Papers by Nathan Rothschild
Phanerochaete chrysosporium immobilized on polyurethane foam cubes in a nonimmersed liquid cultur... more Phanerochaete chrysosporium immobilized on polyurethane foam cubes in a nonimmersed liquid culture system and maintained under different carbon-to-nitrogen (C/N) ratios and levels. Lignin peroxidase (LIP) activity was obtained in cultures exposed to air when the C/N ratio was low (7.47), i.e., when nitrogen levels were high (C/N ؍ 56/45 mM) or carbon levels were low (C/N ؍ 5.6/4.5 mM). At the low C/N ratio, the fungus was carbon starved and did not produce extracellular polysaccharides. At a high C/N ratio (153), i.e., under conditions of excess carbon (nitrogen limitation) (C/N ؍ 56/2.2 mM), cultures exposed to air produced large amounts of polysaccharide, and LIP activity was detected only in cultures exposed to pure oxygen. Under high-nitrogen conditions, LIP production was 1,800 U/liter in cultures exposed to pure oxygen and 1,300 U/liter in cultures exposed to air, with H1 and H2 being the main isoenzymes. The oxygen level did not significantly alter the isoenzyme profile, nor did low-carbon conditions. The formation of manganese peroxidase was generally less affected by the oxygen level than that of LIP but was considerably reduced by a low C/N ratio. The effects of oxygen level and C/N ratio on the synthesis of glyoxal oxidase paralleled their effects on LIP synthesis except in the case of high nitrogen, which totally suppressed glyoxal oxidase activity.
The extracellular lignin peroxidase (LIP) protein profile of the fungus Phanerochaete chrysospori... more The extracellular lignin peroxidase (LIP) protein profile of the fungus Phanerochaete chrysosporium, grown in nonimmersed liquid culture under conditions of excess nitrogen, changed markedly with culture age. At peak LIP activity (day 4), the heme-protein profile in the extracellular fluid, analyzed by anion-exchange highpressure liquid chromatography, was characterized by a predominance of the LIP isozymes H1 and H2, small amounts of H6 and H8, and other minor peaks, designated Ha and Hb. On day 5, the level of H1 increased and it became the dominant isozyme, with a corresponding decrease in the level of H2. Moreover, the relative levels of H6 and H8 decreased with corresponding increases in Ha and Hb levels. This change in LIP profile occurred extracellularly and resulted from the enzymatic dephosphorylation of LIP isozymes. An enzymatic fraction responsible for LIP isozyme dephosphorylation, termed LIP dephosphorylating (LpD) fraction, was partially purified from the culture fluid. Incubation of the LpD fraction with 32 P-labeled H2, H6, H8, and H10 isozymes separated from nitrogen-limited cultures resulted in the formation of the dephosphorylated isozymes H1, Ha, Hb, and Hc, respectively. Dephosphorylation did not significantly change the catalytic properties of the LIP isozymes with veratryl alcohol as a substrate. LIP dephosphorylation is therefore suggested to be a posttranslational modification process catalyzed extracellularly by the LpD activity.
Biochemical Pharmacology, 1999
Reactivators of organophosphate (OP)-inhibited cholinesterases (ChEs) are believed to give rise t... more Reactivators of organophosphate (OP)-inhibited cholinesterases (ChEs) are believed to give rise to phosphorylated oximes (POX) that reinhibit the enzyme. Diethylphosphoryl oximes (DEP-OX) that were generated in situ were demonstrated in the past to be unstable, yet were more potent inhibitors of acetylcholinesterase (AChE) than the parent OPs. In view of the inconsistencies among reported results, and the potential toxicity of POXs, it seemed important to characterize authentic DEP-OXs, and to evaluate their interference with reactivation of diethylphosphoryl-ChE (DEP-ChE) conjugates. To this end, the diethylphosphoric acid esters of 1-methyl-2-pyridinium carboxaldehyde oxime (DEP-2PAM) and 1-methyl-4 pyridinium carboxaldehyde oxime (DEP-4PAM) were synthesized and chemically defined. The half-lives of DEP-2PAM and DEP-4PAM in 10 mM Tris buffer, pH 7.8, at 29 degrees were found to be 10 and 980 sec, respectively. The two DEP-OXs inhibited ChEs with the following ranking order: for DEP-2PAM, human butyrylcholinesterase (HuBChE, k(i) = 2.03 x 10(9) M(-1) min(-1)) > mouse AChE (MoAChE) approximately equal to fetal bovine serum AChE (FBS-AChE) approximately equal to equine BChE (EqBChE); for DEP-4PAM, HuBChE (k(i) = 0.71 x 10(9) M(-1) min(-1)) > EqBChE > MoAChE > FBS-AChE. A dialkylarylphosphate hydrolase (phosphotriesterase; PTE) from Pseudomonas sp. catalyzed the hydrolysis of DEP-4PAM with k(cat)/Km = 3.56 x 10(7) M(-1) min(-1) and Km = 0.78 mM. Reactivation of DEP-ChEs was enhanced by PTE when 4-PAM-based oximes were used as reactivators, whereas reactivation with 2-PAM-based oximes was not affected by PTE. This observation is attributed primarily to the short half-life of DEP-OXs derived from the latter oximes. Relatively low doses of PTE can detoxify large quantities of DEP-OXs rapidly, and thereby augment the efficacy of antidotes that contain the oxime function in position 4 of the pyridine ring.
Archives of Biochemistry and Biophysics, 1999
The lignin peroxidase (LIP) isozyme profile of the white-rot fungus Phanerochaete chrysosporium c... more The lignin peroxidase (LIP) isozyme profile of the white-rot fungus Phanerochaete chrysosporium changes markedly with culture age. This change occurs extracellularly and results from enzymatic dephosphorylation of LIP isozymes. In this study, a novel mannose 6-phosphatase (M6Pase) from extracellular culture fluid filtrate of P. chrysosporium, shown to be responsible for the extracellular postranslational modification of LIP, was purified and characterized. In vitro incubation of the purified M6Pase with purified LIP isozyme H2 resulted in its conversion to isozyme H1, with an equimolar release of orthophosphate. Using different sugar phosphates as substrate, the enzyme exhibited narrow specificity, showing activity mostly for mannose 6-phosphate (K m ؍ 0.483 mM). The enzyme displayed a molecular mass of 82 kDa, as determined by gel filtration, and 40.4 and 39.1 kDa, on SDS-PAGE, suggesting that the native form is a dimer. The N-terminal sequence of the enzyme has no homology with that of other reported phosphatases. M6Pase is a metaloprotein with manganese and cobalt as the preferred metal ions. It is N-glycosylated proteins with an isoelectric point of 4.7-4.8 and a pH optimum of 5. Based on its characteristics, M6Pase from P. chrysosporium seems to be a unique phosphatase responsible for posttranslation modification of LIP isozymes.
Phanerochaete chrysosporium were studied by using shallow stationary cultures grown in the presen... more Phanerochaete chrysosporium were studied by using shallow stationary cultures grown in the presence of limited or excess N. When no Mn was added, LIP was formed in both N-limited and N-excess cultures exposed to air, but no LIP activity was observed at Mn concentrations greater than 13 mg/liter. In oxygen-flushed, N-excess cultures, LIP was formed at all Mn concentrations, and the peak LIP activity values in the extracellular fluid were nearly identical in the presence of Mn concentrations ranging from 3 to 1,500 mg/liter. When the availability of oxygen to cultures exposed to air was increased by growing the fungus under nonimmersed liquid conditions, higher levels of Mn were needed to suppress LIP formation compared with the levels needed in shallow stationary cultures. The composition of LIP isozymes was affected by the levels of N and Mn. Addition of veratryl alcohol to cultures exposed to air did not eliminate the suppressive effect of Mn on LIP formation. A deficiency of Mn in N-excess cultures resulted in lower biomass and a lower rate of glucose consumption than in the presence of Mn. In addition, almost no activity of the antioxidant enzyme Mn superoxide dismutase was observed in Mn-deficient, N-excess cultures, but the activity of this enzyme increased as the Mn concentration increased from 3 to 13 mg/liter. No Zn/Cu superoxide dismutase activity was observed in N-excess cultures regardless of the Mn concentration.
Enzyme and Microbial Technology, 2002
The gene structure and expression of the antioxidant enzyme manganese-containing superoxide dismu... more The gene structure and expression of the antioxidant enzyme manganese-containing superoxide dismutase (MnSOD1) of the white-rot fungus Phanerochaete chrysosporium was characterized. The MnSOD1 protein was purified from liquid cultures of P. chrysosporium and the amino acid sequences of four of its tryptic fragments were determined and used to clone a 3.2-kb genomic fragment encompassing the entire gene of MnSOD1. Sequence analysis of the new gene, MnSOD1, revealed more than 80% identity with the MnSOD genes from the white-rot fungi Ganoderma resinaceum, Ganoderma microsporum and Amauroderma rude, forming a clearly discernible cluster. The expression of MnSOD1 was measured as a function of culture age, and a good correlation between transcript level and enzyme activity was observed, with a peak at 72 and 96 h of incubation, respectively. Interestingly, as for P. chrysosporium, the only SOD activity detectable in linear cultures of other white-rot fungi tested (Pleurotus florida, Lentinus edodes, Trametes versicolor, Ganoderma lucidum) was cyanide-insensitive SOD activity defined as MnSOD. (C.G. Dosoretz). metabolism, subjecting the fungus to a ROS-rich environment. Oxygen toxicity has been reported to be the principal cause of a major loss in organization of ultrastructure of cells from cultures of P. chrysosporium that had been exposed to pure oxygen . Numerous chlamydospores are developed when LIP synthesis is triggered , and cells with impaired mitochondrial function express LIP, probably in response to accumulating ROS . Similar levels of LIP activity were obtained in oxygenated cultures of P. chrysosporium as in manganese-deficient cultures under atmospheric air . In the latter, no manganese-containing superoxide dismutase (MnSOD) activity was detected, suggesting that both cultures are saturated by the ROS needed to trigger LIP expression. In addition to the ROS-rich environment to which the fungus is subjected during lignin degradation, the fungus itself develops mechanisms for the production of a main ROS, hydrogen peroxide (H 2 O 2 ), and is further exposed to high concentrations of the toxic aromatic radical intermediates generated.
Journal of Biotechnology, 1995
The influence of extracellular ligninolytic fluid addition on lignin peroxidase (LIP), Mn-peroxid... more The influence of extracellular ligninolytic fluid addition on lignin peroxidase (LIP), Mn-peroxidase (MNP) and glyoxal oxidase (GLOX) activity in free and immobilized (polyurethane foam) batch cultures of Phanerochaete chrysosporium was examined. Addition was carried out in several proportions during growth (day 0) and idiophasic phase (day 4). A decrease in the activity of added LIP was observed for both free pellets and immobilized cultures. In both cases, a de novo LIP activity was slightly lower than in control cultures (without addition). MNP and GLOX activity was not significantly altered by the addition of ligninolytic extracellular fluid. LIP and MNP isoenzymes were detected in different degrees, showing a typical behaviour of N-limited cultures of P. chrysosporium monitored at 409 nm. During late secondary metabolism in immobilized cultures, a second peak of ligninolytic activity was obtained, for which the level of LIP activity was similar to the first one, but glyoxal oxidase activity was 3-fold higher. Isoenzyme profile for this second peak was typical of C-limitation, being H2 isoenzyme the predominant hemeprotein for all assays. Total protein profile (monitored at 280 nm) also showed another peak at 1.5-min elution volume, whose area was quite similar to the one obtained for H2. The proteins eluted at 1.5 min were isolated and analyzed by SDS-electrophoresis, detecting a band displaying proteolytic activity. 0168-1656/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0168-1656(95)00029-l
Biochemical Pharmacology, 1998
Reactivation of inhibited acetylcholinesterase (AChE) is essential for rapid recovery after organ... more Reactivation of inhibited acetylcholinesterase (AChE) is essential for rapid recovery after organophosphate (OP) poisoning. However, following administration of an oxime reactivator, such as pralidoxime mesylate (P2S), in patients poisoned with certain diethylphosphorothioate pesticides, no reactivation is observed, presumably due to reinhibition by circulating anti-cholinesterase OPs. Pretreatment alone with organophosphorus hydrolases (OPH) that are capable of rapidly hydrolyzing OPs was demonstrated, in animals, to confer significant protection against OP toxicity. One strategy to augment the potentially therapeutic scope of OPHs is a combined post-exposure treatment consisting of a drug(s) commonly used against OP toxicity and a suitable hydrolase. In this study, we examined the in vitro ability of OPH from Pseudomonas sp. (OPHps) to prevent reinhibition of P2S-reactivated AChE by excess OPs. The kinetic parameters of the reactivation of a series of diethylphosphoryl-AChE (DEPOAChE) conjugates, obtained by the use of various diethylphosphates, were determined and compared with the rates of reactivation in the presence of OPHps, with and without the OP inhibitors in the reactivation medium. Extrapolation of the in vitro results to in vivo conditions suggests that an OPHps concentration as low as 1 g/mL blood would result in a 100-fold decrease in the concentration of circulating anti-AChE pesticides within less than one blood-circulation time, thereby minimizing reinhibition of the reactivated enzyme. Thus, for DEP-based pesticides, the combination of P2S-OPH treatment can significantly improve clinical recovery after OP intoxication. In addition, it is shown here for the first time that an OPH can effectively hydrolyze quaternary ammonium-containing OPs. This indicates that hydrolysis of phosphorylated oximes, toxic side products of oxime treatment, may also be accelerated by OPHs. BIOCHEM PHARMACOL 55;2: 159 -168, 1998.
Enzyme and Microbial Technology, 2002
The extracellular ligninolytic enzymatic activity of the fungus Irpex lacteus (Polyporus tulipife... more The extracellular ligninolytic enzymatic activity of the fungus Irpex lacteus (Polyporus tulipiferae) grown in nonimmersed liquid culture under nitrogen limitation was characterized. Laccase (LAC), manganese-dependent lignin peroxidase (MNP) and lignin peroxidase (LIP) activities were detected and their formation followed a temporal sequence peaking at day 4, 7 and 11, respectively. Three LIP isozymes, designated LIP1, LIP2 and LIP3, were isolated from manganese-depleted cultures, and exhibited molecular masses of 41, 41 and 44 kDa, respectively, pIs of 5.0, 4.9 and <4.6, respectively, and to 4-5% glycosylation. All isozymes were active towards a range of phenolic substrates, with veratryl alcohol being the preferred one. The kinetics constants obtained for veratryl alcohol and H 2 O 2 were comparable to those of LIPs from other white-rot fungi. The N-terminal sequences of LIP1 and LIP2 were identical and presented 64% similarity with LIP3, and were closely related to the amino acid sequences of LIP isozymes from Phlebia radiata (LIG III), Trametes versicolor (LIG C) and Phanerochaete chrysosporium (GLG2, GLG3 and GLG4). Sequence alignment with other reported LIPs revealed fewer identical or similar residues. Taken together, these data suggest that despite similarity in catalytic properties, kinetics parameters and spectral characteristics, there seems to be high degree of diversity among LIPs of different white-rot fungi.
Applied Microbiology and Biotechnology, 2000
Growth parameters, ligninolytic enzyme activities and ability to degrade polycyclic aromatic hydr... more Growth parameters, ligninolytic enzyme activities and ability to degrade polycyclic aromatic hydrocarbons by the fungus Irpex lacteus were characterized and compared with those of other white rot fungi capable of rapid decolorization of poly R-478 and Remazol Brilliant Blue R dyes. I. lacteus was able to grow on mineral and complex media and efficiently colonized sterile and non-sterile soil by exploratory mycelium growing from a wheat straw inoculum. In shallow stationary cultures growing on high nitrogen mineral medium containing 45 mM ammonium as nitrogen source, the fungus produced lignin peroxidase (LIP), Mn-dependent peroxidase (MnP) and laccase simultaneously, the respective maximal activities of 70, 970 and 36 U/l being attained around day 18. Growing in nitrogen-limited medium (2.4 mM ammonium), no LIP was formed and levels of MnP and laccase decreased significantly. During growth in sterile soil, the fungus synthesized LIP and laccase but not MnP. I. lacteus efficiently removed three- and four-ringed PAHs from liquid media and artificially spiked soil. The variety of ligninolytic enzymes, robust growth, capability of soil colonization and resistance to inhibitory action of soil bacteria make I. lacteus a suitable fungal organism for use in bioremediation.
Uploads
Papers by Nathan Rothschild