Anthony J. Herlt

Multiple-Site Labeling of Proteins with Unnatural Amino Acids**

The advent of efficient systems using genetic encoding for the site-specific incorporation of unnatural amino acids (UAA) into proteins has opened countless new possibilities for studying the structure, dynamics, and interactions of proteins. In particular, orthogonal pairs of amber suppressor tRNA (MjtRNA) and tyrosyl-tRNA synthetase (MjTyrRS) of Methanocaldococcus jannaschii have been evolved that specifically recognize amber stop codons for the incorporation of over 40 different UAAs. Although these systems can produce mutant protein with yields as high as for the wildtype, the protein yields are strongly context dependent. Thus, the presence of a single amber stop codon in the gene of a target protein can lead to unacceptably poor expression yields. Many efforts have been directed at biasing the competition between the Escherichia coli release factor RF1 that recognizes the amber stop codon and the suppressor tRNA in favor of the production of full-length protein. Thus, the incorporation of UAAs can be enhanced by omission of RF1 from a cell-free (CF) synthesis system reconstituted from the individually purified enzyme components. CF systems were also used to eliminate RF1 with anti-RF1 antibodies, or by deploying an RNA aptamer against RF1. Unfortunately, all these approaches are affordable only for small-scale sample preparations, and protein yields can be compromised by antibodies, while aptamers still yield truncated protein as the predominant product. Heat-shock inactivation of a thermosensitive mutant of RF1 in a low-yield E. coli CF system increased the incorporation efficiencies of UAAs to at most 75% (< 50% in most cases), while the fidelity of translation was compromised by prolonged heat treatment. Depletion of tagged RF1 from a cell-free S30 extract by affinity chromatography was reported, but the identity of the tag was not revealed and anti-RF1 antibodies were required for complete elimination. In a different approach, truncation of the ribosomal protein L11 was shown to weaken the binding of RF1, but the resulting protein yields with UAAs were only moderately enhanced. Initial efforts to produce RF1-deficient E. coli strains led to compromised strains that depended on unnatural amino acids for growth (making protein expression expensive) or strains that were not entirely independent of RF1. Recently, an enhanced version of release factor RF2 enabled the production of an RF1-free E. coli DH10b strain and the incorporation of UAAs at multiple sites in vivo, but the protein yields obtained with UAAs were significantly reduced compared with those obtained with the natural amino acid (tyrosine). In view of the cost of many of the most attractive UAAs and the difficulty to tailor the concentrations of the aminoacyl-tRNA synthetase (RS) and suppressor tRNA in in vivo expression systems to the requirements of different UAAs and incorporation sites, we developed a continuous exchange cell-free (CECF) system that allows facile, inexpensive, and complete removal of the release factor RF1 from an S30 extract derived from the widely used high-yielding and protease-deficient E. coli strain BL21 Star (DE3) (Invitrogen). The approach relies on replacing wild-type RF1 by a mutant with a C-terminal affinity tag consisting of three consecutive chitin-binding domains (RF1-CBD3) for selective removal by filtration through a chitin column after production of an S30 extract in the usual way. The chitin-binding-domain tag allows the removal of RF1 under conditions that maintain the full activity of the S30 extract and at the same time delivers dramatically improved incorporation yields of difficult UAAs at difficult positions, suppresses the production of truncated protein, and allows the incorporation of UAAs at multiple sites in the same protein. Conveniently, the modified strain is fully compatible with protein expression from pET vectors, which are the most frequently used vectors for protein production in structural biology. The yields of wild-type protein obtained with chitintreated (S30 ) and untreated (S30) extracts were indistinguishable from the yields obtained with S30 extracts prepared from the original BL21 Star (DE3) strain (Figure S3 in the Supporting Information). To assess and optimize the expression yields with UAAs without having to purify the proteins, we used the MjtRNA/MjTyrRS pair evolved for incorporation of the fluorescent UAA l-(7-hydroxy-coumarin-4-yl)ethylglycine (Hco). Optimization employed the West Nile virus NS2B-NS3 protease (WNVpro), a 27 kDa protein that is also an established drug target. Optimization of the concentration of aminoacyl-tRNA syn[*] Dr. K. V. Loscha, A. J. Herlt, Dr. R. Qi, Dr. T. Huber, Dr. K. Ozawa, Prof. G. Otting Research School of Chemistry, The Australian National University Canberra ACT 0200 (Australia) E-mail: gottfried.otting@anu.edu.au Homepage: http://rsc.anu.edu.au/~go/

Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino Acids

The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and to probe the role of peripheral active site residues in nonchemical steps of the catalytic cycle (substrate binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with unnatural L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino acids, as well as leucine, phenylalanine, and tryptophan. Kinetic analysis suggests that the 7-hydroxyl group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product release step of substrate turnover as a result of its electrostatic repulsion of the negatively charged 4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficient catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to the difficulty in improving this native activity through screening hundreds of thousands of mutants with natural amino acids. These results demonstrate that designer amino acids provide easy access to new and valuable sequence and functional space for the engineering and evolution of existing enzyme functions.

Characterization of the phenylurea hydrolases A and B: founding members of a novel amidohydrolase subgroup.

Mycobacterium brisbanense strain JK1, a bacterium capable of degrading the herbicide diuron, was isolated from herbicide-exposed soil. A gene/enzyme system with diuron hydrolase activity was isolated from this strain and named PUH (phenylurea hydrolase) B (puhB/PuhB) because of its close similarity to the previously characterized PUH A (puhA/PuhA). Both PUHs were heterologously expressed, purified and characterized. The PUHs were found to oligomerize as hexamers in solution, with each monomer containing a mononuclear Zn2+ active site. Sequence analysis showed that these enzymes belong to the metal-dependent amidohydrolase superfamily, although they contain a hitherto unreported Asn-X-His metal-binding motif and appear to form a novel sub-group within this superfamily. The effects of temperature and solvent on the enzymes were characterized. Determination of the kinetic parameters of the PUHs was used alongside Brønsted plots to develop a plausible catalytic mechanism, which is similar to that used by urease. In addition to the primary PUH activity, both enzymes are catalytically promiscuous, efficiently hydrolysing esters, carbamates and phosphotriesters. In fact, an analogue of diuron, in which the C-N bond was replaced by a C-O bond, was found to be turned over as efficiently as diuron, suggesting that the substrate specificity is predominantly determined by steric factors. The discovery of PuhA and PuhB on separate continents, and the absence of any other close homologues in the available sequence databases, poses a challenging question regarding the evolutionary origins of these enzymes.

Structure-Based Rational Design of a Phosphotriesterase

ABSTRACT In silico substrate docking of both stereoisomers of the pesticide chlorfenvinphos (CVP) in the phosphotriesterase from Agrobacterium radiobacter identified two residues (F131 and W132) that prevent productive substrate binding and cause stereospecificity. A variant (W131H/F132A) was designed that exhibited ca. 480-fold and 8-fold increases in the rate of Z-CVP and E-CVP hydrolysis, respectively, eliminating stereospecificity.

DNA binding of the anti-cancer platinum complex trans-[{Pt(NH3)2Cl}2μ-dpzm]2+

The DNA binding of the dinuclear platinum complex trans-[{Pt(NH3)2Cl}2μ-dpzm]2+ (di-Pt), linked with the 4,4′-dipyrazolylmethane (dpzm) linker, was examined by 1H and 195Pt NMR and transcription assays. At 60 °C, di-Pt reacts with guanosine two-fold slower (t1/2: 1 h) than cisplatin (t1/2: 0.5 h). With adenosine, the di-Pt complex reacts much slower (t1/2: 7 h) forming a range of different adducts through the N7 and either the N1 or N3 positions of the nucleoside. From 1H NMR analysis of the major product of the reaction of di-Pt with the oligonucleotide d(ATGCAT)2, purified by HPLC, it was determined that the dinuclear platinum complex can readily form a 1,2-GG interstrand DNA cross-link. Transcription assays using di-Pt and the lac UV5 promoter indicated that the metal complex forms an array of adducts vastly different from cisplatin. The two greatest blockages occurred at adenine residues, with possible interstrand and intrastrand AA and AG adducts being formed. These results indicate that unlike other platinum based anti-cancer agents, di-Pt binds to DNA with a preference for adenine bases.

A new approach to the synthesis of ‘chiral’ glycine

Abstract The stereospecific coordination of N-benzylglycinate ion in ΔR-(N-benzylglycinato)bis(ethylene-diamine) cobalt(III) chloride has been determined by X-ray crystallographic analysis, rotatory dispersion and 1H NMR spectroscopy. The chiral glycinato-N and Co centres influence the relative rates of exchange of the diastereotopic glycine methylene protons in basic solution (pH 10·5 with Na3PO4inD2O) and a synthesis supposedly of S-(N-benzyl)- 2-2H glycinate ion ≈ 80% optical purity) has been achieved.

Using a Genetically Encoded Fluorescent Amino Acid as a Site-Specific Probe to Detect Binding of Low-Molecular-Weight Compounds

Development of enzyme inhibitors requires an activity assay for the identification of hits and lead compounds. To determine dissociation constants in a straightforward manner, we explored the use of a genetically encoded fluorescent amino acid for site-specific tagging of the target protein. The unnatural amino acid 7-(hydroxy-coumarin-4-yl) ethylglycine (Hco) was site-specifically incorporated in the target protein by cell-free protein synthesis using an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase pair. Using the West Nile virus nonstructural protein 2B-nonstructural protein 3 protease as the target protein, the fluorescence of Hco-tagged samples proved to be exquisitely sensitive to the presence of inhibitors and small ligand molecules if they bind in the vicinity of the Hco residue. No significant change in fluorescence was observed when the ligand-binding site was far from the Hco residue. Hco-tagged proteins thus combine outstanding sensitivity with accurate information on the site of binding, making Hco labeling an attractive tool in drug discovery.

The absolute configuration of juvenile hormone III bisepoxide

The absolute configuration of juvenile hormone III bisepoxide, the putative characteristic juvenile hormone of higher dipteran insects, has been established as 2E,6S,7S,10R by HPLC and cyclodextrin-modified CE comparison of synthetic stereoisomers with biosynthetically 3H-labelled hormone from the Australian sheep blowfly Lucilia cuprina.

Cyclopentanoids from phenol—9 : 3-Alkyl- and 3-alkenyl-5-hydroxycyclopent-2-enones☆

Abstract An efficient and versatile synthesis of 3-alkyl- and 3-alkenyl-5-hydroxycyclopent-2-enones is described. The key intermediate, 4-(t-butyldimethylsilyloxy)-3-methoxycyclopent-2-enone (5), is prepared in five steps from phenol. 1,2-Addition of various organolithium and Grignard reagents yields tertiary alcohol intermediates which afford the title compounds after solvolysis and desilylation.

Condensation of formaldehyde with chelated glycine and ethylenediamine: a new macrocycle synthesis; X-ray structures of [α-hydroxymethylserine-bis(ethylenediamine)cobalt(III)]2+ and [α-hydroxymethylserine-1,4,8,11-tetra-aza-6,13-dioxacyclotetradecanecobalt(III)]2+ ions

Condensation of formaldehyde with glycinatobis-(ethylenediamine)cobalt(III) ion yields in turn [α-hydroxymethylserinebis(ethylenediamine)cobalt(III)]2+ and the macrocycle [α-hydroxymethylserine-1,4,8,11-tetra-aza-6,13-dioxacyclotetradecanecobalt(III)]2+, the structures of which have been established by X-ray structural analysis.