Low modularity of aminoacyl-tRNA substrates in polymerization by the ribosome.

Aminoacyl-transfer RNAs contain four standardized units: amino acids, an invariant 3'-terminal CCA, trinucleotide anticodons and tRNA bodies. The degree of interchangeability of the three variable modules is poorly understood, despite its role in evolution and the engineering of translation to incorporate unnatural amino acids. Here, a purified translation system is used to investigate effects of various module swaps on the efficiency of multiple ribosomal incorporations of unnatural aminoacyl-tRNA substrates per peptide product. The yields of products containing three to five adjacent l-amino acids with unnatural side chains are low and cannot be improved by optimization or explained simply by any single factor tested. Though combinations of modules that allow quantitative single unnatural incorporations are found readily, finding combinations that enable efficient synthesis of products containing multiple unnatural amino acids is challenging. This implies that assaying multiple, as opposed to single, incorporations per product is a more stringent assay of substrate activity. The unpredictability of most results illustrates the multifactorial nature of substrate recognition and the value of synthetic biology for testing our understanding of translation. Data indicate that the degree of interchangeability of the modules of aminoacyl-tRNAs is low.

Crystal structure of a NifS-like protein from Thermotoga maritima: implications for iron sulphur cluster assembly.

NifS-like proteins are ubiquitous, homodimeric, proteins which belong to the alpha-family of pyridoxal-5'-phoshate dependent enzymes. They are proposed to donate elementary sulphur, generated from cysteine, via a cysteinepersulphide intermediate during iron sulphur cluster biosynthesis, an important albeit not well understood process. Here, we report on the crystal structure of a NifS-like protein from the hyperthermophilic bacterium Thermotoga maritima (tmNifS) at 2.0 A resolution. The tmNifS is structured into two domains, the larger bearing the pyridoxal-5'-phosphate-binding active site, the smaller hosting the active site cysteine in the middle of a highly flexible loop, 12 amino acid residues in length. Once charged with sulphur the loop could possibly deliver S(0) directly to regions far remote from the protein. Based on the three-dimensional structures of the native as well as the substrate complexed form and on spectrophotometric results, a mechanism of sulphur activation is proposed. The His99, which stacks on top of the pyridoxal-5'-phosphate co-factor, is assigned a crucial role during the catalytic cycle by acting as an acid-base catalyst and is believed to have a pK(a) value depending on the co-factor redox state.

Enzymatic synthesis of peptides containing unnatural amino acids.

Several proteases were studied as potential catalysts for the enzymatic synthesis of oligopeptides containing the unnatural amino acid allylglycine, the overall objective being the synthesis of a reactive tetrapeptide that could be chemically polymerized into a potentially biocompatible or biodegradable material. Commercially available enzymes were screened for esterase activity toward the methyl ester of the amino acid allylglycine (DL-AgOMe) to identify potential catalysts for dipeptide synthesis. Proteases from Aspergillus oryzae and Aspergillus sojae, pronase E and protease Nagarse synthesized the protected dipeptide Cbz-allylglycine-phenylalaninamide (Cbz-L-Ag-L-PheNH2) from Cbz-DL-AgOMe and L-PheNH2. However, the same enzymes were not able to catalyze the synthesis of Cbz-phenylalanine-allylglycine ethyl ester (Cbz-L-Phe-L-AgOEt). Thus, although these enzymes could use allylglycine as the acyl donor they could not employ it as the acyl acceptor in peptide synthesis. In contrast, chymotrypsin was able to use allylglycine ethyl ester (DL-AgOEt) as the acyl acceptor in the synthesis of Cbz-L-Phe-L-AgOEt, but was not able to synthesize Cbz-L-Ag-L-PheNH2. The two dipeptides, Cbz-allylglycine-phenylalanine and phenylalanine-allylglycine ethyl ester, served as substrates for the thermolysin-catalyzed synthesis of the tetrapeptide Cbz-L-Ag-L-Phe-L-Phe-L-AgOEt.

A metabolic alkene reporter for spatiotemporally controlled imaging of newly synthesized proteins in Mammalian cells.

The nonsymmetrical spatial distribution of newly synthesized proteins in animal cells plays a central role in many cellular processes. Here, we report that a simple alkene tag, homoallylglycine (HAG), was co-translationally incorporated into a recombinant protein as well as endogenous, newly synthesized proteins in mammalian cells with high efficiency. In conjunction with a photoinduced tetrazole-alkene cycloaddition reaction ("photoclick chemistry"), this alkene tag further served as a bioorthogonal chemical reporter both for the selective protein functionalization in vitro and for a spatiotemporally controlled imaging of the newly synthesized proteins in live mammalian cells. This two-step metabolic alkene tagging-photocontrolled chemical functionalization approach may offer a potentially useful tool to study the role of spatiotemporally regulated protein synthesis in mammalian cells.

Metabolism of allylglycine and cis-crotylglycine by Pseudomonas putida (arvilla) mt-2 harboring a TOL plasmid.

Spontaneous mutants which acquired the ability to utilize d-allylglycine (d-2-amino-4-pentenoic acid) and dl-cis-crotylglycine (dl-2-amino-cis-4-hexenoic acid) but not l-allylglycine or dl-trans-crotylglycine could be readily isolated from Pseudomonas putida mt-2 (PaM1). Derivative strains of PaM1 putatively cured of the TOL (pWWO) plasmid were incapable of forming mutants able to utilize the amino acids for growth; however, this ability could be regained by conjugative transfer of the TOL (pWWO) plasmid from a wild-type strain of mt-2 or of the TOL (pDK1) plasmid from a related strain of P. putida (HS1), into cured recipients. dl-Allylglycine-grown cells of one spontaneous mutant (PaM1000) extensively oxidized dl-allylglycine and dl-cis-crotylglycine, whereas only a limited oxidation was observed toward l-allylglycine and dl-trans-crotylglycine. Cell extracts prepared from PaM1000 cells contained high levels of 2-keto-4-hydroxyvalerate aldolase and 2-keto-4-pentenoic acid hydratase, the latter enzyme showing higher activity toward 2-keto-cis-4-hexenoic acid than toward the trans isomer. Levels of other enzymes of the TOL degradative pathway, including toluate oxidase, catechol-2,3-oxygenase, 2-hydroxymuconic semialdehyde hydrolase, and 2-hydroxymuconic semialdehyde dehydrogenase, were also found to be elevated after growth on allylglycine. Whole cells of a putative cured strain, PaM3, accumulated 2-keto-4-pentenoic acid from d-allylglycine, which was shown to be rapidly degraded by cell extracts of PaM1000 grown on dl-allylglycine. These same cell extracts were also capable of catalyzing the dehydrogenation of d- but not l-allylglycine and were further found to metabolize the amino acid completely to pyruvate and acetaldehyde. Differential centrifugation of crude cell extracts localized d-allylglycine dehydrogenase activity to membrane fractions. The results are consistent with a catabolic pathway for d-allylglycine and dl-cis-crotylglycine involving the corresponding keto-enoic acids as intermediates, the further metabolism of which is effected by the action of TOL plasmid-encoded enzymes.

Reaction mechanism of Escherichia coli cystathionine gamma-synthase: direct evidence for a pyridoxamine derivative of vinylglyoxylate as a key intermediate in pyridoxal phosphate dependent gamma-elimination and gamma-replacement reactions.

Cystathionine gamma-synthase catalyzes a pyridoxal phosphate dependent synthesis of cystathionine from O-succinyl-L-homoserine (OSHS) and L-cysteine via a gamma-replacement reaction. In the absence of L-cysteine, OSHS undergoes an enzyme-catalyzed, gamma-elimination reaction to form succinate, alpha-ketobutyrate, and ammonia. Since elimination of the gamma-substituent is necessary for both reactions, it is reasonable to assume that the replacement and elimination reaction pathways diverge from a common intermediate. Previously, this partitioning intermediate has been assigned to a highly conjugated alpha-iminovinylglycine quininoid (Johnston et al., 1979a). The experiments reported herein support an alternative assignment for the partitioning intermediate. We have examined the gamma-replacement and gamma-elimination reactions of cystathionine gamma-synthase via rapid-scanning stopped-flow and single-wavelength stopped-flow UV-visible spectroscopy. The gamma-elimination reaction is characterized by a rapid decrease in the amplitude of the enzyme internal aldimine spectral band at 422 nm with a concomitant appearance of a new species which absorbs in the 300-nm region. A 485-nm species subsequently accumulates in a much slower relaxation. The gamma-replacement reaction shows a red shift of the 422-nm peak to 425 nm which occurs in the experiment dead time (approximately 3 ms). This relaxation is followed by a decrease in absorbance at 425 nm that is tightly coupled to the appearance of a species which absorbs in the 300-nm region. Reaction of the substrate analogues L-alanine and L-allylglycine with cystathionine gamma-synthase results in bleaching of the 422-nm absorbance and the appearance of a 300-nm species. In the absence of L-cysteine, L-allylglycine undergoes facile proton exchange; in the presence of L-cysteine, L-allylglycine undergoes a gamma-replacement reaction to form a new amino acid, gamma-methylcystathionine. No long-wavelength-absorbing species accumulate during either of these reactions. These results establish that the partitioning intermediate is an alpha-imino beta,gamma-unsaturated pyridoxamine derivative with lambda max congruent to 300 nm and that the 485-nm species which accumulates in the elimination reaction is not on the replacement pathway.

Unique binding of a novel synthetic inhibitor, N-[3-[4-[4-(amidinophenoxy)carbonyl]phenyl]-2-methyl-2-propenoyl]- N-allylglycine methanesulfonate, to bovine trypsin, revealed by the crystal structure of the complex.

Trypsin and N-[3-[4-[4-(amidinophenoxy)carbonyl]phenyl]-2-methyl-2-propenoyl]- N-allylglycine methanesulfonate (1), a newly designed and orally active synthetic trypsin inhibitor, were cocrystallized. The space group of the crystal is P2(1)2(1)2(1) with cell constants a = 63.74 A, b = 63.08 A, and c = 69.38 A, which is nearly identical to that of the orthorhombic crystal of guanidinobenzoyltrypsin. The structure was refined to a crystallographic residual R = 0.176. The refined model of the 1-trypsin complex provides the structural basis for the reaction mechanism of 1. On the basis of the present X-ray results, it is proposed that the potent inhibitory activity of 1 is mainly due to the formation of an acylated trypsin through an "inverse substrate mechanism" and its low rate of deacylation.

Substrate specificity of L-delta-(alpha-aminoadipoyl)-L-cysteinyl-D-valine synthetase from Cephalosporium acremonium: demonstration of the structure of several unnatural tripeptide products.

Potential substrates for L-delta-(alpha-aminoadipoyl)-L-(cysteinyl)-D-valine (ACV) synthetase were initially identified using both the amino-acid-dependent ATP<-->pyrophosphate exchange reaction catalysed by the enzyme and the incorporation of 14C-radiolabelled cysteine and valine into potential peptide products. S-Carboxymethylcysteine was an effective substitute for alpha-aminoadipate and both allylglycine and vinylglycine could substitute for cysteine, indicating that the thiol group of cysteine is not essential for peptide formation. L-allo-Isoleucine but not L-isoleucine substituted effectively for valine. The structures of the presumed peptide products derived from these amino acids were confirmed by combined use of electrospray-ionization m.s. (e.s.m.s.) and 1H n.m.r. These results clearly indicate that, in common with other peptide synthetases, but in contrast with ribosomal peptide synthesis, ACV synthetase has a relatively broad substrate specificity.

Proconvulsant, convulsant and other actions of the D- and L-stereoisomers of allylglycine in the photosensitive baboon, Papio papio.

The effects of the intravenous or intracerebroventricular injection of the stereoisomers, and the racemic mixture, of allylglycine (2-amino-pent-4-enoic acid) have been studied in baboons, Papio papio, with photosensitive epilepsy. Enhancement of the natural syndrome of photosensitivy epilepsy is seen 1-12 h (maximally at 3-8 h) after L-allyglycine, 100 mg/kg, intravenously, or D,L-allyglycine, 200 mg/kg, intravenously. Such enhancement is seen with a slower onset, and to a lesser, and more variable, extent after D-allyglycine, 500-750 mg/kg, intravenously. Brief focal or generalised seizures occurred (in the absence of intermittent photic stimulation) after L-allyglycine, 150-200 mg/kg, intravenously. This effect is similar to that previously observed after D,L-allyglycine, 300-400 mg/kg. D-Allyglycine, 780 mg/kg, intravenously produced episodes of vertical nystagmus with increased extensor motor tone, but no 'spontaneous' seizures. Intracerebroventricular injection of L-allylglycine, D-allyglycine or D,L-allyglycine, 100 mg in 1 ml saline, did not modify the natural syndrome of photosensitive epilepsy. D-Allylglycine, or D,L-allyglycine, 100 mg intracerebroventricularly, after 1-2 h gave rise to a syndrome with vomiting, sustained vertical nystagmus, and intermittent extensor spasms. The results are interpreted in terms of regional differences in the metabolism of the two isomers to active compounds that can inhibit glutamic acid decarboxylase. D-Allylglycine is active only at the brain stem and cerebellum because D-amino acid oxidase is largely confined to these brain areas.