Yasuhiro Kashima

A Gene Encoding Phosphatidylethanolamine N-Methyltransferase from Acetobacter aceti and Some Properties of its Disruptant

Phosphatidylcholine (PC) is a major component of membranes not only in eukaryotes, but also in several bacteria, including Acetobacter. To identify the PC biosynthetic pathway and its role in Acetobacter sp., we have studied Acetobacter aceti IFO3283, which is characterized by high ethanol oxidizing ability and high resistance to acetic acid. The pmt gene of A. aceti, encoding phosphatidylethanolamine N-methyltransferase (Pmt), which catalyzes methylation of phosphatidylethanolamine (PE) to PC, has been cloned and sequenced. One recombinant plasmid that complemented the PC biosynthesis was isolated from a gene library of the genomic DNA of A. aceti. The pmt gene encodes a polypeptide with molecular mass of either 25125, 26216, or 29052 for an about 27-kDa protein. The sequence of this gene showed significant similarity (44.3% identity in the similar sequence region) with the Rhodobacter sphaeroides pmtA gene which is involved in PE N-methylation. When the pmt gene was expressed in E. coli, which lacks PC, the Pmt activity and PC formation were clearly demonstrated. A. aceti strain harboring an interrupted pmt allele, pmt::Km, was constructed. The pmt disruption was confirmed by loss of Pmt and PC, and by Southern blot analyses. The null pmt mutant contained no PC, but tenfold more PE and twofold more phosphatidylglycerol (PG). The pmt disruptant did not show any dramatic effects on growth in basal medium supplemented with ethanol, but the disruption caused slow growth in basal medium supplemented with acetate. These results suggest that the lack of PC in the A. aceti membrane may be compensated by the increases of PE and PG by an unknown mechanism, and PC in A. aceti membrane is related to its acetic acid tolerance.

Purification and Characterization of the First Archaeal Glutamate Decarboxylase from Pyrococcus horikoshii

Glutamate decarboxylase (GAD) from the archaeon Pyrococcus horikoshii was successfully expressed and purified, with the aim of developing a hyperthermostable GAD for industrial applications. Its biochemical properties were different from those reported for other GADs. The enzyme had broad substrate specificity, and its optimum pH and temperature were pH 8.0 and >97 °C.

Purification and characterization of intracellular esterases related to ethylacetate formation in Acetobacter pasteurianus

Acetobacter sp. produce ethylacetate during acetic acid fermentation. We found that high ethylacetate-producing strains contained more intracellular esterase than low ethylacetate-producing strains. Acetobacter pasteurianus N-23, a high ethylacetate-producing strain, formed two major intracellular esterases (esterase-1 and esterase-2) that were regulated by ethanol. These two esterases were purified to homogeneity by means of hydrophobic interaction, ion exchange, gel filtration, and hydroxyapatite chromatographies. The two esterases hydrolyzed various esters, but only esterase-1 showed ethylacetate synthesis activity at pH 3. The molecular weights of esterase-1 and esterase-2 were 72 kDa and 44 kDa, respectively, as determined by gel filtration, and 38 kDa and 42 kDa, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Esterase-1 was inhibited by cysteine enzyme inhibitors such as diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. These esterases may play some role in the production of ethylacetate by Acetobacter sp. during vinegar fermentation.

Role of intracellular esterases in the production of esters by Acetobacter pasteurianus

Esters are the major flavor compounds produced by Acetobacter sp. during vinegar production. The two genes encoding the esterases in the bacteria were disrupted, and the effects of the disruptions studied. When cultured in the presence of ethanol, the est1 gene-disrupted mutant (DE1K) did not produce any ethyl acetate or isoamyl acetate. However, the disruption of est2 did not affect the ester production. Ethyl acetate production by N-23 (pME122P) and DE1K (pME122P), which contain est1, was 1.7-fold higher than that by the wild type, N-23. On analyzing the relationship between ethyl acetate production and the extracellular ethanol and acetic acid concentrations, we found that the highest amount of ethyl acetate was produced when the molar ratio of ethanol and acetic acid was 1:1. These results indicate that the ester production by Acetobacter sp. is mostly catalyzed by the intracellular esterase, esterase-1, with ethanol and acetic acid used as the substrates.

Characterization and crystal structure of the thermophilic ROK hexokinase from Thermus thermophilus

We characterized and determined the crystal structure of a putative glucokinase/hexokinase from Thermus thermophilus that belongs to the ROK (bacterial repressors, uncharacterized open reading frames, and sugar kinases) family. The protein possessed significant enzymatic activity against glucose and mannose, with V(max) values of 260 and 68 μmol·min(-1)·mg(-1) protein, respectively. Therefore, we concluded that the enzyme is a hexokinase. However, the hexokinase showed little catalytic capacity for galactose and fructose. Circular dichroism measurements indicated that the enzyme was structurally stable at 90°C. The crystal structure of the enzyme was determined at a resolution of 2.02 Å, with R(cryst) and R(free) values of 18.1% and 22.6%, respectively. The polypeptide structure was divided into large and small domains. The ROK consensus sequences 1 and 2 were included in the large domain. The cysteine-rich consensus sequence 2 folded into a zinc finger, and the bound zinc was confirmed by both electron density and X-ray absorption fine structure (XAFS) spectrum. The overall structure was a homotetramer that consisted of a dimer of dimers. The accessible surface area buried by the association of the dimers into the tetrameric structures was significantly higher in the T. thermophilus enzyme than in a homologous tetrameric ROK sugar kinase.

Crystallization and preliminary crystallographic analysis of a putative glucokinase/hexokinase from Thermus thermophilus.

Glucokinase/hexokinase catalyzes the phosphorylation of glucose to glucose 6-phosphate, which is the first step of glycolysis. The open reading frame TTHA0299 of the extreme thermophile Thermus thermophilus encodes a putative glucokinase/hexokinase which contains the consensus sequence for proteins from the repressors, open reading frames and sugar kinases family. In this study, the glucokinase/hexokinase from T. thermophilus was purified and crystallized using polyethylene glycol 8000 as a precipitant. Diffraction data were collected and processed to 2.02 Å resolution. The crystal belonged to space group P2(1), with unit-cell parameters a = 70.93, b = 138.14, c = 75.16 Å, β = 95.41°.