Bacterial oxygenases: potential, features and new investigation methods.

Bacterial oxygenases, the enzymes involved in the breakdown pathways of aromatic and aliphatic hydrocarbons, have aroused great interest within the scientific community for their potential applications in a number of different fields: environmental (bioremediation and biomonitoring), biotechnological (green chemistry) and biomedical (drug production). The aims of this study were 1) to clone new bacterial oxygenases using conventional methods and further develop new screening techniques and 2) to assess the ability of the newly-cloned enzyme(s), of toluene/o-xilene monooxygenase (ToMO) and of some of its mutants, to exploit various types of aromatic substrate on which the activity of these enzymes has never previously been assessed. Using conventional techniques, Pseudomonas sp. N1 genes coding for naphthalene dioxygenase (NDO) and Sphingobium sp. PhS genes, putatively coding for Phenanthrene dioxygenase (PhDO), were cloned; of the two enzymes only NDO appeared to be functional. ToMO is an enzyme complex formed from six different subunits, ABCDEF; subunit A (TouA) is part of the terminal hydroxylase and contains the active site. In this study, in addition to wild type enzymes, the TouA mutants D211A and D211A/E214G, previously created by this laboratory, and TouA E214G, already described in the literature, were also used. The ability of ToMO and TouA mutants, and of NDO to hydroxylate different aromatic compounds other than their natural substrates (toluene and naphthalene respectively) was evaluated by biotransformation assays using, as substrates, the following molecules: 1,2,3-trimethoxybenzene, anisole, benzophenone, bibenzyl, biphenyl, nitrobenzene, quinoline, and trans-stilbene. These compounds were chosen for the environmental, biotechnological and pharmacological importance of their hydroxylated derivatives. To this end, ToMO, TouA mutants and NDO were expressed in the heterologous host E. coli JM109. For both enzymes, the products of bioconversion were identified by GC-MS and, using the appropriate standard of reference, quantified by HPLC analysis. ToMO proved active in all substrates tested. The mutant E214G, like the wild type, was capable of oxidising all the compounds tested: in almost all the biotransformations both the mutant form and the wild type behaved in the same way in that they both produced the same isomers in similar proportions. In terms of catalytic efficiency, the mutant E214G was found to be most powerful: the mutants D211A and D211A/E214G were capable of hydroxylating only a few of the substrates used (1,2,3-trimethoxybenzene, anisole, benzophenone, biphenyl) with very low yields. However, considering the fact that strains expressing the mutants D211A e D211A/E214G showed impaired growth and expression problems, probably attributable to unwanted mutations introduced during mutagenesis, these data cannot be considered definitive. The ability of NDO to perform dihydroxylation processes was confirmed but, in addition, monohydroxylation reactions (quinoline) and dealkylation reactions (anisole and 1,2,3-trimethoxybenzene) were observed. The quantitative analysis of the biotransformation products showed that the enzyme efficiency decreased with the increasing steric footprint of the substrate. The failure of nitrobenzene to be transformed suggests that, in addition to steric footprint, other factors such as the presence of deactivating substituent groups plays a key role in the catalytic process of this enzyme. Both NDO and ToMO have shown a good potential in the field of biotransformation and further studies could lead to the production of more useful and efficient enzyme variants. During an internship at the University of Granada in Spain, a new protocol, based on the SIGEX (Substrate Induced Gene Expression) system, was developed to screen bacterial genomic libraries. This method, which is normally used to screen metagenomic libraries, was adapted to analyse libraries created from a single bacterial genome. With this technique it is possible to perform a rapid screening on the basis of a signal emitted from a reporter gene included in the construct used in creating the library. Using a previously constructed library, this approach allowed, in a short time, 60 clones to be isolated that had a high probability of containing naphthalene-dependent regulatory genes. The sequencing of the obtained clones, a necessary step in order to validate the protocol, in currently in progress.