The enzyme norcoclaurine synthase (NCS) catalyzes the stereospecific Pictet-Spengler cyclization between dopamine and 4-hydroxyphenylacetaldehyde, the key step in the benzylisoquinoline alkaloid biosynthetic pathway. The crystallographic structure of norcoclaurine synthase from Thalictrum flavum in its complex with dopamine substrate and the nonreactive substrate analogue 4-hydroxybenzaldehyde has been solved at 2.1 Å resolution. NCS shares no common features with the functionally correlated "Pictet-Spenglerases" that catalyze the first step of the indole alkaloids pathways and conforms to the overall fold of the Bet v1-like protein. The active site of NCS is located within a 20-Å -long catalytic tunnel and is shaped by the side chains of a tyrosine, a lysine, an aspartic, and a glutamic acid. The geometry of the amino acid side chains with respect to the substrates reveals the structural determinants that govern the mechanism of the stereoselective Pictet-Spengler cyclization, thus establishing an excellent foundation for the understanding of the finer details of the catalytic process. Site-directed mutations of the relevant residues confirm the assignment based on crystallographic findings.

Pseudomonas stutzeri OX1 meta pathway genes for toluene and o-xylene catabolism were analyzed, and loci encoding phenol hydroxylase, catechol 2,3-dioxygenase, 2-hydroxymuconate semialdehyde dehydrogenase, and 2-hydroxymuconate semialdehyde hydrolase were mapped. Phenol hydroxylase converted a broad range of substrates, as it was also able to transform the nongrowth substrates 2,4-dimethylphenol and 2,5-dimethylphenol into 3,5-dimethylcatechol and 3,6-dimethylcatechol, respectively, which, however, were not cleaved by catechol 2,3-dioxygenase. The identified gene cluster displayed a gene order similar to that of the Pseudomonas sp. strain CF600 dmp operon for phenol catabolism and was found to be coregulated by the tou operon activator TouR. A hypothesis about the evolution of the toluene and o-xylene catabolic pathway in P. stutzeri OX1 is discussed.In bacteria, aerobic catabolic pathways for aromatic hydrocarbon degradation can schematically be divided into two major biochemical steps. First, early reactions, the so-called upper pathways or peripheral routes, channel the hydrocarbons towards the formation of partially oxidized aromatic intermediates. Then, dihydroxylated aromatic molecules that can undergo the cleavage of the ring are produced and further processed to give compounds that can enter the tricarboxylic acid cycle. Whereas a wide variety of very different peripheral routes for the oxidation of many different aromatic hydrocarbons exists, only a limited number of dihydroxylated compounds that can be cleaved and productively processed to enter the tricarboxylic acid cycle are known.A good example of this is represented by the diversity of the known toluene catabolic pathways. Toluene is oxidized through different routes: via progressive oxidation of the methyl group (TOL pathway) (6), via dioxygenation (25), or via monooxygenations of the aromatic ring in different positions (18,22,31). Most of these pathways give rise to (methyl)catechols further processed through meta cleavage pathways. At least in one strain, Pseudomonas mendocina KR1, protocatechuate is produced and then cleaved in intradiol position (27). The genes coding for upper and lower pathways may be clustered in one (32), two (6), or more (18, 29) operons, independently but coordinately regulated.The combination of different upper operons with one or more lower operons can thus increase not only the number of pathways through which a certain molecule can be degraded but also the range of substrates utilized for growth (10), and it is recognized as a mode for the evolution of new catabolic pathways (23, 28).Pseudomonas stutzeri OX1 is able to utilize toluene and o-xylene as the sole carbon and energy sources. For both compounds the degradation proceeds through two successive monooxygenations of the aromatic nucleus catalyzed by toluene-o-xylene monooxygenase (ToMO) followed by extradiol ring cleavage (3). Here we investigate the organization of genes involved in the further degradation of toluene and oxylene derivatives produced by the act...

The regulation of the tou operon of Pseudomonas stutzeri OX1, for degradation of toluene and o-xylene via phenolic intermediates, has been faithfully reconstructed in vitro with purified proteins. The set-up included the prokaryotic enhancer-binding protein TouR, the σ 54 -dependent P ToMO promoter and the σ 54 -containing RNA polymerase. With this system we prove that direct binding of 2-methylphenol (o-cresol) to TouR is the only regulatory step for activation of P ToMO in response to aromatic effectors, thereby ruling out the involvement of other factors or a need for protein processing. In addition, we found that while TouR failed entirely to activate P ToMO in the absence of inducers, the protein had per se a very significant ATPase activity, which was only moderately increased by o-cresol addition. The results presented here support the view that TouR-like proteins are particularly suitable as evolutionary assets to endow recently evolved pathways for the degradation of environmental pollutants with an optimal degree of transcriptional regulation.

Toluene–o-xylene monooxygenase is an enzymatic complex, encoded by the touABCDEF genes, responsible for the early stages of toluene and o-xylene degradation inPseudomonas stutzeri OX1. In order to identify the loci involved in the transcriptional regulation of the tou gene cluster, deletion analysis and complementation studies were carried out with Pseudomonas putida PaW340 as a heterologous host harboring pFB1112, a plasmid that allowed regulated expression, inducible by toluene and o-xylene and their corresponding phenols, of the toluene–o-xylene monooxygenase. A locus encoding a positive regulator, designated touR, was mapped downstream from the tou gene cluster. TouR was found to be similar to transcriptional activators of aromatic compound catabolic pathways belonging to the NtrC family and, in particular, to DmpR (83% similarity), which controls phenol catabolism. By using atouA-C2,3O fusion reporter system and by primer extension analysis, a TouR cognate promoter (PToMO ) was mapped, which showed the typical −24 TGGC, −12 TTGC sequences characteristic of ς54-dependent promoters and putative upstream activating sequences. By using the reporter system described, we found that TouR responds to mono- and dimethylphenols, but not the corresponding methylbenzenes. In this respect, the regulation of theP. stutzeri system differs from that of other toluene or xylene catabolic systems, in which the hydrocarbons themselves function as effectors. Northern analyses indicated low transcription levels oftou structural genes in the absence of inducers. Basal toluene–o-xylene monooxygenase activity may thus transform these compounds to phenols, which then trigger the TouR-mediated response.