Acetaldehyde inhibits retinoic acid biosynthesis to mediate alcohol teratogenicity.

Alcohol consumption during pregnancy induces Fetal Alcohol Spectrum Disorder (FASD), which has been proposed to arise from competitive inhibition of retinoic acid (RA) biosynthesis. We provide biochemical and developmental evidence identifying acetaldehyde as responsible for this inhibition. In the embryo, RA production by RALDH2 (ALDH1A2), the main retinaldehyde dehydrogenase expressed at that stage, is inhibited by ethanol exposure. Pharmacological inhibition of the embryonic alcohol dehydrogenase activity, prevents the oxidation of ethanol to acetaldehyde that in turn functions as a RALDH2 inhibitor. Acetaldehyde-mediated reduction of RA can be rescued by RALDH2 or retinaldehyde supplementation. Enzymatic kinetic analysis of human RALDH2 shows a preference for acetaldehyde as a substrate over retinaldehyde. RA production by hRALDH2 is efficiently inhibited by acetaldehyde but not by ethanol itself. We conclude that acetaldehyde is the teratogenic derivative of ethanol responsible for the reduction in RA signaling and induction of the developmental malformations characteristic of FASD. This competitive mechanism will affect tissues requiring RA signaling when exposed to ethanol throughout life.

Overcoming an optimization plateau in the directed evolution of highly efficient nerve agent bioscavengers.

Improving an enzyme's initially low catalytic efficiency with a new target substrate by an order of magnitude or two may require only a few rounds of mutagenesis and screening or selection. However, subsequent rounds of optimization tend to yield decreasing degrees of improvement (diminishing returns) eventually leading to an optimization plateau. We aimed to optimize the catalytic efficiency of bacterial phosphotriesterase (PTE) toward V-type nerve agents. Previously, we improved the catalytic efficiency of wild-type PTE toward the nerve agent VX by 500-fold, to a catalytic efficiency (kcat/KM) of 5 × 106 M-1 min-1. However, effective in vivo detoxification demands an enzyme with a catalytic efficiency of >107 M-1 min-1. Here, following eight additional rounds of directed evolution and the computational design of a stabilized variant, we evolved PTE variants that detoxify VX with a kcat/KM ≥ 5 × 107 M-1 min-1 and Russian VX (RVX) with a kcat/KM ≥ 107 M-1 min-1. These final 10-fold improvements were the most time consuming and laborious, as most libraries yielded either minor or no improvements. Stabilizing the evolving enzyme, and avoiding tradeoffs in activity with different substrates, enabled us to obtain further improvements beyond the optimization plateau and evolve PTE variants that were overall improved by >5000-fold with VX and by >17 000-fold with RVX. The resulting variants also hydrolyze G-type nerve agents with high efficiency (GA, GB at kcat/KM > 5 × 107 M-1 min-1) and can thus serve as candidates for broad-spectrum nerve-agent prophylaxis and post-exposure therapy using low enzyme doses.

Kinetic characterization and regulation of the human retinaldehyde dehydrogenase 2 enzyme during production of retinoic acid.

Retinoic acid (RA) is an important regulator of embryogenesis and tissue homoeostasis. Perturbation of RA signalling causes developmental disorders, osteoarthritis, schizophrenia and several types of tumours. RA is produced by oxidation of retinaldehyde from vitamin A. The main enzyme producing RA in the early embryo is retinaldehyde dehydrogenase 2 (RALDH2, ALDH1A2). In the present study we describe in depth the kinetic properties and regulation of the human RALDH2 (hRALDH2) enzyme. We show that this enzyme produces RA using in vivo and in vitro assays. We studied the naturally occurring all-trans-, 9-cis- and 13-cis-retinaldehyde isomers as substrates of hRALDH2. Based on the values measured for the Michaelis-Menten constant Km and the maximal rate Vmax, in vitro hRALDH2 displays the same catalytic efficiency for their oxidation. We characterized two known inhibitors of the vertebrate RALDH2 and determined their kinetic parameters on hRALDH2. In addition, RA was studied as a possible inhibitor of hRALDH2 and a regulator of its activity. We show that hRALDH2 is not inhibited by its oxidation product, all-trans-RA, suggesting the absence of a negative feedback regulatory loop. Expression of the Raldh2 gene is known to be regulated by RA itself, suggesting that the main regulation of the hRALDH2 activity level is transcriptional.

Spanning high-dimensional expression space using ribosome-binding site combinatorics.

Protein levels are a dominant factor shaping natural and synthetic biological systems. Although proper functioning of metabolic pathways relies on precise control of enzyme levels, the experimental ability to balance the levels of many genes in parallel is a major outstanding challenge. Here, we introduce a rapid and modular method to span the expression space of several proteins in parallel. By combinatorially pairing genes with a compact set of ribosome-binding sites, we modulate protein abundance by several orders of magnitude. We demonstrate our strategy by using a synthetic operon containing fluorescent proteins to span a 3D color space. Using the same approach, we modulate a recombinant carotenoid biosynthesis pathway in Escherichia coli to reveal a diversity of phenotypes, each characterized by a distinct carotenoid accumulation profile. In a single combinatorial assembly, we achieve a yield of the industrially valuable compound astaxanthin 4-fold higher than previously reported. The methodology presented here provides an efficient tool for exploring a high-dimensional expression space to locate desirable phenotypes.