Alkyl gallates inhibit serine O-acetyltransferase in bacteria and enhance susceptibility of drug-resistant Gram-negative bacteria to antibiotics.

A principal concept in developing antibacterial agents with selective toxicity is blocking metabolic pathways that are critical for bacterial growth but that mammalian cells lack. Serine O-acetyltransferase (CysE) is an enzyme in many bacteria that catalyzes the first step in l-cysteine biosynthesis by transferring an acetyl group from acetyl coenzyme A (acetyl-CoA) to l-serine to form O-acetylserine. Because mammalian cells lack this l-cysteine biosynthesis pathway, developing an inhibitor of CysE has been thought to be a way to establish a new class of antibacterial agents. Here, we demonstrated that alkyl gallates such as octyl gallate (OGA) could act as potent CysE inhibitors in vitro and in bacteria. Mass spectrometry analyses indicated that OGA treatment markedly reduced intrabacterial levels of l-cysteine and its metabolites including glutathione and glutathione persulfide in Escherichia coli to a level similar to that found in E. coli lacking the cysE gene. Consistent with the reduction of those antioxidant molecules in bacteria, E. coli became vulnerable to hydrogen peroxide-mediated bacterial killing in the presence of OGA. More important, OGA treatment intensified susceptibilities of metallo-ß-lactamase-expressing Gram-negative bacteria (E. coli and Klebsiella pneumoniae) to carbapenem. Structural analyses showed that alkyl gallate bound to the binding site for acetyl-CoA that limits access of acetyl-CoA to the active site. Our data thus suggest that CysE inhibitors may be used to treat infectious diseases caused by drug-resistant Gram-negative bacteria not only via direct antibacterial activity but also by enhancing therapeutic potentials of existing antibiotics.

Killing Two Birds with One Stone: Discovery of Dual Inhibitors of Oxygen and Fumarate Respiration in Zoonotic Parasite, Echinococcus multilocularis.

Ascofuranone (AF), a meroterpenoid isolated from various filamentous fungi, including Acremonium egyptiacum, has been reported as a potential lead candidate for drug development against parasites and cancer. In this study, we demonstrated that AF and its derivatives are potent anthelminthic agents, particularly against Echinococcus multilocularis, which is the causative agent of alveolar echinococcosis. We measured the inhibitory activities of AF and its derivatives on the mitochondrial aerobic and anaerobic respiratory systems of E. multilocularis larvae. Several derivatives inhibited complex II (succinate:quinone reductase [SQR]; IC50 = 0.037 to 0.135 µM) and also complex I to III (NADH:cytochrome c reductase; IC50 = 0.008 to 0.401 µM), but not complex I (NADH:quinone reductase), indicating that mitochondrial complexes II and III are the targets. In particular, complex II inhibition in the anaerobic pathway was notable because E. multilocularis employs NADH:fumarate reductase (fumarate respiration), in addition to NADH oxidase (oxygen respiration), resulting in complete shutdown of ATP synthesis by oxidative phosphorylation. A structure-activity relationship study of E. multilocularis complex II revealed that the functional groups of AF are essential for inhibition. Binding mode prediction of AF derivatives to complex II indicated potential hydrophobic and hydrogen bond interactions between AF derivatives and amino acid residues within the quinone binding site. Ex vivo culture assays revealed that AF derivatives progressively reduced the viability of protoscoleces under both aerobic and anaerobic conditions. These findings confirm that AF and its derivatives are the first dual inhibitors of fumarate and oxygen respiration in E. multilocularis and are potential lead compounds in the development of anti-echinococcal drugs.

Dephospho-Coenzyme A Kinase Is an Exploitable Drug Target against Plasmodium falciparum: Identification of Selective Inhibitors by High-Throughput Screening of a Large Chemical Compound Library.

Malaria is a mosquito-borne fatal infectious disease that affects humans and is caused by Plasmodium parasites, primarily Plasmodium falciparum. Widespread drug resistance compels us to discover novel compounds and alternative drug discovery targets. The coenzyme A (CoA) biosynthesis pathway is essential for the malaria parasite P. falciparum. The last enzyme in CoA biosynthesis, dephospho-CoA kinase (DPCK), is essential to the major life cycle development stages but has not yet been exploited as a drug target in antimalarial drug discovery. We performed a high-throughput screen of a 210,000-compound library using recombinant P. falciparum DPCK (PfDPCK). A high-throughput enzymatic assay using a 1,536-well platform was developed to identify potential PfDPCK inhibitors. PfDPCK inhibitors also inhibited parasite growth in a P. falciparum whole-cell asexual blood-stage assay in both drug-sensitive and drug-resistant strains. Hit compounds were selected based on their potency in cell-free (PfDPCK) and whole-cell (Pf3D7 and PfDd2) assays, selectivity over the human orthologue (HsCOASY) and no cytotoxicity (HepG2). The compounds were ranked using a multiparameter optimization (MPO) scoring model, and the specific binding and the mechanism of inhibition were investigated for the most promising compounds.

Characterization and application of l-methionine γ-lyase Q349S mutant enzyme with an enhanced activity toward l-homocysteine.

l-Methionine γ-lyse (MGL), a pyridoxal 5'-phosphate-dependent enzyme, catalyzes the α,γ-elimination of l-methionine (l-Met) and l-homocysteine (l-Hcy) to produce α-keto acids, thiols, and ammonia. Previously, various mutant enzymes of Pseudomonas putida MGL (PpMGL) were prepared to identify a homocysteine (Hcy)-specific enzyme that would assist the diagnosis of homocystinuria. Among the mutat enzymes the Q349S mutant exhibited high degradation activity toward l-Hcy. In the present study, PpMGL Q349S was characterized; the results suggested that it could be applied to determine the amount of l-Hcy. Compared to the wild-type PpMGL, specific activities of the Q349S mutant with l-Hcy and l-Met were 1.5 and 0.7 times, respectively. Additionally, we confirmed that l-Hcy in plasma samples could be accurately detected using the Q349S mutant by preincubating it with cysteine desulfurase (CsdA). Furthermore, we determined the X-ray crystal structure of PpMGL Q349S l-Met or l-Hcy complexes Michaelis complex, germinal diamine, and external aldimine at 2.25-2.40 Å. These 3D structures showed that the interaction partner of the ß-hydroxyl group of Thr355 in the wild-type PpMGL was changed to the carboxyl group of the Hcy-PLP external aldimine in the Q349S mutant. The interaction of Ser349 and Arg375 was different between l-Met and l-Hcy recognition, indicating that it was important for the recognition of the carboxyl group of the substrate.

The ASCT/SCS cycle fuels mitochondrial ATP and acetate production in Trypanosoma brucei.

Acetate:succinate CoA transferase (ASCT) is a mitochondrial enzyme that catalyzes the production of acetate and succinyl-CoA, which is coupled to ATP production with succinyl-CoA synthetase (SCS) in a process called the ASCT/SCS cycle. This cycle has been studied in Trypanosoma brucei (T. brucei), a pathogen of African sleeping sickness, and is involved in (i) ATP and (ii) acetate production and proceeds independent of oxygen and an electrochemical gradient. Interestingly, knockout of ASCT in procyclic form (PCF) of T. brucei cause oligomycin A-hypersensitivity phenotype indicating that ASCT/SCS cycle complements the deficiency of ATP synthase activity. In bloodstream form (BSF) of T. brucei, ATP synthase works in reverse to maintain the electrochemical gradient by hydrolyzing ATP. However, no information has been available on the source of ATP, although ASCT/SCS cycle could be a potential candidate. Regarding mitochondrial acetate production, which is essential for fatty acid biosynthesis and growth of T. brucei, ASCT or acetyl-CoA hydrolase (ACH) are known to be its source. Despite the importance of this cycle, direct evidence of its function is lacking, and there are no comprehensive biochemical or structural biology studies reported so far. Here, we show that in vitro-reconstituted ASCT/SCS cycle is highly specific towards acetyl-CoA and has a higher kcat than that of yeast and bacterial ATP synthases. Our results provide the first biochemical basis for (i) rescue of ATP synthase-deficient phenotype by ASCT/SCS cycle in PCF and (ii) a potential source of ATP for the reverse reaction of ATP synthase in BSF.

Selective Cytotoxicity of Dihydroorotate Dehydrogenase Inhibitors to Human Cancer Cells Under Hypoxia and Nutrient-Deprived Conditions.

Human dihydroorotate dehydrogenase (HsDHODH) is a key enzyme of pyrimidine de novo biosynthesis pathway. It is located on the mitochondrial inner membrane and contributes to the respiratory chain by shuttling electrons to the ubiquinone pool. We have discovered ascofuranone (1), a natural compound produced by Acremonium sclerotigenum, and its derivatives are a potent class of HsDHODH inhibitors. We conducted a structure-activity relationship study and have identified functional groups of 1 that are essential for the inhibition of HsDHODH enzymatic activity. Furthermore, the binding mode of 1 and its derivatives to HsDHODH was demonstrated by co-crystallographic analysis and we show that these inhibitors bind at the ubiquinone binding site. In addition, the cytotoxicities of 1 and its potent derivatives 7, 8, and 9 were studied using human cultured cancer cells. Interestingly, they showed selective and strong cytotoxicity to cancer cells cultured under microenvironment (hypoxia and nutrient-deprived) conditions. The selectivity ratio of 8 under this microenvironment show the most potent inhibition which was over 1000-fold higher compared to that under normal culture condition. Our studies suggest that under microenvironment conditions, cancer cells heavily depend on the pyrimidine de novo biosynthesis pathway. We also provide the first evidence that 1 and its derivatives are potential lead candidates for drug development which target the HsDHODH of cancer cells living under a tumor microenvironment.

Glycerol kinase of African trypanosomes possesses an intrinsic phosphatase activity.

BACKGROUND: In general, glycerol kinases (GKs) are transferases that catalyze phospho group transfer from ATP to glycerol, and the mechanism was suggested to be random bi-bi. The reverse reaction i.e. phospho transfer from glycerol 3-phosphate (G3P) to ADP is only physiologically feasible by the African trypanosome GK. In contrast to other GKs the mechanism of Trypanosoma brucei gambiense glycerol kinase (TbgGK) was shown to be in an ordered fashion, and proceeding via autophosphorylation. From the unique reaction mechanism of TbgGK, we envisaged its potential to possess phosphatase activity in addition to being a kinase. METHODS: Our hypothesis was tested by spectrophotometric and LC-MS/MS analyses using paranitrophenyl phosphate (pNPP) and TbgGK's natural substrate, G3P respectively. Furthermore, protein X-ray crystallography and site-directed mutagenesis were performed to examine pNPP binding, catalytic residues, and the possible reaction mechanism. RESULTS: In addition to its widely known and expected phosphotransferase (class II) activity, TbgGK can efficiently facilitate the hydrolytic cleavage of phosphoric anhydride bonds (a class III property). This phosphatase activity followed the classical Michaelis-Menten pattern and was competitively inhibited by ADP and G3P, suggesting a common catalytic site for both activities (phosphatase and kinase). The structure of the TGK-pNPP complex, and structure-guided mutagenesis implicated T276 to be important for the catalysis. Remarkably, we captured a crystallographic molecular snapshot of the phosphorylated T276 reaction intermediate. CONCLUSION: We conclude that TbgGK has both kinase and phosphatase activities. GENERAL SIGNIFICANCE: This is the first report on a bifunctional kinase/phosphatase enzyme among members of the sugar kinase family.

Structural and mechanistic insights into homocysteine degradation by a mutant of methionine γ-lyase based on substrate-assisted catalysis.

Methionine γ-lyse (MGL) catalyzes the α, γ-elimination of l-methionine and its derivatives as well as the α, ß-elimination of l-cysteine and its derivatives to produce α-keto acids, volatile thiols, and ammonia. The reaction mechanism of MGL has been characterized by enzymological studies using several site-directed mutants. The Pseudomonas putida MGL C116H mutant showed drastically reduced degradation activity toward methionine while retaining activity toward homocysteine. To understand the underlying mechanism and to discern the subtle differences between these substrates, we analyzed the crystal structures of the reaction intermediates. The complex formed between the C116H mutant and methionine demonstrated that a loop structure (Ala51-Asn64) in the adjacent subunit of the catalytic dimer cannot approach the cofactor pyridoxal 5'-phosphate (PLP) because His116 disrupts the interaction of Asp241 with Lys240, and the liberated side chain of Lys240 causes steric hindrance with this loop. Conversely, in the complex formed between C116H mutant and homocysteine, the thiol moiety of the substrate conjugated with PLP offsets the imidazole ring of His116 via a water molecule, disrupting the interaction of His116 and Asp241 and restoring the interaction of Asp241 with Lys240. These structural data suggest that the Cys116 to His mutation renders the enzyme inactive toward the original substrate, but activity is restored when the substrate is homocysteine due to substrate-assisted catalysis.

The hyperthermophilic cystathionine γ-synthase from the aerobic crenarchaeon Sulfolobus tokodaii: expression, purification, crystallization and structural insights.

Cystathionine γ-synthase (CGS; EC 2.5.1.48), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the formation of cystathionine from an L-homoserine derivative and L-cysteine in the first step of the transsulfuration pathway. Recombinant CGS from the thermoacidophilic archaeon Sulfolobus tokodaii (StCGS) was overexpressed in Escherichia coli and purified to homogeneity by heat treatment followed by hydroxyapatite and gel-filtration column chromatography. The purified enzyme shows higher enzymatic activity at 353 K under basic pH conditions compared with that at 293 K. Crystallization trials yielded three crystal forms from different temperature and pH conditions. Form I crystals (space group P21; unit-cell parameters a = 58.4, b = 149.3, c = 90.2 Å, ß = 108.9°) were obtained at 293 K under acidic pH conditions using 2-methyl-2,4-pentanediol as a precipitant, whereas under basic pH conditions the enzyme crystallized in form II at 293 K (space group C2221; unit-cell parameters a = 117.7, b = 117.8, c = 251.3 Å) and in form II' at 313 K (space group C2221; unit-cell parameters a = 107.5, b = 127.7, c = 251.1 Å) using polyethylene glycol 3350 as a precipitant. X-ray diffraction data were collected to 2.2, 2.9 and 2.7 Šresolution for forms I, II and II', respectively. Structural analysis of these crystal forms shows that the orientation of the bound PLP in form II is significantly different from that in form II', suggesting that the change in orientation of PLP with temperature plays a role in the thermophilic enzymatic activity of StCGS.

Structural Insights into the Molecular Design of Flutolanil Derivatives Targeted for Fumarate Respiration of Parasite Mitochondria.

Recent studies on the respiratory chain of Ascaris suum showed that the mitochondrial NADH-fumarate reductase system composed of complex I, rhodoquinone and complex II plays an important role in the anaerobic energy metabolism of adult A. suum. The system is the major pathway of energy metabolism for adaptation to a hypoxic environment not only in parasitic organisms, but also in some types of human cancer cells. Thus, enzymes of the pathway are potential targets for chemotherapy. We found that flutolanil is an excellent inhibitor for A. suum complex II (IC50 = 0.058 µM) but less effectively inhibits homologous porcine complex II (IC50 = 45.9 µM). In order to account for the specificity of flutolanil to A. suum complex II from the standpoint of structural biology, we determined the crystal structures of A. suum and porcine complex IIs binding flutolanil and its derivative compounds. The structures clearly demonstrated key interactions responsible for its high specificity to A. suum complex II and enabled us to find analogue compounds, which surpass flutolanil in both potency and specificity to A. suum complex II. Structures of complex IIs binding these compounds will be helpful to accelerate structure-based drug design targeted for complex IIs.

Molecular basis for the reverse reaction of African human trypanosomes glycerol kinase.

The glycerol kinase (GK) of African human trypanosomes is compartmentalized in their glycosomes. Unlike the host GK, which under physiological conditions catalyzes only the forward reaction (ATP-dependent glycerol phosphorylation), trypanosome GK can additionally catalyze the reverse reaction. In fact, owing to this unique reverse catalysis, GK is potentially essential for the parasites survival in the human host, hence a promising drug target. The mechanism of its reverse catalysis was unknown; therefore, it was not clear if this ability was purely due to its localization in the organelles or whether structure-based catalytic differences also contribute. To investigate this lack of information, the X-ray crystal structure of this protein was determined up to 1.90 Å resolution, in its unligated form and in complex with three natural ligands. These data, in conjunction with results from structure-guided mutagenesis suggests that the trypanosome GK is possibly a transiently autophosphorylating threonine kinase, with the catalytic site formed by non-conserved residues. Our results provide a series of structural peculiarities of this enzyme, and gives unexpected insight into the reverse catalysis mechanism. Together, they provide an encouraging molecular framework for the development of trypanosome GK-specific inhibitors, which may lead to the design of new and safer trypanocidal drug(s).

Structures of cyanobacteriochromes from phototaxis regulators AnPixJ and TePixJ reveal general and specific photoconversion mechanism.

Cyanobacteriochromes are cyanobacterial tetrapyrrole-binding photoreceptors that share a bilin-binding GAF domain with photoreceptors of the phytochrome family. Cyanobacteriochromes are divided into many subclasses with distinct spectral properties. Among them, putative phototaxis regulators PixJs of Anabaena sp. PCC 7120 and Thermosynechococcus elongatus BP-1 (denoted as AnPixJ and TePixJ, respectively) are representative of subclasses showing red-green-type and blue/green-type reversible photoconversion, respectively. Here, we determined crystal structures for the AnPixJ GAF domain in its red-absorbing 15Z state (Pr) and the TePixJ GAF domain in its green-absorbing 15E state (Pg). The overall structure of these proteins is similar to each other and also similar to known phytochromes. Critical differences found are as follows: (i) the chromophore of AnPixJ Pr is phycocyanobilin in a C5-Z,syn/C10-Z,syn/C15-Z,anti configuration and that of TePixJ Pg is phycoviolobilin in a C10-Z,syn/C15-E,anti configuration, (ii) a side chain of the key aspartic acid is hydrogen bonded to the tetrapyrrole rings A, B and C in AnPixJ Pr and to the pyrrole ring D in TePixJ Pg, (iii) additional protein-chromophore interactions are provided by subclass-specific residues including tryptophan in AnPixJ and cysteine in TePixJ. Possible structural changes following the photoisomerization of the chromophore between C15-Z and C15-E are discussed based on the X-ray structures at 1.8 and 2.0-Å resolution, respectively, in two distinct configurations.

The role of amino acid residues in the active site of L-methionine γ-lyase from Pseudomonas putida.

Cys116, Lys240*, and Asp241* (asterisks indicate residues from the second subunit of the active dimer) at the active site of L-methionine γ-lyase of Pseudomonas putida (MGL_Pp) are highly conserved among heterologous MGLs. In a previous study, we found that substitution of Cys116 for His led to a drastic increase in activity toward L-cysteine and a decrease in that toward L-methionine. In this study, we examined some properties of the C116H mutant by kinetic analysis and 3D structural analysis. We assumed that substitution of Cys116 for His broke the original hydrogen-bond network and that this induced a significant effect of Tyr114 as a general acid catalyst, possibly due to the narrow space in the active site. The C116H mutant acquired a novel ß-elimination activity and lead a drastic conformation change in the histidine residue at position 116 by binding the substrate, suggesting that this His residue affects the reaction specificity of C116H. Furthermore, we suggest that Lys240* is important for substrate recognition and structural stability and that Asp241* is also involved in substrate specificity in the elimination reaction. Based on this, we suggest that the hydrogen-bond network among Cys116, Lys240*, and Asp241* contributes to substrate specificity that is, to L-methionine recognition at the active site in MGL_Pp.

X-ray crystal structure of the light-independent protochlorophyllide reductase.

Photosynthetic organisms adopt two different strategies for the reduction of the C17 = C18 double bond of protochlorophyllide (Pchlide) to form chlorophyllide a, the direct precursor of chlorophyll a (refs 1-4). The first involves the activity of the light-dependent Pchlide oxidoreductase, and the second involves the light-independent (dark-operative) Pchlide oxidoreductase (DPOR). DPOR is a nitrogenase-like enzyme consisting of two components, L-protein (a BchL dimer) and NB-protein (a BchN-BchB heterotetramer), which are structurally related to nitrogenase Fe protein and MoFe protein, respectively. Here we report the crystal structure of the NB-protein of DPOR from Rhodobacter capsulatus at a resolution of 2.3A. As expected, the overall structure is similar to that of nitrogenase MoFe protein: each catalytic BchN-BchB unit contains one Pchlide and one iron-sulphur cluster (NB-cluster) coordinated uniquely by one aspartate and three cysteines. Unique aspartate ligation is not necessarily needed for the cluster assembly but is essential for the catalytic activity. Specific Pchlide-binding accompanies the partial unwinding of an alpha-helix that belongs to the next catalytic BchN-BchB unit. We propose a unique trans-specific reduction mechanism in which the distorted C17-propionate of Pchlide and an aspartate from BchB serve as proton donors for C18 and C17 of Pchlide, respectively. Intriguingly, the spatial arrangement of the NB-cluster and Pchlide is almost identical to that of the P-cluster and FeMo-cofactor in nitrogenase MoFe-protein, illustrating that a common architecture exists to reduce chemically stable multibonds of porphyrin and dinitrogen.

Structural basis of carbohydrate transfer activity by human UDP-GalNAc: polypeptide alpha-N-acetylgalactosaminyltransferase (pp-GalNAc-T10).

Mucin-type O-glycans are important carbohydrate chains involved in differentiation and malignant transformation. Biosynthesis of the O-glycan is initiated by the transfer of N-acetylgalactosamine (GalNAc) which is catalyzed by UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (pp-GalNAc-Ts). Here we present crystal structures of the pp-GalNAc-T10 isozyme, which has specificity for glycosylated peptides, in complex with the hydrolyzed donor substrate UDP-GalNAc and in complex with GalNAc-serine. A structural comparison with uncomplexed pp-GalNAc-T1 suggests that substantial conformational changes occur in two loops near the catalytic center upon donor substrate binding, and that a distinct interdomain arrangement between the catalytic and lectin domains forms a narrow cleft for acceptor substrates. The distance between the catalytic center and the carbohydrate-binding site on the lectin beta sub-domain influences the position of GalNAc glycosylation on GalNAc-glycosylated peptide substrates. A chimeric enzyme in which the two domains of pp-GalNAc-T10 are connected by a linker from pp-GalNAc-T1 acquires activity toward non-glycosylated acceptors, identifying a potential mechanism for generating the various acceptor specificities in different isozymes to produce a wide range of O-glycans.

Structure determination of GGA-GAE and gamma1-ear in complex with peptides: crystallization of low-affinity complexes in membrane traffic.

Crystallization of protein-protein complexes is an important step in the studies of biological functions of proteins. However, weak and transient, even though specific, interactions often present difficulties in crystallization of protein complexes due to the heterogeneity of the sample mixture. For example, the gamma1-ear domain of the AP-1 complex and the GAE domain of GGA1, responsible for the interaction with accessory proteins involved in vesicular transport, are known to interact with target proteins with affinities of the order of 1-100 microM. Such low affinities have hampered crystallization trials of the complexes. To overcome this problem, the gamma1-ear and GAE domains were first co-crystallized with excess amounts of the peptides. Co-crystals of both domains were obtained and the complex structures were determined at 2.5-2.9 A resolution. Based on the crystal packing of gamma1-ear and the cognate peptide, gamma1-ear fused with a peptide tag at the N-terminus was prepared. The peptide-tagged gamma1-ear readily crystallized and the crystal diffracted far better, 1.9-2.2 A resolution, compared with the co-crystallized complex, giving significantly more details without affecting the overall complex structure.