stability defects cause a deficiency of human glutathione transferase omega 1

The polymorphic deletion of Glu-155 from human glutathione transferase omega1 (GSTO1-1) occurs in most populations. Although the recombinant ΔGlu-155 enzyme expressed in Escherichia coli is active, the deletion causes a deficiency of the active enzyme in vivo. The crystal structure and the folding/unfolding kinetics of the ΔGlu-155 variant were determined in order to investigate the cause of the rapid loss of the enzyme in human cells. The crystal structure revealed altered packing around the Glu-155 deletion, an increase in the predicted solvent-accessible area and a corresponding reduction in the buried surface area. This increase in solvent accessibility was consistent with an elevated Stern-Volmer constant. The unfolding of both the wild type and ΔGlu-155 enzyme in urea is best described by a three-state model, and there is evidence for the more pronounced population of an intermediate state by the ΔGlu-155 enzymes. Studies using intrinsic fluorescence revealed a free energy change around 14.4 kcal/mol for the wild type compared with around 8.6 kcal/mol for the ΔGlu-155 variant, which indicates a decrease in stability associated with the Glu-155 deletion. Urea induced unfolding of the wild type GSTO1-1 was reversible through an initial fast phase followed by a second slow phase. In contrast, the ΔGlu-155 variant lacks the slow phase, indicating a refolding defect. It is possible that in some conditions in vivo, the increased solvent-accessible area and the low stability of the ΔGlu-155 variant may promote its unfolding, whereas the refolding defect limits its refolding, resulting in GSTO1-1 deficiency.     

4-hydroxyphenylpyruvate dioxygenase catalysis: identification of catalytic residues and production of a hydroxylated intermediate shared with a structurally unrelated enzyme

4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxyphenylpyruvate (HPP) into homogentisate. HPPD is the molecular target of very effective synthetic herbicides. HPPD inhibitors may also be useful in treating life-threatening tyrosinemia type I and are currently in trials for treatment of Parkinson disease. The reaction mechanism of this key enzyme in both plants and animals has not yet been fully elucidated. In this study, using site-directed mutagenesis supported by quantum mechanical/molecular mechanical theoretical calculations, we investigated the role of catalytic residues potentially interacting with the substrate/intermediates. These results highlight the following: (i) the central role of Gln-272, Gln-286, and Gln-358 in HPP binding and the first nucleophilic attack; (ii) the important movement of the aromatic ring of HPP during the reaction, and (iii) the key role played by Asn-261 and Ser-246 in C1 hydroxylation and the final ortho-rearrangement steps (numbering according to the Arabidopsis HPPD crystal structure 1SQD). Furthermore, this study reveals that the last step of the catalytic reaction, the 1,2 shift of the acetate side chain, which was believed to be unique to the HPPD activity, is also catalyzed by a structurally unrelated enzyme.     

Structural Basis for a Bispecific NADP+ and CoA Binding Site in an Archaeal Malonyl-Coenzyme A Reductase

Autotrophic members of the Sulfolobales (crenarchaeota) use the 3-hydroxypropionate/4-hydroxybutyrate cycle to assimilate CO₂ into cell material. The product of the initial acetyl-CoA carboxylation with CO₂, malonyl-CoA, is further reduced to malonic semialdehyde by an NADPH-dependent malonyl-CoA reductase (MCR); the enzyme also catalyzes the reduction of succinyl-CoA to succinic semialdehyde onwards in the cycle. Here, we present the crystal structure of Sulfolobus tokodaii malonyl-CoA reductase in the substrate-free state and in complex with NADP⁺ and CoA. Structural analysis revealed an unexpected reaction cycle in which NADP⁺ and CoA successively occupy identical binding sites. Both coenzymes are pressed into an S-shaped, nearly superimposable structure imposed by a fixed and preformed binding site. The template-governed cofactor shaping implicates the same binding site for the 3′- and 2′-ribose phosphate group of CoA and NADP⁺, respectively, but a different one for the common ADP part: the β-phosphate of CoA aligns with the α-phosphate of NADP⁺. Evolution from an NADP⁺ to a bispecific NADP⁺ and CoA binding site involves many amino acid exchanges within a complex process by which constraints of the CoA structure also influence NADP⁺ binding. Based on the paralogous aspartate-β-semialdehyde dehydrogenase structurally characterized with a covalent Cys-aspartyl adduct, a malonyl/succinyl group can be reliably modeled into MCR and discussed regarding its binding mode, the malonyl/succinyl specificity, and the catalyzed reaction. The modified polypeptide surrounding around the absent ammonium group in malonate/succinate compared with aspartate provides the structural basis for engineering a methylmalonyl-CoA reductase applied for biotechnical polyester building block synthesis.     

Modulating O2 reactivity in a fungal flavoenzyme: involvement of aryl-alcohol oxidase Phe-501 contiguous to catalytic histidine

Aryl-alcohol oxidase (AAO) is a flavoenzyme responsible for activation of O₂ to H₂O₂ in fungal degradation of lignin. The AAO crystal structure shows a buried active site connected to the solvent by a hydrophobic funnel-shaped channel, with Phe-501 and two other aromatic residues forming a narrow bottleneck that prevents the direct access of alcohol substrates. However, ligand diffusion simulations show O₂ access to the active site following this channel. Site-directed mutagenesis of Phe-501 yielded a F501A variant with strongly reduced O₂ reactivity. However, a variant with increased reactivity, as shown by kinetic constants and steady-state oxidation degree, was obtained by substitution of Phe-501 with tryptophan. The high oxygen catalytic efficiency of F501W, ∼2-fold that of native AAO and ∼120-fold that of F501A, seems related to a higher O₂ availability because the turnover number was slightly decreased with respect to the native enzyme. Free diffusion simulations of O₂ inside the active-site cavity of AAO (and several in silico Phe-501 variants) yielded >60% O₂ population at 3–4 Å from flavin C4a in F501W compared with 44% in AAO and only 14% in F501A. Paradoxically, the O₂ reactivity of AAO decreased when the access channel was enlarged and increased when it was constricted by introducing a tryptophan residue. This is because the side chain of Phe-501, contiguous to the catalytic histidine (His-502 in AAO), helps to position O₂ at an adequate distance from flavin C4a (and His-502 Nϵ). Phe-501 substitution with a bulkier tryptophan residue resulted in an increase in the O₂ reactivity of this flavoenzyme.     

The catalytic aspartate is protonated in the Michaelis complex formed between trypsin and an in vitro evolved substrate-like inhibitor: a refined mechanism of serine protease action

The mechanism of serine proteases prominently illustrates how charged amino acid residues and proton transfer events facilitate enzyme catalysis. Here we present an ultrahigh resolution (0.93 Å) x-ray structure of a complex formed between trypsin and a canonical inhibitor acting through a substrate-like mechanism. The electron density indicates the protonation state of all catalytic residues where the catalytic histidine is, as expected, in its neutral state prior to the acylation step by the catalytic serine. The carboxyl group of the catalytic aspartate displays an asymmetric electron density so that the O_δ2–C_γ bond appears to be a double bond, with O_δ2 involved in a hydrogen bond to His-57 and Ser-214. Only when Asp-102 is protonated on O_δ1 atom could a density functional theory simulation reproduce the observed electron density. The presence of a putative hydrogen atom is also confirmed by a residual mF_obs − DF_calc density above 2.5 σ next to O_δ1. As a possible functional role for the neutral aspartate in the active site, we propose that in the substrate-bound form, the neutral aspartate residue helps to keep the pK_a of the histidine sufficiently low, in the active neutral form. When the histidine receives a proton during the catalytic cycle, the aspartate becomes simultaneously negatively charged, providing additional stabilization for the protonated histidine and indirectly to the tetrahedral intermediate. This novel proposal unifies the seemingly conflicting experimental observations, which were previously seen as either supporting the charge relay mechanism or the neutral pK_a histidine theory.     

Crystallographic, kinetic, and spectroscopic study of the first ligninolytic peroxidase presenting a catalytic tyrosine

Trametes cervina lignin peroxidase (LiP) is a unique enzyme lacking the catalytic tryptophan strictly conserved in all other LiPs and versatile peroxidases (more than 30 sequences available). Recombinant T. cervina LiP and site-directed variants were investigated by crystallographic, kinetic, and spectroscopic techniques. The crystal structure shows three substrate oxidation site candidates involving His-170, Asp-146, and Tyr-181. Steady-state kinetics for oxidation of veratryl alcohol (the typical LiP substrate) by variants at the above three residues reveals a crucial role of Tyr-181 in LiP activity. Moreover, assays with ferrocytochrome c show that its ability to oxidize large molecules (a requisite property for oxidation of the lignin polymer) originates in Tyr-181. This residue is also involved in the oxidation of 1,4-dimethoxybenzene, a reaction initiated by the one-electron abstraction with formation of substrate cation radical, as described for the well known Phanerochaete chrysosporium LiP. Detailed spectroscopic and kinetic investigations, including low temperature EPR, show that the porphyrin radical in the two-electron activated T. cervina LiP is unstable and rapidly receives one electron from Tyr-181, forming a catalytic protein radical, which is identified as an H-bonded neutral tyrosyl radical. The crystal structure reveals a partially exposed location of Tyr-181, compatible with its catalytic role, and several neighbor residues probably contributing to catalysis: (i) by enabling substrate recognition by aromatic interactions; (ii) by acting as proton acceptor/donor from Tyr-181 or H-bonding the radical form; and (iii) by providing the acidic environment that would facilitate oxidation. This is the first structure-function study of the only ligninolytic peroxidase described to date that has a catalytic tyrosine.     

Cytochrome P450-type hydroxylation and epoxidation in a tyrosine-liganded hemoprotein, catalase-related allene oxide synthase

The ability of hemoproteins to catalyze epoxidation or hydroxylation reactions is usually associated with a cysteine as the proximal ligand to the heme, as in cytochrome P450 or nitric oxide synthase. Catalase-related allene oxide synthase (cAOS) from the coral Plexaura homomalla, like catalase itself, has tyrosine as the proximal heme ligand. Its natural reaction is to convert 8R-hydroperoxy-eicosatetraenoic acid (8R-HPETE) to an allene epoxide, a reaction activated by the ferric heme, forming product via the Fe(IV)-OH intermediate, Compound II. Here we oxidized cAOS to Compound I (Fe(V)=O) using the oxygen donor iodosylbenzene and investigated the catalytic competence of the enzyme. 8R-hydroxyeicosatetraenoic acid (8R-HETE), the hydroxy analog of the natural substrate, normally unreactive with cAOS, was thereby epoxidized stereospecifically on the 9,10 double bond to form 8R-hydroxy-9R,10R-trans-epoxy-eicosa-5Z,11Z,14Z-trienoic acid as the predominant product; the turnover was 1/s using 100 μm iodosylbenzene. The enantiomer, 8S-HETE, was epoxidized stereospecifically, although with less regiospecificity, and was hydroxylated on the 13- and 16-carbons. Arachidonic acid was converted to two major products, 8R-HETE and 8R,9S-eicosatrienoic acid (8R,9S-EET), plus other chiral monoepoxides and bis-allylic 10S-HETE. Linoleic acid was epoxidized, whereas stearic acid was not metabolized. We conclude that when cAOS is charged with an oxygen donor, it can act as a stereospecific monooxygenase. Our results indicate that in the tyrosine-liganded cAOS, a catalase-related hemoprotein in which a polyunsaturated fatty acid can enter the active site, the enzyme has the potential to mimic the activities of typical P450 epoxygenases and some capabilities of P450 hydroxylases.     

Hydrogen peroxide elimination from C4a-hydroperoxyflavin in a flavoprotein oxidase occurs through a single proton transfer from flavin N5 to a peroxide leaving group

C4a-hydroperoxyflavin is found commonly in the reactions of flavin-dependent monooxygenases, in which it plays a key role as an intermediate that incorporates an oxygen atom into substrates. Only recently has evidence for its involvement in the reactions of flavoprotein oxidases been reported. Previous studies of pyranose 2-oxidase (P2O), an enzyme catalyzing the oxidation of pyranoses using oxygen as an electron acceptor to generate oxidized sugars and hydrogen peroxide (H(2)O(2)), have shown that C4a-hydroperoxyflavin forms in P2O reactions before it eliminates H(2)O(2) as a product (Sucharitakul, J., Prongjit, M., Haltrich, D., and Chaiyen, P. (2008) Biochemistry 47, 8485-8490). In this report, the solvent kinetic isotope effects (SKIE) on the reaction of reduced P2O with oxygen were investigated using transient kinetics. Our results showed that D(2)O has a negligible effect on the formation of C4a-hydroperoxyflavin. The ensuing step of H(2)O(2) elimination from C4a-hydroperoxyflavin was shown to be modulated by an SKIE of 2.8 ± 0.2, and a proton inventory analysis of this step indicates a linear plot. These data suggest that a single-proton transfer process causes SKIE at the H(2)O(2) elimination step. Double and single mixing stopped-flow experiments performed in H(2)O buffer revealed that reduced flavin specifically labeled with deuterium at the flavin N5 position generated kinetic isotope effects similar to those found with experiments performed with the enzyme pre-equilibrated in D(2)O buffer. This suggests that the proton at the flavin N5 position is responsible for the SKIE and is the proton-in-flight that is transferred during the transition state. The mechanism of H(2)O(2) elimination from C4a-hydroperoxyflavin is consistent with a single proton transfer from the flavin N5 to the peroxide leaving group, possibly via the formation of an intramolecular hydrogen bridge.     

Mannheimia haemolytica leukotoxin-induced increase in leukotriene B4 production by bovine neutrophils is mediated by a sustained and excessive increase in intracellular calcium concentration

Isolated bovine neutrophils were used to study the relationship between the duration and magnitude of the Mannheimia haemolytica leukotoxin-induced increase in intracellular calcium concentration and leukotriene B4 synthesis. In contrast to recombinant human C5a, which caused a transient, small increase in intracellular calcium concentration and no effects on leukotriene B4 synthesis, exposure of neutrophils to leukotoxin resulted in a rapid, sustained, large increase in intracellular calcium concentration, followed by leukotriene B4 synthesis. This leukotoxin-induced response was similar to those produced by the calcium ionophore, A23187, and phorbol myristate acetate, which also caused significant leukotriene B4 production. Manipulation of the duration and magnitude of leukotoxin- and A23187-induced intracellular calcium concentration increase confirmed that a high and sustained intracellular calcium concentration was necessary to stimulate production of leukotriene B4, which is believed to play an important role in the pathogenesis of pulmonary M. haemolytica infection.     

Rigidity of wedge loop in PACSIN 3 protein is a key factor in dictating diameters of tubules

BAR (Bin/amphiphysin/Rvs) domain-containing proteins participate in cellular membrane remodeling. The F-BAR proteins normally generate low curvature tubules. However, in the PACSIN subfamily, the F-BAR domain from PACSIN 1 and 2 can induce both high and low curvature tubules. We found that unlike PACSIN 1 and 2, PACSIN 3 could only induce low curvature tubules. To elucidate the key factors that dictate the tubule curvature, crystal structures of all three PACSIN F-BAR domains were determined. A novel type of lateral interaction mediated by a wedge loop is observed between the F-BAR neighboring dimers. Comparisons of the structures of PACSIN 3 with PACSIN 1 and 2 indicate that the wedge loop of PACSIN 3 is more rigid, which influences the lateral interactions between assembled dimers. We further identified the residues that affect the rigidity of the loop by mutagenesis and determined the structures of two PACSIN 3 wedge loop mutants. Our results suggest that the rigidity-mediated conformations of the wedge loop correlate well with the various crystal packing modes and membrane tubulations. Thus, the rigidity of the wedge loop is a key factor in dictating tubule diameters.     

Lysine221 is the general base residue of the isochorismate synthase from Pseudomonas aeruginosa (PchA) in a reaction that is diffusion limited

The isochorismate synthase from Pseudomonas aeruginosa (PchA) catalyzes the conversion of chorismate to isochorismate, which is subsequently converted by a second enzyme (PchB) to salicylate for incorporation into the salicylate-capped siderophore pyochelin. PchA is a member of the MST family of enzymes, which includes the structurally homologous isochorismate synthases from Escherichia coli (EntC and MenF) and salicylate synthases from Yersinia enterocolitica (Irp9) and Mycobacterium tuberculosis (MbtI). The latter enzymes generate isochorismate as an intermediate before generating salicylate and pyruvate. General acid–general base catalysis has been proposed for isochorismate synthesis in all five enzymes, but the residues required for the isomerization are a matter of debate, with both lysine221 and glutamate313 proposed as the general base (PchA numbering). This work includes a classical characterization of PchA with steady state kinetic analysis, solvent kinetic isotope effect analysis and by measuring the effect of viscosogens on catalysis. The results suggest that isochorismate production from chorismate by the MST enzymes is the result of general acid–general base catalysis with a lysine as the base and a glutamic acid as the acid, in reverse protonation states. Chemistry is determined to not be rate limiting, favoring the hypothesis of a conformational or binding step as the slow step.     

Stabilization of C4a-hydroperoxyflavin in a two-component flavin-dependent monooxygenase is achieved through interactions at flavin N5 and C4a atoms

p-Hydroxyphenylacetate (HPA) 3-hydroxylase is a two-component flavin-dependent monooxygenase. Based on the crystal structure of the oxygenase component (C₂), His-396 is 4.5 Å from the flavin C4a locus, whereas Ser-171 is 2.9 Å from the flavin N5 locus. We investigated the roles of these two residues in the stability of the C4a-hydroperoxy-FMN intermediate. The results indicated that the rate constant for C4a-hydroperoxy-FMN formation decreased ∼30-fold in H396N, 100-fold in H396A, and 300-fold in the H396V mutant, compared with the wild-type enzyme. Lesser effects of the mutations were found for the subsequent step of H₂O₂ elimination. Studies on pH dependence showed that the rate constant of H₂O₂ elimination in H396N and H396V increased when pH increased with pK_a >9.6 and >9.7, respectively, similar to the wild-type enzyme (pK_a >9.4). These data indicated that His-396 is important for the formation of the C4a-hydroperoxy-FMN intermediate but is not involved in H₂O₂ elimination. Transient kinetics of the Ser-171 mutants with oxygen showed that the rate constants for the H₂O₂ elimination in S171A and S171T were ∼1400-fold and 8-fold greater than the wild type, respectively. Studies on the pH dependence of S171A with oxygen showed that the rate constant of H₂O₂ elimination increased with pH rise and exhibited an approximate pK_a of 8.0. These results indicated that the interaction of the hydroxyl group side chain of Ser-171 and flavin N5 is required for the stabilization of C4a-hydroperoxy-FMN. The double mutant S171A/H396V reacted with oxygen to directly form the oxidized flavin without stabilizing the C4a-hydroperoxy-FMN intermediate, which confirmed the findings based on the single mutation that His-396 was important for formation and Ser-171 for stabilization of the C4a-hydroperoxy-FMN intermediate in C₂.     

Glutathione degradation by the alternative pathway (DUG pathway) in Saccharomyces cerevisiae is initiated by (Dug2p-Dug3p)2 complex, a novel glutamine amidotransferase (GATase) enzyme acting on glutathione

The recently identified, fungi-specific alternative pathway of glutathione degradation requires the participation of three genes, DUG1, DUG2, and DUG3. Dug1p has earlier been shown to function as a Cys-Gly-specific dipeptidase. In the present study, we describe the characterization of Dug2p and Dug3p. Dug3p has a functional glutamine amidotransferase (GATase) II domain that is catalytically important for glutathione degradation as demonstrated through mutational analysis. Dug2p, which has an N-terminal WD40 and a C-terminal M20A peptidase domain, has no peptidase activity. The previously demonstrated Dug2p-Dug3p interaction was found to be mediated through the WD40 domain of Dug2p. Dug2p was also shown to be able to homodimerize, and this was mediated by its M20A peptidase domain. In vitro reconstitution assays revealed that Dug2p and Dug3p were required together for the cleavage of glutathione into glutamate and Cys-Gly. Purification through gel filtration chromatography confirmed the formation of a Dug2p-Dug3p complex. The functional complex had a molecular weight that corresponded to (Dug2p-Dug3p)(2) in addition to higher molecular weight oligomers and displayed Michaelis-Menten kinetics. (Dug2p-Dug3p)(2) had a K(m) for glutathione of 1.2 mm, suggesting a novel GATase enzyme that acted on glutathione. Dug1p activity in glutathione degradation was found to be restricted to its Cys-Gly peptidase activity, which functioned downstream of the (Dug2p-Dug3p)(2) GATase. The DUG2 and DUG3 genes, but not DUG1, were derepressed by sulfur limitation. Based on these studies and the functioning of GATases, a mechanism is proposed for the functioning of the Dug proteins in the degradation of glutathione.