DEGRADATION OF 3-NITROPHENOL BY PSEUDOMONAS-PUTIDA B2 OCCURS VIA 1,2,4-BENZENETRIOL

Growth of Pseudomonas putida B2 in chemostat cultures on a mixture of 3-nitrophenol and glucose induced 3-nitrophenol and 1,2,4-benzenetriol -dependent oxygen uptake activities. Anaerobic incubations of cell sus pensions with 3-nitrophenol resulted in complete conversion of the sub strate to ammonia and 1,2,4-benzenetriol. This indicates that P. putid a B2 degrades 3-nitrophenol via 1,2,4-benzenetriol, via a pathway invo lving a hydroxylaminolyase. Involvement of this pathway in nitroaromat ic metabolism has previously only been found for degradation of 4-nitr obenzoate. Reduction of 3 nitrophenol by cell-free extracts was strict ly NADPH-dependent. Attempts to purify the enzymes responsible for 3-n itrophenol metabolism were unsuccessful, because their activities were extremely unstable. 3-Nitrophenol reductase was therefore characteriz ed in cell-free extracts. The enzyme had a sharp pH optimum at pH 7 an d a temperature optimum at 25 degrees C. At 30 degrees C, reductase ac tivity was completely destroyed within one hour, while at 0 degrees C, the activity in cell-free extracts was over 100-fold more stable. The K-m values for NADPH and 3-nitrophenol were estimated at 0.17 mM and below 2 mu M, respectively. The substrate specificity of the reductase activity was very broad: all 17 nitroaromatics tested were reduced by cell-free extracts. However, neither intact cells nor cell-free extra cts could convert a set of synthesized hydroxylaminoaromatic compounds to the corresponding catechols and ammonia. Apparently, the hydroxyla minolyase of P. putida B2 has a very narrow substrate specificity, ind icating that this organism is not a suitable biocatalyst for the indus trial production of catechols from nitroaromatics.