R.D. Stenner

Significance While the bottom-up design of enzymes appears to be an intractably complex problem, a minimal approach that combines elementary, de novo-designed proteins with intrinsically reactive cofactors offers a simple means to rapidly access sophisticated catalytic mechanisms. Not only is this method proven in the reproduction of powerful oxidative chemistry of the natural peroxidase enzymes, but we show here that it extends to the efficient, abiological—and often asymmetric—formation of strained cyclopropane rings, nitrogen–carbon and carbon–carbon bonds, and the ring expansion of a simple cyclic molecule to form a precursor for NAD+, a fundamentally important biological cofactor. That the enzyme also functions in vivo paves the way for its incorporation into engineered biosynthetic pathways within living organisms.

The alcohol-O-acyltransferases are bisubstrate enzymes that catalyse the transfer of acyl chains from an acyl-coenzyme A (CoA) donor to an acceptor alcohol. In the industrial yeast Saccharomyces cerevisiae this reaction produces acyl esters that are an important influence on the flavour of fermented beverages and foods. There is also a growing interest in using acyltransferases to produce bulk quantities of acyl esters in engineered microbial cell factories. However, the structure and function of the alcohol-O-acyltransferases remain only partly understood. Here, we recombinantly express, purify and characterize Atf1p, the major alcohol acetyltransferase from S. cerevisiae. We find that Atf1p is promiscuous with regard to the alcohol cosubstrate but that the acyltransfer activity is specific for acetyl-CoA. Additionally, we find that Atf1p is an efficient thioesterase in vitro with specificity towards medium-chain-length acyl-CoAs. Unexpectedly, we also find that mutating the supposed catalytic histidine (H191) within the conserved HXXXDG active site motif only moderately reduces the thioesterase activity of Atf1p. Our results imply a role for Atf1p in CoA homeostasis and suggest that engineering Atf1p to reduce the thioesterase activity could improve product yields of acetate esters from cellular factories. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.

As coronavirus disease 2019 (COVID-19) persists, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) emerge, accumulating spike (S) glycoprotein mutations. S receptor binding domain (RBD) comprises a free fatty acid (FFA)-binding pocket. FFA binding stabilizes a locked S conformation, interfering with virus infectivity. We provide evidence that the pocket is conserved in pathogenic β-coronaviruses (β-CoVs) infecting humans. SARS-CoV, MERS-CoV, SARS-CoV-2, and VOCs bind the essential FFA linoleic acid (LA), while binding is abolished by one mutation in common cold-causing HCoV-HKU1. In the SARS-CoV S structure, LA stabilizes the locked conformation, while the open, infectious conformation is devoid of LA. Electron tomography of SARS-CoV-2-infected cells reveals that LA treatment inhibits viral replication, resulting in fewer deformed virions. Our results establish FFA binding as a hallmark of pathogenic β-CoV infection and replication, setting the stage for FFA-based antiviral strategies to overcome COVID-19.

The hepatocarcinogenicity of trichloroethylene (TRI) in mice has been attributed to a metabolite, trichloroacetate (TCA). Rats of various strains appear to be resistant to TRI-induced hepatocarcinogenesis and produce lower peak concentrations of TCA. Mice, however, also form significant amounts of another carcinogenic metabolite, dichloroacetate (DCA). The present study was conducted to investigate the interspecies differences in the metabolism of TRI between the mouse, rat, and dog and to gain further insight into the role metabolic factors may play in the apparent species specificity of liver tumor induction by TRI. Fischer 344 rats and beagle dogs were dosed orally with TRI and blood was analyzed for TRI, DCA, TCA, and trichloroethanol (TCE). Data on the metabolism of TRI in mice have been previously published. Limited data are available on the metabolism of TRI in humans. Dogs produce higher peak concentrations and have a larger area under the concentration-time curve (AUC) for TCA as compared to rats given similar doses of TRI. Dichloroacetate was not found in measurable concentrations, that is, above 4 nmol/ml, the minimal quantifiable concentration, in the blood of either rats or dogs. Appreciable concentrations of DCA were found in the blood of mice administered TRI in previous studies. Trichloroethanol was found to be present in the blood, urine, and bile, primarily as the glucuronide conjugate. In all species, peak TCA concentrations were observed beyond the disappearance of TRI. The AUC for TCE glucuronide is consistent with its acting as a precursor for TCA and probably contributes to the continued increase in TCA concentration after TRI disappears from the system. Investigations into the binding of TCA to plasma constituents in the rat, dog, mouse, and human suggest that binding also plays a role in species differences in the distribution and elimination of TCA.