Characterization of three γ-glutamyltranspeptidases from Pseudomonas aeruginosa PAO1.

The Pseudomonas aeruginosa strain, PAO1, has three putative γ-glutamyltranspeptidase (GGT) genes: ggtI, ggtII, and ggtIII. In this study, the expression of each of these genes in P. aeruginosa PAO1 was analyzed, and the properties of the corresponding GGT proteins were investigated. This is the first report on biochemical characterization of GGT paralogs from Pseudomonas species. The crude extracts prepared from P. aeruginosa PAO1 exhibited hydrolysis and transpeptidation activities of 17.3 and 65.0 mU/mg, respectively, and the transcription of each gene to mRNA was confirmed by RT-PCR. All genes were cloned, and the expression plasmids constructed were introduced into an Escherichia coli expression system. Enzyme activity of the expressed protein of ggtI (PaGGTI) was not detected in the system, while the enzyme activities of the expressed proteins derived from ggtII and ggtIII (PaGGTII and PaGGTIII, respectively) were detected. However, the enzyme activity of PaGGTII was very low and easily decreased. PaGGTII with C-terminal his-tag (PaGGTII25aa) showed increased activity and stability, and the purified enzyme consisted of a large subunit of 40 kDa and a small subunit of 28 kDa. PaGGTIII consisted of a large subunit of 37 kDa and a small subunit of 24 kDa. The maximum hydrolysis and transpeptidation activities of PaGGTII25aa were obtained at 40ºC-50ºC, and the maximum hydrolysis and transpeptidation activities of PaGGTIII were obtained at 50ºC-60ºC. These enzymes retained approximately 80% of their hydrolysis and transpeptidation activities after incubation at 50ºC for 10 min, reflecting good stability. Both PaGGTII25aa and PaGGTIII showed higher activities of hydrolysis and transpeptidation in the alkali range than in the acidic range. However, they were highly stable at a wide pH range (5-10.5).

Initial Step of Selenite Reduction via Thioredoxin for Bacterial Selenoprotein Biosynthesis.

Many organisms reductively assimilate selenite to synthesize selenoprotein. Although the thioredoxin system, consisting of thioredoxin 1 (TrxA) and thioredoxin reductase with NADPH, can reduce selenite and is considered to facilitate selenite assimilation, the detailed mechanism remains obscure. Here, we show that selenite was reduced by the thioredoxin system from Pseudomonas stutzeri only in the presence of the TrxA (PsTrxA), and this system was specific to selenite among the oxyanions examined. Mutational analysis revealed that Cys33 and Cys36 residues in PsTrxA are important for selenite reduction. Free thiol-labeling assays suggested that Cys33 is more reactive than Cys36. Mass spectrometry analysis suggested that PsTrxA reduces selenite via PsTrxA-SeO intermediate formation. Furthermore, an in vivo formate dehydrogenase activity assay in Escherichia coli with a gene disruption suggested that TrxA is important for selenoprotein biosynthesis. The introduction of PsTrxA complemented the effects of TrxA disruption in E. coli cells, only when PsTrxA contained Cys33 and Cys36. Based on these results, we proposed the early steps of the link between selenite and selenoprotein biosynthesis via the formation of TrxA-selenium complexes.

Crystal structure of the catalytic unit of thermostable GH87 α-1,3-glucanase from Streptomyces thermodiastaticus strain HF3-3.

α-1,3-Glucan is a homopolymer composed of D-glucose (Glc) and it is an extracellular polysaccharide found in dental plaque due to Streptococcus species. α-1,3-Glucanase from Streptomyces thermodiastaticus strain HF3-3 (Agl-ST) has been identified as a thermostable α-1,3-glucanase, which is classified into glycoside hydrolase family 87 (GH87) and specifically hydrolyzes α-1,3-glucan with an endo-action. The enzyme has a potential to inhibit the production of dental plaque and to be used for biotechnological applications. Here we show the structure of the catalytic unit of Agl-ST determined at 1.16 Å resolution using X-ray crystallography. The catalytic unit is composed of two modules, a ß-sandwich fold module, and a right-handed ß-helix fold module, which resembles other structural characterized GH87 enzymes from Bacillus circulans str. KA-304 and Paenibacillus glycanilyticus str. FH11, with moderate sequence identities between each other (approximately 27% between the catalytic units). However, Agl-ST is smaller in size and more thermally stable than the others. A disulfide bond that anchors the C-terminal coil of the ß-helix fold, which is expected to contribute to thermal stability only exists in the catalytic unit of Agl-ST.

Prevention of oral biofilm formation and degradation of biofilm by recombinant α-1,3-glucanases from Streptomyces thermodiastaticus HF3-3.

The genes encoding α-1,3-glucanases (Agls; AglST1 and AglST2) from Streptomyces thermodiastaticus HF3-3 were cloned and were then expressed in Escherichia coli Rosetta-gami B (DE3). We purified the resultant histidine (His)-tagged α-1,3-glucanases (recombinant enzymes, rAglST1 and rAglST2). Both the recombinant enzymes were similar to the wild-type enzymes. We examined the effects of rAglST1 and rAglST2 on the formation and degradation of biofilms on glass plates with Streptococcus mutans NRBC 13955 by evaluating the biofilm content (%), release of reducing sugar (mM), release of S. mutans (log CFU/mL), and the biofilm structure using laser scanning microscopy (LSM). The results showed that after incubation for 16 h, rAglST1 and rAglST2 reduced the formation of biofilm to 52% and 49% of the control, respectively. The result may reflect the fact that the concentration of the reducing sugar and the number of S. mutans cells in the rAglATs-added medium were higher than in the control medium. After an 8-h treatment with rAglST1 and rAglST2, biofilms decreased to less than 60% of the control. The number of S. mutans cells in the reaction mixture gradually increased during the incubation period. The enzymes can degrade the biofilms that were pre-formed on the glass plate by more than 50% after a 30-min incubation in the presence of toothpaste ingredients (1% w/v of sodium fluoride, benzethonium chloride, and sodium dodecyl sulfate) at 50°C. Our study showed that rAglST1 and rAglST2 have advantageous properties for dental care applications.

A Novel Lysophosphatidic Acid Acyltransferase of Escherichia coli Produces Membrane Phospholipids with a cis-vaccenoyl Group and Is Related to Flagellar Formation.

Lysophosphatidic acid acyltransferase (LPAAT) introduces fatty acyl groups into the sn-2 position of membrane phospholipids (PLs). Various bacteria produce multiple LPAATs, whereas it is believed that Escherichia coli produces only one essential LPAAT homolog, PlsC-the deletion of which is lethal. However, we found that E. coli possesses another LPAAT homolog named YihG. Here, we show that overexpression of YihG in E. coli carrying a temperature-sensitive mutation in plsC allowed its growth at non-permissive temperatures. Analysis of the fatty acyl composition of PLs from the yihG-deletion mutant (∆yihG) revealed that endogenous YihG introduces the cis-vaccenoyl group into the sn-2 position of PLs. Loss of YihG did not affect cell growth or morphology, but ∆yihG cells swam well in liquid medium in contrast to wild-type cells. Immunoblot analysis showed that FliC was highly expressed in ∆yihG cells, and this phenotype was suppressed by expression of recombinant YihG in ∆yihG cells. Transmission electron microscopy confirmed that the flagellar structure was observed only in ∆yihG cells. These results suggest that YihG has specific functions related to flagellar formation through modulation of the fatty acyl composition of membrane PLs.

Lysophosphatidic acid acyltransferase from the thermophilic bacterium Thermus thermophilus HB8 displays substrate promiscuity.

Lysophosphatidic acid acyltransferase is a phospholipid biosynthetic enzyme that introduces a fatty acyl group into the sn-2 position of phospholipids. Its substrate selectivity is physiologically important in defining the physicochemical properties of lipid membranes and modulating membrane protein function. However, it remains unclear how these enzymes recognize various fatty acids. Successful purification of bacterial lysophosphatidic acid acyltransferases (PlsCs) was recently reported and has paved a path for the detailed analysis of their reaction mechanisms. Here, we purified and characterized PlsC from the thermophilic bacterium Thermus thermophilus HB8. This integral membrane protein remained active even after solubilization and purification and showed reactivity toward saturated, unsaturated, and methyl-branched fatty acids, although branched-chain acyl groups are the major constituent of phospholipids of this bacterium. Multiple sequence alignment revealed the N-terminal end of the enzyme to be shorter than that of PlsCs with defined substrate selectivity, suggesting that the shortened N-terminus confers substrate promiscuity. ABBREVIATIONS: ACP: acyl carrier protein; CAPS: N-cyclohexyl-3-aminopropanesulfonic acid; CoA: coenzyme A; CYMAL-6: 6-cyclohexyl-1-hexyl-ß-D-maltoside; DDM: n-dodecyl-ß-D-maltoside; DTNB: 5,5´-dithiobis(2-nitrobenzoic acid); EPA: eicosapentaenoic acid; G3P: glycerol 3-phosphate; HEPES: N-2-hydroxyethylpiperazine-N´-2-ethanesulfonic acid; LPA: lysophosphatidic acid; MS: mass spectrometry; PA: phosphatidic acid.

A novel 1-acyl-sn-glycerol-3-phosphate O-acyltransferase homolog for the synthesis of membrane phospholipids with a branched-chain fatty acyl group in Shewanella livingstonensis Ac10.

1-Acyl-sn-glycerol-3-phosphate O-acyltransferase (PlsC) plays an essential role in the formation of phosphatidic acid, a precursor of various membrane phospholipids (PLs), in bacteria by catalyzing the introduction of an acyl group into the sn-2 position of lysophosphatidic acid. Various bacteria produce more than one PlsC. However, the physiological significance of the occurrence of multiple PlsCs is poorly understood. A psychrotrophic bacterium, Shewanella livingstonensis Ac10, which produces eicosapentaenoic acid at low temperatures, has five putative PlsCs (PlsC1-5). We previously showed that PlsC1 is responsible for the production of PLs containing an eicosapentaenoyl group. Here, we characterized another putative PlsC of this bacterium named PlsC4. We generated a plsC4-disrupted mutant and found that PLs containing 13:0 found in the parental strain were almost completely absent in the mutant. The loss of these PLs was suppressed by introduction of a plsC4-expression plasmid. PLs containing 15:0 were also drastically decreased by plsC4 disruption. Gas chromatography-mass spectrometry analysis of fatty acyl methyl esters derived from PLs of the parental strain showed that the 13:0 and 15:0 groups were an 11-methyllauroyl group and a 13-methylmyristoyl group, respectively. Phospholipase A2 treatment revealed that these fatty acyl groups were linked to the sn-2 position of PLs. Thus, PlsC4 is a new type of PlsC homolog that is responsible for the synthesis of PLs containing a branched-chain fatty acyl group at the sn-2 position and plays a clearly different role from that of PlsC1 in vivo.