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Tracking the localization and dynamics of the intracellular bioactive lipid phosphatidic acid (PA) is important for understanding diverse biological phenomena. Although several PA sensors have been developed, better ones are still needed for comprehensive PA detection in cells. We recently found that α-synuclein (α-Syn) selectively and strongly bound to PA in vitro. Here, we revealed that the N-terminal region of α-Syn (α-Syn-N) specifically bound to PA, with a dissociation constant of 6.6 μm. α-Syn-N colocalized with PA-producing enzymes, diacylglycerol kinase (DGK) β at the plasma membrane (PM), myristoylated DGKζ at the Golgi apparatus, phorbol ester-stimulated DGKγ at the PM, and phospholipase D2 at the PM and Golgi but not with the phosphatidylinositol-4,5-bisphosphate-producing enzyme in COS-7 cells. However, α-Syn-N failed to colocalize with them in the presence of their inhibitors and/or their inactive mutants. These results indicate that α-Syn-N specifically binds to cellular PA and can be applied as an excellent PA sensor.

Specific inhibitors of diacylglycerol kinase (DGK) ζ can be promising anticancer medications via the activation of cancer immunity. Although the detection of cellular activities of target enzymes is essential for drug screening in addition to in vitro assays, it is difficult to detect the activity of DGKζ in cells. In the present study, we generated AcGFP-DGKζ cDNA with a consensus N-myristoylation sequence at the 5' end (Myr-AcGFP-DGKζ) to target DGKζ to membranes. Using liquid chromatography (LC)-tandem mass spectrometry (MS/MS) (LC-MS/MS), we showed that Myr-AcGFP-DGKζ, but not AcGFP-DGKζ without the myristoylation sequence, substantially augmented the levels of several phosphatidic acid (PtdOH) species. In contrast to Myr-AcGFP-DGKζ, its inactive mutant did not exhibit an increase in PtdOH production, indicating that the increase in PtdOH production was DGK activity-dependent. This method will be useful in chemical compound selection for the development of drugs targeting DGKζ and can be applicable to various soluble (nonmembrane bound) lipid-metabolizing enzymes, including other DGK isozymes.

Chitinases are generally composed of multiple domains; a catalytic domain and one or more additional domains that are not absolutely required but may modify the chitinolytic activity. The LinChi78 chitinase from Listeria innocua has a catalytic domain (CatD), a fibronectin type III-like (FnIII) domain, a chitin-binding domain (ChBD), and an unknown-function region (UFR) located between the CatD and FnIII domains. The UFR is 146 amino acid residues in length and does not have a homologous domain in the Conserved Domain Database. We performed a functional analysis of these domains and the UFR using several C-terminally and internally deleted mutants of LinChi78. Hydrolysis of an artificial substrate was almost unaffected by deletion of the ChBD and/or the FnIII domain, although the ChBD-deleted enzymes were approximately 30% less active toward colloidal chitin than LinChi78. On the other hand, deletion of the UFR led to an extensive loss of chitinase activity toward an artificial substrate as well as polymeric substrates. Upon further analysis, we found that the GKQTI stretch, between the 567th (G) and 571th (I) amino acid residues, in the UFR is critical for LinChi78 activity and demonstrated that Gln569 and Ile571 play central roles in eliciting this activity. Taken together, these results indicated that LinChi78 has a unique catalytic region composed of a typical CatD and an additional region that is essential for activity. Characterization of the unique catalytic region of LinChi78 will improve our understanding of GH18 chitinases.

Mouse acidic mammalian chitinase (AMCase) degrades chitin with highest efficiency at pH 2.0 and is active up to pH 8.0. Here, we report that mouse AMCase also exhibits transglycosylation activity under neutral conditions. We incubated natural and artificial chitin substrates with mouse AMCase at pH 2.0 or 7.0 and analyzed the resulting oligomers using an improved method of fluorescence assisted carbohydrate electrophoresis. Mouse AMCase produces primarily dimers of N-acetyl-D-glucosamine [(GlcNAc)2] under both pH conditions while generating transglycosylated (GlcNAc)3 primarily at pH 7.0 and at lower levels at pH 2.0. These results indicate that mouse AMCase catalyzes hydrolysis as well as transglycosylation and suggest that this enzyme can play a novel role under physiological conditions in peripheral tissues, such as the lungs. This article is protected by copyright. All rights reserved.

Two putative chitinase genes, lin0153 and lin1996, from the nonpathogenic bacterium Listeria innocua were expressed in Escherichia coli, and the gene products were characterized. The genes were close homologs of chitinases from the pathogenic bacterium Listeria monocytogenes, in which chitinases and chitin-binding proteins play important roles in pathogenesis in mice-infection models. The purified recombinant enzymes that are different in size, LinChi78 (lin0153 product) and LinChi35 (lin1996 product)-with molecular masses of 82 and 38 kDa, including vector-derived additional sequences, respectively-exhibited optimum catalytic activity under neutral and acidic conditions at 50 °C, respectively, and were stable over broad pH (4-11) and temperature (4-40 °C) ranges. LinChi35 displayed higher k cat and K M values for 4-nitrophenyl N,N-diacetyl-β-D-chitobioside [4NP-(GlcNAc)2] than LinChi78. Both enzymes produced primarily dimers from colloidal chitin as a substrate. However, LinChi78 and LinChi35 could hydrolyze oligomeric substrates in a processive exo- and nonprocessive endo-manner, respectively, and showed different reactivity toward oligomeric substrates. Both enzymes could bind chitin beads but were different in their binding ability toward crystalline α-chitin and cellulose. The structure-function relationships of these chitinases are discussed in reference to other bacterial chitinases.

Two glucoamylase-like genes, TVN1315 and Ta0286 , from the archaea Thermoplasma volcanium and T. acidophilum , respectively, were expressed in Escherichia coli . The gene products, TVN1315 and Ta0286, were identified as archaeal trehalases. These trehalases belong to the CAZy database family GH15, although they have putative (α/α) 6 barrel catalytic domain structures similar to those of GH37 and GH65 family trehalases from other organisms. These newly identified trehalases function within a narrow range of acidic pH values (pH 3.2 to 4.0) and at high temperatures (50 to 60°C), and these enzymes display K m values for trehalose higher than those observed for typical trehalases. These enzymes were inhibited by validamycin A; however, the inhibition constants ( K i ) were higher than those of other trehalases. Three TVN1315 mutants, corresponding to E408Q, E571Q, and E408Q/E571Q mutations, showed reduced activity, suggesting that these two glutamic acid residues are involved in trehalase catalysis in a manner similar to that of glucoamylase. To date, TVN1315 and Ta0286 are the first archaeal trehalases to be identified, and this is the first report of the heterologous expression of GH15 family trehalases. The identification of these trehalases could extend our understanding of the relationships between the structure and function of GH15 family enzymes as well as glycoside hydrolase family enzymes; additionally, these enzymes provide insight into archaeal trehalose metabolism.

Two archaeal trehalase-like genes, Saci1250 and Saci1816, belonging to glycoside hydrolase family 15 (GH15) from the acidophilic Crenarchaeon Sulfolobus acidocaldarius were expressed in Escherichia coli. The gene products showed trehalose-hydrolyzing activities, and the names SaTreH1 and SaTreH2 were assigned to Saci1816 and Saci1250 gene products, respectively. These newly identified enzymes functioned within a narrow range of acidic pH values at elevated temperatures, which is similar to the behavior of Euryarchaeota Thermoplasma trehalases. SaTreH1 displayed high KM and kcat values, whereas SaTreH2 had lower KM and kcat values despite a high degree of identity in their primary structures. A mutation analysis indicated that two glutamic acid residues in SaTreH1, E374 and E574, may be involved in trehalase catalysis because SaTreH1 E374Q and E574Q showed greatly reduced trehalose-hydrolyzing activities. Additional mutations substituting G573 and H575 residues with serine and glutamic acid residues, respectively, to mimic the TVN1315 sequence resulted in a decrease in trehalase activity and thermal stability. Taken together, the results indicated that Crenarchaea trehalases adopt active site structures that are similar to Euryarchaeota enzymes but have distinct molecular features. The identification of these trehalases could extend our understanding of the relationships between the structure and function of GH15 trehalases as well as other family enzymes and will provide insights into archaeal trehalose metabolism.

Bacillus thuringiensis is a Gram-positive soil bacterium that is known to be a bacterial biopesticide that produces insecticidal proteins called crystal proteins (Cry). In the insecticidal process, chitinases are suggested to perforate the peritrophic membrane barrier to facilitate the invasion of the Cry proteins into epithelial membranes. A chitinase gene from B. thuringiensis was successfully expressed in a soluble form in Escherichia coli, and the gene product was purified and characterized. The purified recombinant enzyme, BthChi74, hydrolyzed an artificial substrate, 4-nitrophenyl N,N′-diacetyl-β-d-chitobioside [4NP-(GlcNAc)2], and the natural substrates, colloidal chitin and crystalline α-chitin, but it did not hydrolyze cellulose. BthChi74 exhibited catalytic activity under a weakly acidic to neutral pH range at 50 °C, and it was stable over a wide pH range for 24 h. Differential scanning fluorimetry (DSF) indicated a protein melting temperature (T m) of 63.6 °C. Kinetic analysis revealed k cat and K M values of 1.5 s−1 and 159 μM, respectively, with 4NP-(GlcNAc)2 as a substrate. BthChi74 produced (GlcNAc)2 and GlcNAc from colloidal chitin and α-chitin as substrates, but the activity toward the latter was lower than that toward the former. BthChi74 could bind similarly to chitin beads, crystalline α-chitin, and cellulose through a unique family 2 carbohydrate-binding module (CBM2). The structure–function relationships of BthChi74 are discussed in relation to other chitinases, such as Listeria chitinase, which possesses a family 5 carbohydrate-binding module (CBM5).

Clostridium sp. G0005 glucoamylase (CGA) is composed of a β-sandwich domain (BD), a linker, and a catalytic domain (CD). In the present study, CGA was expressed in Escherichia coli as inclusion bodies when the N-terminal region (39 amino acid residues) of the BD was truncated. To further elucidate the role of the N-terminal region of the BD, we constructed N-terminally truncated proteins (Δ19, Δ24, Δ29, and Δ34) and assessed their solubility and activity. Although all evaluated proteins were soluble, their hydrolytic activities toward maltotriose as a substrate varied: Δ19 and Δ24 were almost as active as CGA, but the activity of Δ29 was substantially lower, and Δ34 exhibited little hydrolytic activity. Subsequent truncation analysis of the N-terminal region sequence between residues 25 and 28 revealed that truncation of less than 26 residues did not affect CGA activity, whereas truncation of 26 or more residues resulted in a substantial loss of activity. Based on further site-directed mutagenesis and N-terminal sequence analysis, we concluded that the 26XaaXaaTrp28 sequence of CGA is important in exhibiting CGA activity. These results suggest that the N-terminal region of the BD in bacterial GAs may function not only in folding the protein into the correct structure but also in constructing a competent active site for catalyzing the hydrolytic reaction.

The biochemical properties of the maltodextrin-hydrolyzing enzymes of cold-tolerant proteobacterium Caulobacter crescentus CB15 remain to be elucidated, although whose maltodextrin transport systems were well investigated. We cloned the putative glucoamylase of C. crescentus CB15 (CauloGA) gene. The CauloGA gene product that was expressed in E. coli was prone to forming inclusion bodies; however, most of the gene product was expressed in a soluble and active form when it was expressed as a fusion protein with Staphylococcus Protein A. The fusion protein was purified using an IgG Sepharose column and was identified as the active GA. The optimum temperature and pH for the activity of this GA toward maltotriose as a substrate were approximately 40°C and 5.0, respectively, and a differential scanning fluorimetry (DSF) analysis revealed that the melting temperature (Tm) of CauloGA was 42.9°C. The kinetic analyses with maltotriose and other maltodextrins as the substrates indicated that CauloGA has higher kcat and smaller Km values at 30°C with both substrates compared with other GAs at lower substrate concentration. However, the enzyme activities toward the substrates decreased as the substrate concentrations increased at concentrations higher than approximately 10-fold the Km. The function-based identification of thermolabile Caulobacter GA contributes to the understanding of the maltodextrin-degradation system of C. crescentus as well as the bacterial GA's function-structure relationship.