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H M Tan

Publications and source records attributed to H M Tan.

43 records · Page 3Linked to original sources
PubMed1994

The effect of ferredoxin(BED) overexpression on benzene dioxygenase activity in Pseudomonas putida ML2.

The benzene dioxygenase from Pseudomonas putida ML2 is a multicomponent complex comprising a flavoprotein reductase, a ferredoxin, and a terminal iron-sulfur protein (ISP). The catalytic activity of the isolated complex shows a nonlinear relationship with protein concentration in cell extracts, with the limiting factor for activity in vitro being ferredoxin(BED). The relative levels of the three components were analyzed by using 125I-labelled antibodies, and the functional molar ratio of ISP(BED), ferredoxin(BED), and reductase(BED) was shown to be 1:0.9:0.8, respectively. The concentration of ferredoxin(BED) was confirmed by quantitative electron paramagnetic resonance spectroscopy of the 2Fe-2S centers in ferredoxin(BED) and ISP(BED) of whole cells. These results demonstrate that the ferredoxin(BED) component is a limiting factor in dioxygenase activity in vitro. To determine if it is a limiting factor in vivo, a plasmid (pJRM606) overproducing ferredoxin(BED) was introduced into P. putida ML2. The benzene dioxygenase activity of this strain, measured in cell extracts, was fivefold greater than in the wild type, and the activity was linear with protein concentration in cell extracts above 2 mg/ml. Western blotting (immunoblotting) and electron paramagnetic resonance spectroscopic analysis confirmed an elevated level of ferredoxin(BED) protein and active redox centers in the recombinant strain. However, in these cells, the increased level of ferredoxin(BED) had no effect on the overall rate of benzene oxidation by whole cells. Thus, we conclude that ferredoxin(BED) is not limiting at the high intracellular concentration (0.48 mM) found in cells.

Benzene↗
PubMed1993

The Pseudomonas putida ML2 plasmid-encoded genes for benzene dioxygenase are unusual in codon usage and low in G+C content.

Benzene dioxygenase, catalyzing the oxidation of benzene to cis-1,2-dihydroxy-cyclohexa-3,5-diene, comprises four polypeptides that are encoded by plasmid pHMT112 of Pseudomonas putida ML2. In this study, the nucleotide (nt) sequences of four genes encoding this enzyme (bedC1C2BA) were determined, and the amino acid (aa) sequences were deduced. The sequence showed significant homology with the chromosomally encoded benzene dioxygenase and toluene dioxygenase genes (73-77% for nt and 83-99% for aa), but not the plasmid-encoded naphthalene dioxygenase genes (20-26% for nt and 32-36% for aa). A conserved motif (Cys-Xaa-His-15-to-17 aa-Cys-Xaa2-His, where Xaa is any aa), proposed to bind the Rieske-type [2Fe-2S] cluster, was identified in the deduced aa sequence of the iron-sulfur proteins. Three regions were also identified in the flavoprotein which are likely to be involved in FAD and NAD+ binding. The gene order of bedC1C2BA is consistent with most ring-hydroxylating dioxygenases isolated from Pseudomonas. However, the G+C content of 47% is in contrast to the high G+C content of the Pseudomonas chromosome (63%) and other Pseudomonas plasmids (57%), and with its unique codon usage preference this suggests that bedC1C2BA originated from a host derived from a different genus.

Amino Acid Sequence↗
PubMed1993

Molecular analysis of the plasmid-borne bed gene cluster from Pseudomonas putida ML2 and cloning of the cis-benzene dihydrodiol dehydrogenase gene.

Pseudomonas putida ML2 contains a large catabolic plasmid, pHMT112, carrying genes that encode the dioxygenase and dehydrogenase involved in the catabolism of benzene via the ortho or beta-ketoadipate pathway. pHMT112 was derived from a larger and less stable plasmid in P. putida ML2 following growth on succinate as carbon and energy source but was, however, stably maintained in P. putida even in the absence of selection for growth on benzene. Cleavage sites for the restriction endonucleases DraI, XbaI, and BamHI were mapped on the plasmid. A region of the plasmid, downstream of the benzene dioxygenase genes (bedC1C2BA), was found to encode the cis-benzene dihydrodiol dehydrogenase gene (bedD). Recombinant Escherichia coli containing bedC1C2BAD genes was found to express benzene dioxygenase and dehydrogenase activity, indicated by the production of catechol when incubated in the presence of benzene. Hybridization using benzene dioxygenase genes as probes was used to construct a restriction map of the 35.5-kb XhoI-DraI fragment on which the bed operon was located.

Benzene↗
PubMed1990

Cloning and expression of the plasmid-encoded benzene dioxygenase genes from Pseudomonas putida ML2.

Hybridization using heterologous dioxygenase gene probes indicated the presence of the genes encoding the enzyme benzene dioxygenase on a 112 kb plasmid from Pseudomonas putida ML2. They were identified as benzene dioxygenase genes (bed ABC1C2) by cloning in Escherichia coli and analysis of expression by Western blotting using antibodies raised to the four polypeptides of purified benzene dioxygenase.

Cloning, Molecular↗
PubMed1990

Experience with 395 extracorporeal shockwave lithotripsy in the treatment of renal and ureteric calculi.

Three hundred and ninety five cases in 358 consecutive patients (male-232, female-126) with renal and ureteric stones were treated with extracorporeal shockwave lithotripsy (ESWL) from March to November 1988. They either had ESWL alone, or in combination with stone manipulation or debulking percutaneous nephrolithotripsy (PCNL). Seventy five percent of the stones were found in the pelvicalyceal system and 25% in the ureter. Seventy-six percent of the stones were less than 25mm size. Two hundred and ninety (79%) cases were followed up to three months. Two hundred and forty nine (85.9%) cases were stone free and 36 (12.4%) had residual sand less than 3mm size. Five (1.7%) cases failed to fragment with ESWL monotherapy and were salvaged by either percutaneous or ureteroscopic intervention. None of the cases required any open surgery intervention.

Adult↗
PubMed1990

Percutaneous renal surgery for urolithiasis.

Sixty eight consecutive cases of percutaneous renal surgery, percutaneous nephrolithotripsy (PCNL), were performed on 64 patients (male-41, female-23) at the Subang Jaya Medical Centre from April 1988 to July 1989. All the cases were done as a one stage procedure. Fifty eight stones were large renal or staghorn and ten were ureteric. Thirty cases (41%) were stone free after PCNL alone. Thirty eight cases had residual fragments needing extracorporeal shockwave lithotripsy (ESWL). Mean operating time was 109.6 +/- 36.0 minutes. Mean hospital stay was 4.5 +/- 1.8 days. At three months follow-up, 86% of the cases were stone free. The remaining had residual sand (less than 3mm). Minor complications occurred in six patients. None required major surgical intervention post PCNL.

Female↗