Hee-Sung Park

Identification and Characterization of the Nitrobenzene Catabolic Plasmids pNB1 and pNB2 in Pseudomonas putida HS12

Pseudomonas putida HS12, which is able to grow on nitrobenzene, was found to carry two plasmids, pNB1 and pNB2. The activity assay experiments of wild-type HS12(pNB1 and pNB2), a spontaneous mutant HS121(pNB2), and a cured derivative HS124(pNB1) demonstrated that the catabolic genes coding for the nitrobenzene-degrading enzymes, designated nbz, are located on two plasmids, pNB1 and pNB2. The genes nbzA, nbzC, nbzD, and nbzE, encoding nitrobenzene nitroreductase, 2-aminophenol 1,6-dioxygenase, 2-aminomuconic 6-semialdehyde dehydrogenase, and 2-aminomuconate deaminase, respectively, are located on pNB1 (59.1 kb). Meanwhile, the nbzB gene encoding hydroxylaminobenzene mutase, a second-step enzyme in the nitrobenzene catabolic pathway, was found in pNB2 (43.8 kb). Physical mapping, cloning, and functional analysis of the two plasmids and their subclones in Escherichia coli strains revealed in more detail the genetic organization of the catabolic plasmids pNB1 and pNB2. The genes nbzA and nbzB are located on the 1.1-kb SmaI-SnaBI fragment of pNB1 and the 1.0-kb SspI-SphI fragment of pNB2, respectively, and their expressions were not tightly regulated. On the other hand, the genes nbzC, nbzD, and nbzE, involved in the ring cleavage pathway of 2-aminophenol, are localized on the 6.6-kb SnaBI-SmaI fragment of pNB1 and clustered in the order nbzC-nbzD-nbzE as an operon. The nbzCDE genes, which are transcribed in the opposite direction of the nbzA gene, are coordinately regulated by both nitrobenzene and a positive transcriptional regulator that seems to be encoded on pNB2.

A chemical biology route to site-specific authentic protein modifications

Radicals push proteins beyond genes Chemically modifying proteins after their translation can expand their structural and functional roles (see the Perspective by Hofmann and Bode). Two related methods describe how to exploit free radical chemistry to form carbon-carbon bonds between amino acid residues and a selected functional group. Wright et al. added a wide range of functional groups to proteins containing dehydroalanine precursors, with borohydride mediating the radical chemistry. Yang et al. employed a similar approach, using zinc in combination with copper ions. Together, these results will be useful for introducing functionalities and labels to a wide range of proteins. Science, this issue pp. 597 and 623; see also p. 553 Radical chemistry mediated by metal ions allows for the selective chemical modification of a range of proteins. Many essential biological processes are controlled by posttranslational protein modifications. The inability to synthetically attain the diversity enabled by these modifications limits functional studies of many proteins. We designed a three-step approach for installing authentic posttranslational modifications in recombinant proteins. We first use the established O-phosphoserine (Sep) orthogonal translation system to create a Sep-containing recombinant protein. The Sep residue is then dephosphorylated to dehydroalanine (Dha). Last, conjugate addition of alkyl iodides to Dha, promoted by zinc and copper, enables chemoselective carbon-carbon bond formation. To validate our approach, we produced histone H3, ubiquitin, and green fluorescent protein variants with site-specific modifications, including different methylations of H3K79. The methylated histones stimulate transcription through histone acetylation. This approach offers a powerful tool to engineer diverse designer proteins.

A Facile Strategy for Selective Incorporation of Phosphoserine into Histones

Histones are the main protein components of chromatin. They undergo numerous posttranslational modifications, including phosphorylation, acetylation, and methylation, which often affect each other[1, 2]. Such cross-regulation of histone modification is known to play a central role in many physiological processes. Phosphorylation of histone H3 at serine 10 (H3S10) and acetylation of N-terminal lysine residues in histone H3 are representative markers of transcriptional activation in eukaryotic cells. Many in vivo studies have suggested a possible link between these modifications[3-5], but this association is not yet established. Early in vitro studies using small synthetic H3 peptides provided mixed and conflicting results regarding the association between phosphorylation and acetylation[3, 4, 6], and recent peptide experiments have failed to find any correlation[7]. However, since synthetic peptides differ from the physiological substrates that undergo modifications, they can hardly represent the real physiological interactions between chromatin and modifying enzymes. Such peptides often show more than a thousand-fold lower binding abilities for modifying proteins compared to nucleosomal substrates[6]. Inconsistent results and a lack of direct evidence have led to the development of two opposing models (synergistic and independent) for these modifications[6, 8]. Thus, despite extensive studies, the correlation between phosphorylation and acetylation in histone H3 remains unclear.

Controlled and oriented immobilization of protein by site-specific incorporation of unnatural amino acid.

Immobilization of proteins in a functionally active form and proper orientation is crucial for effective surface-based analysis of proteins. Here we present a general method for controlled and oriented immobilization of protein by site-specific incorporation of unnatural amino acid and click chemistry. The utility and potential of this method was demonstrated by applying it to the analysis of interaction between a pathogenic protein DrrA of Legionella pneumophila and its binding partner Rab1 of human. Kinetic analysis of Rab1 binding onto the DrrA-immobilized surfaces using surface plasmon resonance revealed that immobilization of site-specifically biotinylated DrrA results in about 10-fold higher sensitivity in binding assay than the conventional immobilization of DrrA with random orientation. The present method is expected to find wide applications in the fields of the surface-based studies of protein-protein (or ligand) interactions, drug screening, biochip, and single molecule analysis.

Simple and efficient strategy for site-specific dual labeling of proteins for single-molecule fluorescence resonance energy transfer analysis.

Analysis of protein dynamics using single-molecule fluorescence resonance energy transfer (smFRET) is widely used to understand the structure and function of proteins. Nonetheless, site-specific labeling of proteins with a pair of donor and acceptor dyes still remains a challenge. Here we present a general and facile method for site-specific dual labeling of proteins by incorporating two different, readily available, unnatural amino acids (p-acetylphenylalanine and alkynyllysine) for smFRET. We used newly evolved alkynyllysine-specific aminoacyl-tRNA synthetase/tRNA(UCA) and p-acetylphenylalanyl-tRNA synthetase/tRNA(CUA). The utility of our approach was demonstrated by analyzing the conformational change of dual-labeled calmodulin using smFRET measurements. The present labeling approach is devoid of major limitations in conventional cysteine-based labeling. Therefore, our method will significantly increase the repertoire of proteins available for FRET study and expand our ability to explore more complicated molecular dynamics.

Toward understanding phosphoseryl-tRNACys formation: The crystal structure of Methanococcus maripaludis phosphoseryl-tRNA synthetase

A number of archaeal organisms generate Cys-tRNACys in a two-step pathway, first charging phosphoserine (Sep) onto tRNACys and subsequently converting it to Cys-tRNACys. We have determined, at 3.2-Å resolution, the structure of the Methanococcus maripaludis phosphoseryl-tRNA synthetase (SepRS), which catalyzes the first step of this pathway. The structure shows that SepRS is a class II, α4 synthetase whose quaternary structure arrangement of subunits closely resembles that of the heterotetrameric (αβ)2 phenylalanyl-tRNA synthetase (PheRS). Homology modeling of a tRNA complex indicates that, in contrast to PheRS, a single monomer in the SepRS tetramer may recognize both the acceptor terminus and anticodon of a tRNA substrate. Using a complex with tungstate as a marker for the position of the phosphate moiety of Sep, we suggest that SepRS and PheRS bind their respective amino acid substrates in dissimilar orientations by using different residues.

Genetic and Structural Organization of the Aminophenol Catabolic Operon and Its Implication for Evolutionary Process

The aminophenol (AP) catabolic operon in Pseudomonas putida HS12 mineralizing nitrobenzene was found to contain all the enzymes responsible for the conversion of AP to pyruvate and acetyl coenzyme A via extradiol meta cleavage of 2-aminophenol. The sequence and functional analyses of the corresponding genes of the operon revealed that the AP catabolic operon consists of one regulatory gene, nbzR, and the following nine structural genes, nbzJCaCbDGFEIH, which encode catabolic enzymes. The NbzR protein, which is divergently transcribed with respect to the structural genes, possesses a leucine zipper motif and a MarR homologous domain. It was also found that NbzR functions as a repressor for the AP catabolic operon through binding to the promoter region of the gene cluster in its dimeric form. A comparative study of the AP catabolic operon with other meta cleavage operons led us to suggest that the regulatory unit (nbzR) was derived from the MarR family and that the structural unit (nbzJCaCbDGFEIH) has evolved from the ancestral meta cleavage gene cluster. It is also proposed that these two functional units assembled through a modular type gene transfer and then have evolved divergently to acquire specialized substrate specificities (NbzCaCb and NbzD) and catalytic function (NbzE), resulting in the creation of the AP catabolic operon. The evolutionary process of the AP operon suggests how bacteria have efficiently acquired genetic diversity and expanded their metabolic capabilities by modular type gene transfer.

Production of L-DOPA(3,4-dihydroxyphenyl-L-alanine) from benzene by using a hybrid pathway.

As an alternative approach to the production of L-DOPA from a cheap raw material, we constructed a hybrid pathway consisting of toluene dioxygenase, toluene cis-glycol dehydrogenase, and tyrosine phenol-lyase. In this pathway, catechol is formed from benzene through the sequential action of toluene dioxygenase and toluene cis-glycol dehydrogenase, and L-DOPA is synthesized from the resulting catechol in the presence of pyruvate and ammonia by tyrosine phenol-lyase cloned from Citrobacter freundii. When the hybrid pathway was expressed in E. coli, production of L-DOPA was as low as 3 mM in 4 h due to the toxic effect of benzene on the cells. In order to reduce lysis of cells, Pseudomonas aeruginosa was employed as an alternative, which resulted in accumulation of about 14 mM L-DOPA in 9 h, showing a stronger resistance to benzene.

Efficient Single-Molecule Fluorescence Resonance Energy Transfer Analysis by Site-Specific Dual-Labeling of Protein Using an Unnatural Amino Acid

Single-molecule fluorescence resonance energy transfer (smFRET) measurement provides a unique and powerful approach to understand complex biological processes including conformational and structural dynamics of individual biomolecules. For effective smFRET analysis of protein, site-specific dual-labeling with two fluorophores as an energy donor and an acceptor is crucial. Here we demonstrate that site-specific dual-labeling of protein via incorporation of unnatural amino acid provides a clearer picture for the folded and unfolded states of the protein in smFRET analysis than conventional labeling using double cysteines. As a model study, maltose-binding protein (MBP) was dually labeled via incorporation of ρ-azido-l-phenylalanine and cysteine at specific positions, immobilized on a surface, and subjected to smFRET analysis under native and denaturing conditions. The resulting histograms show that site-specific dual-labeling results in a more homogeneous distribution in protein populations, enabling a precise smFRET analysis of protein.

Chemical biology approaches for studying posttranslational modifications

ABSTRACT Posttranslational modification (PTM) is a key mechanism for regulating diverse protein functions, and thus critically affects many essential biological processes. Critical for systematic study of the effects of PTMs is the ability to obtain recombinant proteins with defined and homogenous modifications. To this end, various synthetic and chemical biology approaches, including genetic code expansion and protein chemical modification methods, have been developed. These methods have proven effective for generating site-specific authentic modifications or structural mimics, and have demonstrated their value for in vitro and in vivo functional studies of diverse PTMs. This review will discuss recent advances in chemical biology strategies and their application to various PTM studies.