Functional Identification of APIP as Human mtnB,a Key Enzyme in the Methionine Salvage Pathway

The methionine salvage pathway is widely distributed among some eubacteria, yeast, plants and animals and recycles the sulfur-containing metabolite 5-methylthioadenosine (MTA) to methionine. In eukaryotic cells, the methionine salvage pathway takes place in the cytosol and usually involves six enzymatic activities: MTA phosphorylase (MTAP, EC 2.4.2.28), 5′-methylthioribose-1-phosphate isomerase (mtnA, EC 5.3.1.23), 5′-methylthioribulose-1-phosphate dehydratase (mtnB, EC: 4.2.1.109), 2,3-dioxomethiopentane-1-phosphate enolase/phosphatase (mtnC, EC 3.1.3.77), aci-reductone dioxygenase (mtnD, EC 1.13.11.54) and 4-methylthio-2-oxo-butanoate (MTOB) transaminase (EC 2.6.1.-). The aim of this study was to complete the available information on the methionine salvage pathway in human by identifying the enzyme responsible for the dehydratase step. Using a bioinformatics approach, we propose that a protein called APIP could perform this role. The involvement of this protein in the methionine salvage pathway was investigated directly in HeLa cells by transient and stable short hairpin RNA interference. We show that APIP depletion specifically impaired the capacity of cells to grow in media where methionine is replaced by MTA. Using a Shigella mutant auxotroph for methionine, we confirm that the knockdown of APIP specifically affects the recycling of methionine. We also show that mutation of three potential phosphorylation sites does not affect APIP activity whereas mutation of the potential zinc binding site completely abrogates it. Finally, we show that the N-terminal region of APIP that is missing in the short isoform is required for activity. Together, these results confirm the involvement of APIP in the methionine salvage pathway, which plays a key role in many biological functions like cancer, apoptosis, microbial proliferation and inflammation.     

2-Keto-4-methylthiobutyrate, an intermediate in the methionine salvage pathway, is a good substrate for CtBP1

In the present work, we have studied the kinetic properties of the catalytic domain of CtBP1, a co-repressor belonging to the d-2-hydroxyacid dehydrogenase family and known to reduce pyruvate in the presence of NADH. CtBP1 acted on a variety of alpha-keto acids, for which it displayed biphasic curves with inhibition at elevated concentrations, as observed with other dehydrogenases of the same family. Based on catalytic efficiencies, the best substrate was 2-keto-4-methylthiobutyrate, an intermediate of the methionine salvage pathway. It was about 20-fold better than 2-ketoisocaproate and glyoxylate, and 80-fold better than pyruvate. From these data we conclude that 2-keto-4-methylthiobutyrate may be an important regulator of CtBP activity, possibly linking gene repression to the activity of the methionine salvage and spermine synthesis pathways.     

1+1?=?3: A Fusion of 2 Enzymes in the Methionine Salvage Pathway of Tetrahymena thermophila Creates a Trifunctional Enzyme That Catalyzes 3 Steps in the Pathway

The methionine salvage pathway is responsible for regenerating methionine from its derivative, methylthioadenosine. The complete set of enzymes of the methionine pathway has been previously described in bacteria. Despite its importance, the pathway has only been fully described in one eukaryotic organism, yeast. Here we use a computational approach to identify the enzymes of the methionine salvage pathway in another eukaryote, Tetrahymena thermophila. In this organism, the pathway has two fused genes, MTNAK and MTNBD. Each of these fusions involves two different genes whose products catalyze two different single steps of the pathway in other organisms. One of the fusion proteins, mtnBD, is formed by enzymes that catalyze non-consecutive steps in the pathway, mtnB and mtnD. Interestingly the gene that codes for the intervening enzyme in the pathway, mtnC, is missing from the genome of Tetrahymena. We used complementation tests in yeast to show that the fusion of mtnB and mtnD from Tetrahymena is able to do in one step what yeast does in three, since it can rescue yeast knockouts of mtnB, mtnC, or mtnD. Fusion genes have proved to be very useful in aiding phylogenetic reconstructions and in the functional characterization of genes. Our results highlight another characteristic of fusion proteins, namely that these proteins can serve as biochemical shortcuts, allowing organisms to completely bypass steps in biochemical pathways.     

Purification and Characterization of Methionine:Glyoxylate Aminotransferase from Brassica carinata and Brassica napus

The first step in the biosynthesis of allylglucosinolate from methionine in Brassica is thought to be the transamination of methionine to 2-keto-4-methylthiobutyrate. By using Q-Sepharose and Red Agarose, followed by high resolution anion exchange chromatography and chromatofocussing, a methionine:glyoxylate aminotransferase (MGAT) was purified to homogeneity from leaves of Brassica carinata var R-4218, and approximately 5000-fold from leaves of Brassica napus var Topas. The final purification was accomplished using nondenaturing polyacrylamide gel electrophoresis. The enzyme has a pl of 4.3, a native molecular mass of 230 to 290 kilodaltons, and a subunit molecular mass of approximately 50 kilodaltons. Four isozymes of the enzyme were identified in the six species of Brassica commonly cultivated. Nonglucosinolate producing species had only low levels of MGAT or an MGAT isozyme which was distinctly different from that in Brassica.     

A complete inventory of all enzymes in the eukaryotic methionine salvage pathway

The methionine salvage pathway is universally used to regenerate methionine from 5'-methylthioadenosine, a byproduct of certain reactions involving S-adenosylmethionine. We identified and verified the genes encoding the enzymes of all steps in this cycle in a commonly used eukaryotic model system: the yeast Saccharomyces cerevisiae. The genes encoding 5'-methylthioribose-1-phosphate isomerase and 5'-methylthioribulose-1-phosphate dehydratase are herein named MRI1 and MDE1, respectively. The 5'-methylthioadenosine phosphorylase was verified as Meu1p, the 2,3-dioxomethiopentane-1-phosphate enolase/phosphatase as Utr4p and the aci-reductone dioxygenase as Adi1p. The homologue of the enolase/phosphatase gene, YNL010w, was excluded from its candidate role in the cycle. The methodology used involved auxotrophic growth tests and analysis of intracellular 5'-methylthioadenosine in deletion mutants. The last step, a transamination of 4-methylthio-2-oxobutyrate to yield methionine, was found to be a highly redundant step. It was catalysed by amino acid transaminases, mainly coupled with aromatic and branched chain amino acids as amino donors, but also with proline, lysine and glutamate/glutamine. The aromatic amino acid transaminases, Aro8p and Aro9p, and the branched chain amino acid transaminases, Bat1p and Bat2p, seemed to be the main enzymes exhibiting 4-methylthio-2-oxobutyrate transaminase activity. Bat2p was found to be less specific and used proline, lysine, tyrosine and glutamate as amino donors in addition to the branched chain amino acids. Thus, for the first time, all enzymes of the methionine salvage pathway were identified in a eukaryote.     

Analogs of 1-phosphonooxy-2,2-dihydroxy-3-oxo-5-(methylthio)pentane, an acyclic intermediate in the methionine salvage pathway: a new preparation and characterization of activity with E1 enolase/phosphatase from Klebsiella oxytoca

The methionine salvage pathway allows the in vivo recovery of the methylthio moiety of methionine upon the formation of methylthioadenosine (MTA) from S-adenosylmethionine (SAM). The Fe(II)-containing form of acireductone dioxygenase (ARD) catalyzes the penultimate step in the pathway in Klebsiella oxytoca, the oxidative cleavage of the acireductone 1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene (2) by dioxygen to give formate and 2-oxo-4-(methylthio)butyrate (3). The Ni(II)-bound form (Ni-ARD) catalyzes an off-pathway shunt, forming 3-(methylthio)propionate (4), carbon monoxide, and formate. Acireductone 2 is formed by the action of another enzyme, E1 enolase/phosphatase, on precursor 1-phosphonooxy-2,2-dihydroxy-3-oxo-5-methylthiopentane (1). Simple syntheses of several analogs of 1 are described, and their activity as substrates for E1 enolase/phosphatase characterized. A new bacterial overexpression system and purification procedure for E1, a member of the haloacid dehalogenase (HAD) superfamily, is described, and further characterization of the enzyme presented.     

Roles of RubisCO and the RubisCO-Like Protein in 5-Methylthioadenosine Metabolism in the Nonsulfur Purple Bacterium Rhodospirillum rubrum

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the assimilation of atmospheric CO₂ into organic matter and is thus central to the existence of life on earth. The beginning of the 2000s was marked by the discovery of a new family of proteins, the RubisCO-like proteins (RLPs), which are structural homologs of RubisCO. RLPs are unable to catalyze CO₂ fixation. The RLPs from Chlorobaculum tepidum, Bacillus subtilis, Geobacillus kaustophilus, and Microcystis aeruginosa have been shown to participate in sulfur metabolism. Whereas the precise function of C. tepidum RLP is unknown, the B. subtilis, G. kaustophilus, and M. aeruginosa RLPs function as tautomerases/enolases in a methionine salvage pathway (MSP). Here, we show that the form II RubisCO enzyme from the nonsulfur purple bacterium Rhodospirillum rubrum is also able to function as an enolase in vivo as part of an MSP, but only under anaerobic conditions. However, unlike B. subtilis RLP, R. rubrum RLP does not catalyze the enolization of 2,3-diketo-5-methylthiopentyl-1-phosphate. Instead, under aerobic growth conditions, R. rubrum RLP employs another intermediate of the MSP, 5-methylthioribulose-1-phosphate, as a substrate, resulting in the formation of different products. To further determine the interrelationship between RubisCOs and RLPs (and the potential integration of cellular carbon and sulfur metabolism), the functional roles of both RubisCO and RLP have been examined in vivo via the use of specific knockout strains and complementation studies of R. rubrum. The presence of functional, yet separate, MSPs in R. rubrum under both aerobic (chemoheterotrophic) and anaerobic (photoheterotrophic) growth conditions has not been observed previously in any organism. Moreover, the aerobic and anaerobic sulfur salvage pathways appear to be differentially controlled, with novel and previously undescribed steps apparent for sulfur salvage in this organism.Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) is the key enzyme of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway. This enzyme catalyzes the primary CO₂ fixation reaction and is found in diverse organisms, including plants, most photosynthetic and chemoautotrophic microorganisms, and many archaea (25). On the basis of amino acid sequence similarities, the RubisCO family of proteins has been classified into four groups, i.e., form I, form II, form III, and form IV (Fig. ​(Fig.1).1). The enzymes classified under forms I, II, and III are all able to catalyze the RubisCO reaction, i.e., carboxylation/oxygenation of RuBP. The most recently discovered group of enzymes in the RubisCO family are the form IV or RubisCO-like proteins (RLPs). These proteins have thus far been identified in proteobacteria, cyanobacteria, archaea, and algae (2, 4, 8, 11, 12, 21, 25, 26). RLPs have been further divided into six different subgroups based on sequence similarities within the group: IV-Photo, IV-Nonphoto, IV-YkrW, IV-DeepYrkW, IV-GOS (Global Ocean Sequencing), and IV-AMC (Acid Mine Consortium) (25, 26). Despite sharing a level of sequence similarity with the bonafide RubisCOs, the RLPs are unable to carry out CO₂/O₂ fixation because their sequences contain dissimilar residues at positions analogous to RubisCO''s active-site residues (25). The structures of the Geobacillus kaustophilus and Chlorobaculum tepidum RLPs have now been solved, and there are indeed differences between the tertiary structures of these two proteins and the bonafide RubisCO enzymes (14, 17, 25). Moreover, distinct patterns of active-site residue identities among the different clades of the RLP lineage suggest that these subgroups of RLPs are likely to utilize different substrates and perform dissimilar reactions (23, 25, 26).Open in a separate windowFIG. 1.Summary of the different classes of RubisCO found in nature so far (25). Forms I, II, and III catalyze bonafide CO₂/O₂ fixation reactions by using RuBP as the substrate. Form IV RubisCO (RLP) does not catalyze RuBP-dependent CO₂/O₂ fixation and is divided into six known clades (25), with only representatives of the type IV-YkrW and IV-DeepYkrW subgroups shown to catalyze defined, yet distinct, reactions (Fig. ​(Fig.22).Previous studies performed with the Chlorobaculum tepidum RLP (of the IV-Photo group) gave the first indication that the RLPs may be involved in some aspect of sulfur metabolism (12, 13). This was later substantiated when the precise function was established for the Bacillus subtilis (2), Microcystis aeruginosa (4), and Geobacillus kaustophilus (14) RLPs of the IV-YkrW group. All three proteins catalyze a tautomerase/enolase reaction of a methionine salvage pathway (MSP) in which the substrate 2,3-diketo-5-methylthiopentanyl-1-phosphate (DK-MTP 1P) is converted to 2-hydroxy-3-keto-5-thiomethylpent-1-ene 1-phosphate (HK-MTP 1P) (Fig. ​(Fig.2).2). This reaction is very reminiscent of the enolization of RuBP catalyzed by RubisCO. Moreover, form II RubisCO from Rhodospirillum rubrum was shown to complement an RLP mutant strain of B. subtilis, with the ability to catalyze the identical tautomerase/enolase reaction (2). Interestingly, in addition to the presence of a form II RubisCO gene (cbbM), the genome of R. rubrum also encodes an RLP that clusters with the IV-DeepYkrW group (25). The function of this protein was recently determined, and it was shown to catalyze a distinct reaction that uses 5-methylthioribulose-1-phosphate as the substrate (15). Via an unprecedented 1,3-proton transfer, with two successive 1,2-proton transfers from its substrate, R. rubrum RLP catalyzes the formation of two products, i.e., 1-thiomethyl-d-xylulose-5-phosphate and 1-thiomethyl-d-ribulose-5-phosphate, at a 3:1 ratio (15) (Fig. ​(Fig.2).2). The novel reaction catalyzed by this RLP suggests that R. rubrum likely uses a different pathway to salvage sulfur compounds.Open in a separate windowFIG. 2.Distinct reactions catalyzed by type IV-YkrW (A) and type IV-DeepYkrW (B) classes of form IV RubisCO/RLP, exemplified by the proteins from B. subtilis and R. rubrum, respectively.The presence of an RLP-encoding gene triggered the search for additional genes in the R. rubrum genome that might be homologs of known enzymes that participate in a conventional MSP. Several genes were indeed identified to encode homologs of MSP enzymes. However, to this point there is no experimental evidence for the existence of a functional MSP (21) in R. rubrum. Thus, in this study, we sought to determine the role of the RLP and RubisCO protein in sulfur salvage since each protein catalyzes different reactions and RubisCO is known to be synthesized only under anaerobic conditions (6, 7). Moreover, it is well appreciated that R. rubrum possesses a versatile metabolic capacity and is able to grow under both anaerobic and aerobic conditions, using a variety of carbon sources. The involvement of RLP and RubisCO in sulfur salvage was thus determined and found to be associated with aerobic and anaerobic metabolism, respectively.     

Tetrahymena thermophila and Candida albicans group I intron-derived ribozymes can catalyze the trans-excision-splicing reaction

Group I intron-derived ribozymes can catalyze a variety of non-native reactions. For the trans-excision-splicing (TES) reaction, an intron-derived ribozyme from the opportunistic pathogen Pneumocystis carinii catalyzes the excision of a predefined region from within an RNA substrate with subsequent ligation of the flanking regions. To establish TES as a general ribozyme-mediated reaction, intron-derived ribozymes from Tetrahymena thermophila and Candida albicans, which are similar to but not the same as that from Pneumocystis, were investigated for their propensity to catalyze the TES reaction. We now report that the Tetrahymena and Candida ribozymes can catalyze the excision of a single nucleotide from within their ribozyme-specific substrates. Under the conditions studied, the Tetrahymena and Candida ribozymes, however, catalyze the TES reaction with lower yields and rates [Tetrahymena (k_obs) = 0.14/min and Candida (k_obs) = 0.34/min] than the Pneumocystis ribozyme (k_obs = 3.2/min). The lower yields are likely partially due to the fact that the Tetrahymena and Candida catalyze additional reactions, separate from TES. The differences in rates are likely partially due to the individual ribozymes ability to effectively bind their 3′ terminal guanosines as intramolecular nucleophiles. Nevertheless, our results demonstrate that group I intron-derived ribozymes are inherently able to catalyze the TES reaction.     

An aminotransferase specific for the d-enantiomorph of methionine

An enzyme was isolated from germinating peanut seed and shown to be an aminotransferase specific for the d-enantiomorph of methionine. The keto acid of methionine, α-keto-γ-methylthiobutyrate, was isolated from the reaction mixture and identified. Of the keto acids tested pyruvic acid was the most effective acceptor for the amino group of methionine. A small amount of enzyme was isolated which gave only one band on disc gel electrophoresis.     

The Bacillus subtilis Chemoreceptor McpC Senses Multiple Ligands Using Two Discrete Mechanisms

Bacillus subtilis can perform chemotaxis toward all 20 l-amino acids normally found in proteins. Loss of a single chemoreceptor, McpC, was previously found to reduce chemotaxis to 19 of these amino acids. In this study, we investigated the amino acid-sensing mechanism of McpC. We show that McpC alone can support chemotaxis to 17 of these amino acids to varying degrees. Eleven amino acids were found to directly bind the amino-terminal sensing domain of McpC in vitro. Sequence analysis indicates that the McpC sensing domain exhibits a dual Per-Arnt-Sim (PAS) domain structure. Using this structure as a guide, we were able to isolate mutants that suggest that four amino acids (arginine, glutamine, lysine, and methionine) are sensed by an indirect mechanism. We identified four candidate binding lipoproteins associated with amino acid transporters that may function in indirect sensing: ArtP, GlnH, MetQ, and YckB. ArtP was found to bind arginine and lysine; GlnH, glutamine; MetQ, methionine; and YckB, tryptophan. In addition, we found that ArtP, MetQ, and YckB bind the sensing domain of McpC, suggesting that the three participate in the indirect sensing of arginine, lysine, methionine, and possibly tryptophan as well. Taken together, these results further our understanding of amino acid chemotaxis in B. subtilis and gain insight into how a single chemoreceptor is able to sense many amino acids.     

Defining a kinetic mechanism for l-DOPA 2,3 dioxygenase,a single-domain type I extradiol dioxygenase from Streptomyces lincolnensis

l-DOPA-2,3-dioxygenase from Streptomyces lincolnensis is a single domain type I extradiol dioxygenase of the vicinal oxygen chelate superfamily and catalyzes the second step in the metabolism of the propylhygric acid moiety of the antibiotic, lincomycin. In this report, the kinetic mechanism of l-DOPA dioxygenase is interrogated using stopped-flow in order to determine microscopic rate constants. Pre-steady state, progress curve and steady-state data were combined in a global kinetic analysis using KinTek Explorer in order to define and constrain a kinetic model for the type I l-DOPA dioxygenase. The data are best described by a four step mechanism, in which the cyclization of the enzymatic product is not enzyme catalyzed.     

The role of active-site residues in naphthalene dioxygenase

The three-component naphthalene dioxygenase enzyme system catalyzes the first step in the degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. A member of a large family of bacterial Rieske non-heme iron oxygenases, naphthalene dioxygenase is known to oxidize over 60 different aromatic compounds, and many of the products are enantiomerically pure. The crystal structure of the oxygenase component revealed the enzyme to be an α₃β₃ hexamer and identified the amino acids located near the active site. Site-directed mutagenesis studies have identified the residues involved in electron transfer and those responsible for controlling the regioselectivity and enantioselectivity of the enzyme. The results of these studies suggest that naphthalene dioxygenase can be engineered to catalyze a new and extended range of useful reactions.     

Metabolic pathway promiscuity in the archaeon Sulfolobus solfataricus revealed by studies on glucose dehydrogenase and 2-keto-3-deoxygluconate aldolase

The hyperthermophilic Archaeon Sulfolobus solfataricus metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff pathway. In this pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate (KDG) aldolase then catalyzes the cleavage of 2-keto-3-deoxygluconate to glyceraldehyde and pyruvate. The gene encoding glucose dehydrogenase has been cloned and expressed in Escherichia coli to give a fully active enzyme, with properties indistinguishable from the enzyme purified from S. solfataricus cells. Kinetic analysis revealed the enzyme to have a high catalytic efficiency for both glucose and galactose. KDG aldolase from S. solfataricus has previously been cloned and expressed in E. coli. In the current work its stereoselectivity was investigated by aldol condensation reactions between D-glyceraldehyde and pyruvate; this revealed the enzyme to have an unexpected lack of facial selectivity, yielding approximately equal quantities of 2-keto-3-deoxygluconate and 2-keto-3-deoxygalactonate. The KDG aldolase-catalyzed cleavage reaction was also investigated, and a comparable catalytic efficiency was observed with both compounds. Our evidence suggests that the same enzymes are responsible for the catabolism of both glucose and galactose in this Archaeon. The physiological and evolutionary implications of this observation are discussed in terms of catalytic and metabolic promiscuity.     

Digalactosyldiacylglycerols Isolated from a Brown Alga as Effective Phagostimulants for a Young Abalone

Sinorhizobium fredii USDA191 is a Gram-negative bacterium capable of forming nitrogen-fixing nodules on soybean roots. The USDA191 idhA gene encoding myo-inositol dehydrogenase, an enzyme necessary for myo-inositol utilization, is known to be involved in competitive nodulation and nitrogen fixation. In Bacillus subtilis, myo-inositol dehydrogenase catalyzes the first step of the myo-inositol catabolic pathway. Recently iolE was identified as the gene encoding 2-keto-myo-inositol dehydratase, which catalyzes the second step in the pathway. Here we report the presence of 2-keto-myo-inositol dehydratase activity in free-living USDA191 cells cultured in a medium containing myo-inositol. An iolE ortholog was cloned from USDA191. USDA191 iolE was expressed in Escherichia coli as a His₆-tag fusion and purified to exhibit 2-keto-myo-inositol dehydratase activity. Inactivation of USDA191 iolE led to defective myo-inositol utilization. USDA191 iolE partially complemented a B. subtilis iolE deficient mutant. These results suggest that S. fredii USDA191 utilizes a myo-inositol catabolic pathway, analogous to that of B. subtilis, involving at least idhA and iolE.     

Properties of enzymes involved in D-galactonate catabolism in fungi

Two enzymes catalyze the two step reactions in the D-galactonate nonphosphorolytic catabolic pathway ofAspergillus terreus, namely D-galactonate dehydratase and 2-keto-3-deoxy-D-galactonate (KDGal) aldolase. Maximum enzyme activities were obtained at 40° C and pH 8.0 or at 50° C and pH 7.5 for these two enzymes, respectively. Stability of the two enzymes under different conditions was investigated. From a Lineweaver-Burk plot of the reciprocal of initial velocities and substrate concentrations, apparent K _m values were calculated for D-galactonate, pyruvate and glyceraldehyde and found to be 8.33, 14.28 and 5.55 mM, respectively, in crude cell-free extracts. Results indicated the requirement of magnesium cation for D-galactonate dehydratase activity at an initial concentration of 10^–2 M. The presence of Mg²⁺ in the reaction mixture seems to induce greatly the fitness of the dehydratase with D-galactonate as no activity could be detected with 24-h dialyzed extract in the absence of magnesium cation.     

An internal hydrophobic helical domain of Bacillus subtilis enolase is essential but not sufficient as a non-cleavable signal for its secretion

Many cytoplasmic proteins without a cleavable signal peptide, including enolase, are secreted during the stationary phase in Bacillus subtilis but the molecular mechanism is not yet clear. We previously identified a highly conserved embedded membrane domain in an internal hydrophobic α-helix of enolase that plays an important role in its secretion. In this study, we examined the role of the helix in more detail for the secretion of enolase. Altering this helix by mutations showed that many mutated forms in this domain were not secreted, some of which were not stable as a soluble form in the cytoplasm. On the other hand, mutations on the flanking regions of the helix or the conserved basic residues showed no deleterious effect. Bacillus enolase with the proper hydrophobic helical domain was also exported extracellularly in Escherichia coli, indicating that the requirement of the helix for the secretion of enolase is conserved in these species. GFP fusions with enolase regions showed that the hydrophobic helix domain itself was not sufficient to serve as a functional secretion signal; a minimal length of N-terminus 140 amino acids was required to mediate the secretion of the fused reporter GFP. We conclude that the internal hydrophobic helix of enolase is essential but is not sufficient as a signal for secretion; the intact long N-terminus including the hydrophobic helix domain is required to serve as a non-cleavable signal for the secretion of Bacillus enolase.     

Role of Bacillus subtilis BacB in the Synthesis of Bacilysin

Bacilysin is a non-ribosomally synthesized dipeptide antibiotic that is active against a wide range of bacteria and some fungi. Synthesis of bacilysin (l-alanine-[2,3-epoxycyclohexano-4]-l-alanine) is achieved by proteins in the bac operon, also referred to as the bacABCDE (ywfBCDEF) gene cluster in B. subtilis. Extensive genetic analysis from several strains of B. subtilis suggests that the bacABC gene cluster encodes all the proteins that synthesize the epoxyhexanone ring of l-anticapsin. These data, however, were not consistent with the putative functional annotation for these proteins whereby BacA, a prephenate dehydratase along with a potential isomerase/guanylyl transferase, BacB and an oxidoreductase, BacC, could synthesize l-anticapsin. Here we demonstrate that BacA is a decarboxylase that acts on prephenate. Further, based on the biochemical characterization and the crystal structure of BacB, we show that BacB is an oxidase that catalyzes the synthesis of 2-oxo-3-(4-oxocyclohexa-2,5-dienyl)propanoic acid, a precursor to l-anticapsin. This protein is a bi-cupin, with two putative active sites each containing a bound metal ion. Additional electron density at the active site of the C-terminal domain of BacB could be interpreted as a bound phenylpyruvic acid. A significant decrease in the catalytic activity of a point variant of BacB with a mutation at the N-terminal domain suggests that the N-terminal cupin domain is involved in catalysis.     

MSP hormonal control of the oocyte MAP kinase cascade and reactive oxygen species signaling

The MSP domain is a conserved immunoglobulin-like structure that is important for C. elegans reproduction and human motor neuron survival. C. elegans MSPs are the most abundant proteins in sperm, where they function as intracellular cytoskeletal proteins and secreted hormones. Secreted MSPs bind to multiple receptors on oocyte and ovarian sheath cell surfaces to induce oocyte maturation and sheath contraction. MSP binding stimulates oocyte MPK-1 ERK MAP Kinase (MAPK) phosphorylation, but the function and mechanism are not well understood. Here we show that the Shp class protein-tyrosine phosphatase PTP-2 acts in oocytes downstream of sheath/oocyte gap junctions to promote MSP-induced MPK-1 phosphorylation. PTP-2 functions in the oocyte cytoplasm, not at the cell surface to inhibit multiple RasGAPs, resulting in sustained Ras activation. We also provide evidence that MSP promotes production of reactive oxygen species (ROS), which act as second messengers to augment MPK-1 phosphorylation. The Cu/Zn superoxide dismutase SOD-1, an enzyme that catalyzes ROS breakdown in the cytoplasm, inhibits MPK-1 phosphorylation downstream of or in parallel to ptp-2. Our results support the model that MSP triggers PTP-2/Ras activation and ROS production to stimulate MPK-1 activity essential for oocyte maturation. We propose that secreted MSP domains and Cu/Zn superoxide dismutases function antagonistically to control ROS and MAPK signaling.     

Purification of Prephenate dehydratase from Bacillus subtilis.

Prephenate dehydratase has been purified 10,000-fold from the crude extracts of Bacillus subtilis. The procedure takes advantage of the dissociation of the enzyme to a 55,000-dalton form in the presence of the negative effector, phenylalanine, and its association to a 210,000-dalton form in the presence of the positive effector, methionine. These two forms of the enzyme were separated from the bulk of the other proteins present in the crude extracts by gel filtration alternately in the presence of the two effectors. Sodium dodecyl sulfate electrophoretic analysis showed the enzyme is composed of apparently identical 28,000-dalton polypeptides.     

A Model of the Quaternary Structure of Enolases, Based on Structural and Evolutionary Analysis of the Octameric Enolase from Bacillus subtilis

Purified enolase from Bacillus subtilis has a native mass of approximately 370 kDa. Since B. subtilis enolase was found to have a subunit mass of 46.58 kDa, the quaternary structure of B. subtilis is octameric. The pl for B. subtilis enolase is 6.1, the pH optimum (pH_o) for activity is 8.1–8.2, and the K _m for 2-PGA is approximately 0.67 mM. Using the dimeric Cα structure of yeast dimeric enolase as a guide, these dimers were arranged as a tetramer of dimers to simulate the electron microscopy image processing obtained for the octameric enolase purified from Thermotoga maritima. This arrangement allowed identification of helix J of one dimer (residues 86–96) and the loop between helix L and strand 1 (HL–S1 loop) of another dimer as possible subunit interaction regions. Alignment of available enolase amino acid sequences revealed that in 16 there are two tandem glycines at the C-terminal end of helix L and the HL–S1 loop is truncated by 4–6 residues relative to the yeast polypeptide, two structural features absent in enolases known to be dimers. From these arrangements and alignments it is proposed that the GG tandem at the C-terminal end of helix L and truncation of the HL–S1 loop may play a critical role in octamer formation of enolases. Interestingly, the sequence features associated with dimeric quaternary structure are found in three phylogenetically disparate groups, suggesting that the ancestral enolase was an octamer and that the dimeric structure has arisen independently multiple times through evolutionary history.