Roberta Scognamiglio

Phenol Hydroxylase and Toluene/o-Xylene Monooxygenase from Pseudomonas stutzeri OX1: Interplay between Two Enzymes

ABSTRACT Degradation of aromatic hydrocarbons by aerobic bacteria is generally divided into an upper pathway, which produces dihydroxylated aromatic intermediates by the action of monooxygenases, and a lower pathway, which processes these intermediates down to molecules that enter the citric acid cycle. Bacterial multicomponent monooxygenases (BMMs) are a family of enzymes divided into six distinct groups. Most bacterial genomes code for only one BMM, but a few cases (3 out of 31) of genomes coding for more than a single monooxygenase have been found. One such case is the genome of Pseudomonas stutzeri OX1, in which two different monooxygenases have been found, phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO). We have already demonstrated that ToMO is an oligomeric protein whose subunits transfer electrons from NADH to oxygen, which is eventually incorporated into the aromatic substrate. However, no molecular data are available on the structure and on the mechanism of action of PH. To understand the metabolic significance of the association of two similar enzymatic activities in the same microorganism, we expressed and characterized this novel phenol hydroxylase. Our data indicate that the PH P component of PH transfers electrons from NADH to a subcomplex endowed with hydroxylase activity. Moreover, a regulatory function can be suggested for subunit PH M. Data on the specificity and the kinetic constants of ToMO and PH strongly support the hypothesis that coupling between the two enzymatic systems optimizes the use of nonhydroxylated aromatic molecules by the draining effect of PH on the product(s) of oxidation catalyzed by ToMO, thus avoiding phenol accumulation.

Tuning the Specificity of the Recombinant Multicomponent Toluene o-Xylene Monooxygenase from Pseudomonas sp. Strain OX1 for the Biosynthesis of Tyrosol from 2-Phenylethanol

ABSTRACT Biocatalysis is today a standard technology for the industrial production of several chemicals, and the number of biotransformation processes running on a commercial scale is constantly increasing. Among biocatalysts, bacterial multicomponent monooxygenases (BMMs), a diverse group of nonheme diiron enzymes that activate dioxygen, are of primary interest due to their ability to catalyze a variety of complex oxidations, including reactions of mono- and dihydroxylation of phenolic compounds. In recent years, both directed evolution and rational design have been successfully used to identify the molecular determinants responsible for BMM regioselectivity and to improve their activity toward natural and nonnatural substrates. Toluene o-xylene monooxygenase (ToMO) is a BMM isolated from Pseudomonas sp. strain OX1 which hydroxylates a wide spectrum of aromatic compounds. In this work we investigate the use of recombinant ToMO for the biosynthesis in recombinant cells of Escherichia coli strain JM109 of 4-hydroxyphenylethanol (tyrosol), an antioxidant present in olive oil, from 2-phenylethanol, a cheap and commercially available substrate. We initially found that wild-type ToMO is unable to convert 2-phenylethanol to tyrosol. This was explained by using a computational model which analyzed the interactions between ToMO active-site residues and the substrate. We found that residue F176 is the major steric hindrance for the correct positioning of the reaction intermediate leading to tyrosol production into the active site of the enzyme. Several mutants were designed and prepared, and we found that the combination of different mutations at position F176 with mutation E103G allows ToMO to convert up to 50% of 2-phenylethanol into tyrosol in 2 h.

Conformational analysis of putative regulatory subunit D of the toluene/o-xylene-monooxygenase complex from pseudomonas stutzeri OX1

A gene cluster isolated from Pseudomonas stutzeri OX1 genomic DNA and containing six ORFs codes for toluene/o‐xylene‐monooxygenase. The putative regulatory D subunit was expressed in Escherichia coli and purified. Its protein sequence was verified by mass spectrometry mapping and found to be identical to the sequence predicted on the basis of the DNA sequence. The surface topology of subunit D in solution was probed by limited proteolysis carried out under strictly controlled conditions using several proteases as proteolytic probes. The same experiments were carried out on the homologous P2 component of the multicomponent phenol hydroxylase from Pseudomonas putida CF600. The proteolytic fragments released from both proteins in their native state were analyzed by electrospray mass spectrometry, and the preferential cleavage sites were assessed.

A recombinant ribosome-inactivating protein from the plant Phytolacca dioica L. produced from a synthetic gene

Phytolacca dioica L. leaves produce at least two type‐I ribosome‐inactivating proteins. Each polypeptide chain is subjected to different post‐translational modifications giving rise to PD‐L1 and PD‐L2, and PD‐L3 and PD‐L4, each polypeptide pair having the same primary structure. With the aim of exploiting the cytotoxic properties of these proteins as potential biological phytodrugs, a gene encoding PD‐L4 was designed based on criteria expected to maximize the translation efficiency in tomato. The gene was constructed from 18 oligonucleotides and preliminarily expressed in Escherichia coli, using the T7 promoter system. The protein produced was insoluble and accumulated in inclusion bodies to about 300 mg/l of culture. Ribosome‐inactivating activity was generated by controlled oxidation of the reduced and denatured protein. The recombinant protein was indistinguishable from natural PD‐L4 as isolated from leaves of Phytolacca dioica, in both catalytic activity and primary structure.

PHK from phenol hydroxylase of Pseudomonas sp. OX1. Insight into the role of an accessory protein in bacterial multicomponent monooxygenases

Bacterial multicomponent monooxygenases (BMMs) are members of a wide family of diiron enzymes that use molecular oxygen to hydroxylate a variety of aromatic compounds. The presence of genes encoding for accessory proteins not involved in catalysis and whose role is still elusive, is a common feature of the gene clusters of several BMMs, including phenol hydroxylases and several soluble methane monooxygenases. In this study we have expressed, purified, and partially characterized the accessory component PHK of the phenol hydroxylase from Pseudomonas sp. OX1, a bacterium able to degrade several aromatic compounds. The phenol hydroxylase (ph) gene cluster was expressed in Escherichia coli/JM109 cells in the absence and in the presence of the phk gene. The presence of the phk gene lead to an increase in the hydroxylase activity of whole recombinant cells with phenol. PHK was assessed for its ability to interact with the active hydroxylase complex. Our results show that PHK is neither involved in the catalytic activity of the phenol hydroxylase complex nor required for the assembly of apo-hydroxylase. Our results suggest instead that this component may be responsible for enhancing iron incorporation into the active site of the apo-hydroxylase.

The structure of the O-specific polysaccharide from the lipopolysaccharide of Pseudomonas sp. OX1 cultivated in the presence of the azo dye Orange II

The Gram-negative bacterium Pseudomonas sp. OX1, previously known as Pseudomonas stutzeri OX1, is endowed with a high metabolic versatility. In fact, it is able to utilize a wide range of toxic organic compounds as the only source of carbon and energy for growth. It has been recently observed that, while growing on a glucose-containing liquid medium, Pseudomonas sp. OX1 can reduce azo dyes, ubiquitous pollutants particularly resistant to chemical and physical degradation, with this azoreduction being a process able to generate enough energy to sustain bacterial survival. We have found that, under these conditions, modifications in the primary structure of the O-specific polysaccharide (OPS) within the lipopolysaccharides occur, leading to remarkable changes both in the monosaccharide composition and in the architecture of the repeating unit, with respect to the polysaccharide produced in the absence of azo dyes. In the present paper, we present the complete structure of this O-specific polysaccharide, whose repeating unit is the following: [Formula: see text] This structure is totally different from the one determined from Pseudomonas sp. OX1 grown on rich medium.