Fatty acids are among the major building blocks of living cells, making lipid biosynthesis a potent target for compounds with antibiotic or antineoplastic properties. We present the crystal structure of the 2.6-MDa Saccharomyces cerevisiae fatty acid synthase (FAS) multienzyme in complex with the antibiotic cerulenin, representing, to our knowledge, the first structure of an inhibited fatty acid megasynthase. Cerulenin attacks the FAS ketoacyl synthase (KS) domain, forming a covalent bond to the active site cysteine C1305. The inhibitor binding causes two significant conformational changes of the enzyme. First, phenylalanine F1646, shielding the active site, flips and allows access to the nucleophilic cysteine. Second, methionine M1251, placed in the center of the acyl-binding tunnel, rotates and unlocks the inner part of the fatty acid binding cavity. The importance of the rotational movement of the gatekeeping M1251 side chain is reflected by the cerulenin resistance and the changed product spectrum reported for S. cerevisiae strains mutated in the adjacent glycine G1250. Platensimycin and thiolactomycin are two other potent inhibitors of KSs. However, in contrast to cerulenin, they show selectivity toward the prokaryotic FAS system. Because the flipped F1646 characterizes the catalytic state accessible for platensimycin and thiolactomycin binding, we superimposed structures of inhibited bacterial enzymes onto the S. cerevisiae FAS model. Although almost all side chains involved in inhibitor binding are conserved in the FAS multienzyme, a different conformation of the loop K1413-K1423 of the KS domain might explain the observed low antifungal properties of platensimycin and thiolactomycin.cerulenin ͉ platensimycin ͉ thiolactomycin ͉ fatty acid synthesis ͉ yeast T hree different schemes for de novo synthesis of fatty acids are found in nature. Eukaryotes and advanced prokaryotes generally use the type I fatty acid synthase system (FAS I), composed of complexes of large multifunctional enzymes. Bacteria, in contrast, use the dissociated FAS II system that consists of a set of separate enzymes, each catalyzing one of the reactions of the fatty acid synthase cycle (1). A third system exists in some parasites that use membrane-bound fatty acid elongases for the synthesis of aliphatic chains (2). Despite this considerable variation, the individual reaction steps of fatty acid biosynthesis are essentially conserved in all kingdoms of life. Four basic reactions constitute a single round of elongation. In the first step, an acceptor CoA or acyl carrier protein (ACP) associated acetyl unit is condensed with malonyl-ACP to form ␤-ketobutyryl-ACP, which is subsequently reduced by an NADPH-dependent ketoacyl-ACP reductase. The resulting ␤-hydroxyacyl-ACP is dehydrated to produce enoyl-ACP and finally reduced by an enoyl reductase (ER) to form the saturated acyl-ACP, which can be further elongated in a new cycle [see supporting information (SI) Fig. S1].The importance of the fatty acid biosynthesis pathway makes the FAS sys...

BackgroundProline residues affect protein folding and stability via cis/trans isomerization of peptide bonds and by the Cγ-exo or -endo puckering of their pyrrolidine rings. Peptide bond conformation as well as puckering propensity can be manipulated by proper choice of ring substituents, e.g. Cγ-fluorination. Synthetic chemistry has routinely exploited ring-substituted proline analogs in order to change, modulate or control folding and stability of peptides.Methodology/Principal FindingsIn order to transmit this synthetic strategy to complex proteins, the ten proline residues of enhanced green fluorescent protein (EGFP) were globally replaced by (4R)- and (4S)-fluoroprolines (FPro). By this approach, we expected to affect the cis/trans peptidyl-proline bond isomerization and pyrrolidine ring puckering, which are responsible for the slow folding of this protein. Expression of both protein variants occurred at levels comparable to the parent protein, but the (4R)-FPro-EGFP resulted in irreversibly unfolded inclusion bodies, whereas the (4S)-FPro-EGFP led to a soluble fluorescent protein. Upon thermal denaturation, refolding of this variant occurs at significantly higher rates than the parent EGFP. Comparative inspection of the X-ray structures of EGFP and (4S)-FPro-EGFP allowed to correlate the significantly improved refolding with the Cγ-endo puckering of the pyrrolidine rings, which is favored by 4S-fluorination, and to lesser extents with the cis/trans isomerization of the prolines.Conclusions/SignificanceWe discovered that the folding rates and stability of GFP are affected to a lesser extent by cis/trans isomerization of the proline bonds than by the puckering of pyrrolidine rings. In the Cγ-endo conformation the fluorine atoms are positioned in the structural context of the GFP such that a network of favorable local interactions is established. From these results the combined use of synthetic amino acids along with detailed structural knowledge and existing protein engineering methods can be envisioned as a promising strategy for the design of complex tailor-made proteins and even cellular structures of superior properties compared to the native forms.

Classical enzyme optimization exploits the chemistry confined to the 20 canonical amino acids encoded by the standard genetic code. ‘Genetic code engineering’ allows the global substitution of particular residues with synthetic analogues, endowing proteins with chemical diversity not found in nature. These proteins are congeners of the parent protein because they originate from the same gene sequence, but contain a fraction of noncanonical amino acids. Global substitutions of methionine, proline, phenylalanine, and tyrosine have been carried out with related analogues in Thermoanaerobacter thermohydrosulfuricus lipase. This study represents the first extensive report of an important biocatalyst substituted with a high number of noncanonical amino acids. The generated lipase congeners displayed special features such as enhanced activation, elevated enzyme activity (by up to 25 %) and substrate tolerance (by up to 40 %), and changes in optimal temperature (by up to 20 °C) and pH (by up to 3). These emergent features achieved by genetic code engineering might be important not only for academic research, but also for numerous economical applications in the food, detergent, chemical, pharmaceutical, leather, textile, cosmetic, and paper industries.

Controlling the context: A novel strategy for the in vitro expression and immediate post‐translational membrane insertion of complex membrane proteins into an artificial membrane system is described. In this way problems are circumvented that arise when complex membrane proteins are overexpressed, detergent‐solubilized, and subsequently reconstituted into suitable artificial membrane systems for biophysical characterization. GPCR= G‐protein coupled receptor.

The Friedel–Crafts acylation is commonly used for the synthesis of aryl ketones, and a biocatalytic version, which may benefit from the chemo‐ and regioselectivity of enzymes, has not yet been introduced. Described here is a bacterial acyltransferase which can catalyze Friedel–Crafts C‐acylation of phenolic substrates in buffer without the need of CoA‐activated reagents. Conversions reach up to >99 %, and various C‐ or O‐acyl donors, such as DAPG or isopropenyl acetate, are accepted by this enzyme. Furthermore the enzyme enables a Fries rearrangement‐like reaction of resorcinol derivatives. These findings open an avenue for the development of alternative and selective C−C bond formation methods.

Fluoro amino acids are highly valuable compounds constantly gaining relevance in diverse fields of the biosciences as well as in the pharmaceutical industry. The value of these compounds can be attributed to the properties of the extremely electronegative fluorine atom. This atom forms a highly polarized bond of extraordinary strength with carbon. The formation of the fluorine-carbon bond is challenging: its chemical synthesis demands harsh reaction conditions and to date only one class of enzyme has been found capable of introducing the fluoride ion into an organic compound. Most of these fluorinating enzymes participate in the biosynthesis of 4-fluoro-L-threonine, the only fluoro amino acid of natural origin discovered so far. Despite their scarcity in nature, fluoro amino acids are valuable tools to fluorinate proteins. The fluoro protein variants often show improved stability and folding as well as altered activity and fluorescence characteristics. This review details the biosynthesis of 4-fluoro-L-threonine with a special focus on the fluorinating enzymes. Moreover, we elaborate on the application of fluoro amino acids as building blocks for fluorinated protein variants. Insight into different techniques to incorporate fluoro amino acids into proteins is also provided. We highlight prospects and the current relevance of fluoro amino acids as a tool to engineer proteins with novel traits.

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BackgroundThe suppression of amber stop codons with non-canonical amino acids (ncAAs) is used for the site-specific introduction of many unusual functions into proteins. Specific orthogonal aminoacyl-tRNA synthetase (o-aaRS)/amber suppressor tRNACUA pairs (o-pairs) for the incorporation of ncAAs in S. cerevisiae were previously selected from an E. coli tyrosyl-tRNA synthetase/tRNACUA mutant library. Incorporation fidelity relies on the specificity of the o-aaRSs for their ncAAs and the ability to effectively discriminate against their natural substrate Tyr or any other canonical amino acid.Methodology/Principal FindingsWe used o-pairs previously developed for ncAAs carrying reactive alkyne-, azido-, or photocrosslinker side chains to suppress an amber mutant of human superoxide dismutase 1 in S. cerevisiae. We found worse incorporation efficiencies of the alkyne- and the photocrosslinker ncAAs than reported earlier. In our hands, amber suppression with the ncAA containing the azido group did not occur at all. In addition to the incorporation experiments in S. cerevisiae, we analyzed the catalytic properties of the o-aaRSs in vitro. Surprisingly, all o-aaRSs showed much higher preference for their natural substrate Tyr than for any of the tested ncAAs. While it is unclear why efficiently recognized Tyr is not inserted at amber codons, we speculate that metabolically inert ncAAs accumulate in the cell, and for this reason they are incorporated despite being weak substrates for the o-aaRSs.Conclusions/SignificanceO-pairs have been developed for a whole plethora of ncAAs. However, a systematic and detailed analysis of their catalytic properties is still missing. Our study provides a comprehensive scrutiny of o-pairs developed for the site-specific incorporation of reactive ncAAs in S. cerevisiae. It suggests that future development of o-pairs as efficient biotechnological tools will greatly benefit from sound characterization in vivo and in vitro in parallel to monitoring intracellular ncAA levels.