Kiyofumi Maruyama

Cloning and Characterization of the Genes Encoding Enzymes for the Protocatechuate Meta-degradation Pathway of Pseudomonas ochraceae NGJ1

The 2-pyrone-4,6-dicarboxylate lactonase gene (proL), the protocatechuate 4,5-dioxygenase α and β subunits genes (proOa and proOb), and the 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase gene (proD) were cloned from the chromosomal DNA of Pseudomonas ochraceae NGJ1. These genes were in the order proLOaObD on the DNA, and a possible transcription terminator sequence followed. The proL and proD genes were over-expressed in Escherichia coli, and their gene products were purified for identification, while the expression of proOaOb was at a lower level. The protocatechuate meta-degradation operon was reconstituted with the recombinant plasmids and expressed successfully in E. coli.

Purification and properties of γ-oxalomesaconate hydratase from Pseudomonas ochraceae grown with phthalate

Pseudomonas ochraceae produced inducibly a hydro-lyase which catalyzes the reversible conversion of gamma-oxalomesaconate into (-)-gamma-oxalocitramalate. The enzyme has been purified to homogeneity from the bacteria grown with phthalate. The enzyme was a dimeric protein (pI=4.9) with a Mr of 68,000 and showed a high specificity for gamma-oxalomesaconate (Km=14 microM) and (-)-gamma-oxalocitramalate (Km=6.4 microM). Equilibrium constant for the hydration of gamma-oxalomesaconate at pH 8.0 and 24 degrees C was 2.5. Various thiols activated the enzyme.

Molecular and Catalytic Properties of 2,4′-Dihydroxyacetophenone Dioxygenase from Burkholderia sp. AZ11

The gene dad encoding 2,4′-dihydroxyacetophenone (DHAP) dioxygenase was cloned from Burkholderia sp. AZ11. The initiation codon GTG was converted to ATG for high-level expression of the enzyme in Escherichia coli. The enzyme was moderately thermostable, and the recombinant enzyme was briefly purified. The enzyme (M r=90 kDa) was a homotetramer with a subunit M r of 23 kDa. It contained 1.69 mol of non-heme iron, and had a dark gray color. On anaerobic incubation of it with DHAP, the absorption at around 400 nm increased due to the formation of an enzyme-DHAP complex. Multiple sequence alignment suggested that His77, His79, His115, and Glu96 in the cupin fold were possible metal ligands. The apparent K m for DHAP and the apparent V max were estimated to be 1.60 μM and 6.28 μmol/min/mg respectively. 2-Hydroxyacetophenone was a poor substrate. CuCl2 and HgCl2 strongly inhibited the enzyme, while FeSO4 weakly activated it.

Molecular and Catalytic Properties of Monoacetylphloroglucinol Acetyltransferase from Pseudomonas sp. YGJ3

Monoacetylphloroglucinol (MAPG) acetyltransferase, catalyzing the conversion of MAPG to 2,4-diacetylphloroglucinol (DAPG), was purified from Pseudomonas sp. YGJ3 grown without Cl−. Cl− and pyoluteorin repressed expression of the enzyme. SDS-polyacrylamide gel electrophoresis showed that the purified enzyme (M r=330 kDa) was composed of three subunits of 17, 38, and 43 kDa, and protein sequencing identified these as PhlB, PhlA, and PhlC respectively. The enzyme catalyzed the reversible disproportionation of 2 moles of MAPG to phloroglucinol (PG) and DAPG. The equilibrium constant K (=[DAPG][PG]/[MAPG]2) was estimated to be about 1.0 at 25 °C. A KpnI 20-kb DNA fragment was cloned from the genomic DNA of strain YGJ3, and a 12,598-bp long DNA region containing the phl gene cluster phlACBDEFGHI was sequenced. PCR cloning and expression of the phl genes in Escherichia coli confirmed that expression of phlACB genes produced MAPG ATase.

The Chloride Ion Is an Environmental Factor Affecting the Biosynthesis of Pyoluteorin and 2,4-Diacetylphloroglucinol in Pseudomonas sp. YGJ3

The effects of Cl− on the antibiotics productivity of Pseudomonas sp. YGJ3 were investigated. YGJ3 produced pyoluteorin and 2,4-diacetylphloroglucinol, depending on the concentration of Cl− in the culture medium. Cl− stimulated the biosynthesis of pyoluteorin, thereby repressing 2,4-diacetylphloroglucinol biosynthesis. The cell-free extract from the bacteria grown without Cl− showed high activity of monoacetylphloroglucinol acetyltransferase, an essential enzyme in 2,4-diacetylphloroglucinol biosynthesis.

Role of cysteine residues in 4-oxalomesaconate hydratase from Pseudomonas ochraceae NGJ1.

4-Oxalomesaconate hydratase from Pseudomonas ochraceae NGJ1 is unstable in the absence of reducing reagents such as dithiothreitol, and strongly inhibited by 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). To study the role of cysteine residues in enzyme catalysis, the eight individual cysteine residues of the enzyme were replaced with serine residues by site-directed mutagenesis. The catalytic properties and chemical modification of wild- and mutant type-enzymes by DTNB showed that (i) none of eight cysteine residues was essential for enzyme catalysis; (ii) the inhibition by DTNB was mostly due to modification of Cys-186; (iii) Cys-96 might be another residue reacting with DTNB, and its modification caused an increase in the K m-value for 4-oxalomesaconate; (iv) the other six cysteine residues were inaccessible to DTNB, but susceptible to HgCl2; and (v) only replacement of Cys-186 remarkably improved the stability of the enzyme in the absence of reducing reagent.

Oxidative Degradation of 4-Hydroxyacetophenone in Arthrobacter sp. TGJ4

The 4-hydroxyacetophenone assimilating bacterium Arthrobacter sp. TGJ4 was isolated from a soil sample. The resting cell reaction suggested that the strain cleaved 4-hydroxyacetophenone and its 3-methoxy derivative to the corresponding carboxylic acids and formaldehyde. Some properties of the enzyme catalyzing the cleavage reaction were examined.

Molecular and catalytic properties of 2,4-diacetylphloroglucinol hydrolase (PhlG) from Pseudomonas sp. YGJ3.

Gene phlG encoding 2,4-diacetylphloroglucinol hydrolase was cloned from Pseudomonas sp. YGJ3 and expressed in Escherichia coli. Recombinant PhlG was purified homogeneously. It required 2-mercaptoethanol for stability. K m for 2,4-diacetylphloroglucinol and k cat were determined to be 24 µM and 5.8 s−1 respectively. CoCl2 specifically and significantly activated PhlG.