Search

2-Nitrobenzoate 2-nitroreductase (NbaA) of Pseudomonas fluorescens strain KU-7 is a unique enzyme, transforming 2-nitroben-zoic acid (2-NBA) and 2,4-dinitrobenzoic acid (2,4-DNBA) to the 2-hydroxylamine compounds. Sequence comparison reveals that NbaA contains a conserved cysteine residue at position 141 and two variable regions at amino acids 65 to 74 and 193 to 216. The truncated mutant Δ65-74 exhibited markedly reduced activity toward 2,4-DNBA, but its 2-NBA reduction activity was unaffected; however, both activities were abolished in the Δ193-216 mutant, suggesting that these regions are necessary for the catalysis and specificity of NbaA. NbaA showed different lag times for the reduction of 2-NBA and 2,4-DNBA with NADPH, and the reduction of 2,4-DNBA, but not 2-NBA, failed in the presence of 1 mM dithiothreitol or under anaerobic conditions, indicating oxidative modification of the enzyme for 2,4-DNBA. The enzyme was irreversibly inhibited by 5,5'-dithio-bis-(2-nitrobenzoic acid) and ZnCl2, which bind to reactive thiol/thiolate groups, and was eventually inactivated during the formation of higher-order oligomers at high pH, high temperature, or in the presence of H2O2. SDS-PAGE and mass spectrometry revealed the formation of intermolecular disulfide bonds by involvement of the two cysteines at positions 141 and 194. Site-directed mutagene-sis indicated that the cysteines at positions 39,103,141, and 194 played a role in changing the enzyme activity and specificity toward 2-NBA and 2,4-DNBA. This study suggests that oxidative modifications of NbaA are responsible for the differential specificity for the two substrates and further enzyme inactivation through the formation of disulfide bonds under oxidizing conditions. [ABSTRACT FROM AUTHOR]

We used preS2-S'-β-galactosidase, a three-domain fusion protein that aggregates extensively at 43°C in the cytoplasm of Escherichia coli, to search for multicopy suppressors of protein aggregation and inclusion body formation and took advantage of the known differential solubility of preS2-S'-β-galactosidase at 37 and 43°C to develop a selection procedure for the gene products that would prevent its aggregation in vivo at 43°C. First, we demonstrate that the differential solubility of preS2-S'-β-galactosidase results in a lactose-positive phenotype at 37°C as opposed to a lactose-negative phenotype at 43°C. We searched for multicopy suppressors of preS2-S'-β-galactosidase aggregation by selecting pink lactose-positive colonies on a background of white lactose-negative colonies at 43°C after transformation of bacteria with an E. coli gene bank. We discovered that protein isoaspartate methyltransferase (PIMT) is a multicopy suppressor of preS2-S'-β-galactosidase aggregation at 43°C. Overexpression of PIMT reduces the amount of preS2-S'-β-galactosidase found in inclusion bodies at 43°C and increases its amount in soluble fractions. It reduces the level of isoaspartate formation in preS2-S'-β-galactosidase and increases its thermal stability in E. coil crude extracts without increasing the thermostability of a control protein, citrate synthase, in the same extracts. We could not detect any induction of the heat shock response resulting from PIMT overexpression, as judged from amounts of DnaK and GroEL, which were similar in the PIMT-overproducing and control strains. These results suggest that PIMT might be overburdened in some physiological conditions and that its overproduction may be beneficial in conditions in which protein aggregation occurs, for example, during biotechnological protein overproduction or in protein aggregation diseases. [ABSTRACT FROM AUTHOR]