Unnatural reactions: We report the synthesis of a large variety of N-substituted aspartic acids by addition of structurally diverse amines to fumaric acid; the reactions are catalyzed by a previously engineered variant of methylaspartate ammonia lyase. The additions are highly enantioselective and yield only the L enantiomers of the corresponding amino acid products (>99 % ee). As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
Abstract Macrophage migration inhibitory factor (MIF) is a cytokine found to be associated with chronic obstructive pulmonary disease (COPD). However, there is no consensus on how MIF levels differ in COPD compared to control conditions and there are no reports on MIF expression in lung tissue. Here we studied gene expression of members of the MIF family MIF , D-Dopachrome Tautomerase ( DDT ) and DDT-like ( DDTL ) in a lung tissue dataset with 1087 subjects and identified single nucleotide polymorphisms (SNPs) regulating their gene expression. We found higher MIF and DDT expression in COPD patients compared to non-COPD subjects and found 71 SNPs significantly influencing gene expression of MIF and DDTL . Furthermore, the platform used to measure MIF (microarray or RNAseq) was found to influence the splice variants detected and subsequently the direction of the SNP effects on MIF expression. Among the SNPs found to regulate MIF expression, the major LD block identified was linked to rs5844572, a SNP previously found to be associated with lower diffusion capacity in COPD. This suggests that MIF may be contributing to the pathogenesis of COPD, as SNPs that influence MIF expression are also associated with symptoms of COPD. Our study shows that MIF levels are affected not only by disease but also by genetic diversity (i.e. SNPs). Since none of our significant eSNPs for MIF or DDTL have been described in GWAS for COPD or lung function, MIF expression in COPD patients is more likely a consequence of disease-related factors rather than a cause of the disease.
Methylaspartate ammonia‐lyase (MAL; EC 4.3.1.2) catalyzes the reversible addition of ammonia to mesaconate to yield l ‐ threo ‐(2 S ,3 S )‐3‐methylaspartate and l ‐ erythro ‐(2 S ,3 R )‐3‐methylaspartate as products. In the proposed minimal mechanism for MAL of Clostridium tetanomorphum , Lys‐331 acts as the ( S )‐specific base catalyst and abstracts the 3 S‐ proton from l ‐ threo‐ 3‐methylaspartate, resulting in an enolate anion intermediate. This enolic intermediate is stabilized by coordination to the essential active site Mg 2+ ion and hydrogen bonding to the Gln‐329 residue. Collapse of this intermediate results in the release of ammonia and the formation of mesaconate. His‐194 likely acts as the ( R )‐specific base catalyst and abstracts the 3 R ‐proton from the l ‐ erythro isomer of 3‐methylaspartate, yielding the enolic intermediate. In the present study, we have investigated the importance of the residues Gln‐73, Phe‐170, Gln‐172, Tyr‐356, Thr‐360, Cys‐361 and Leu‐384 for the catalytic activity of C. tetanomorphum MAL. These residues, which are part of the enzyme surface lining the substrate binding pocket, were subjected to site‐directed mutagenesis and the mutant enzymes were characterized for their structural integrity, ability to catalyze the amination of mesaconate, and regio‐ and diastereoselectivity. Based on the observed properties of the mutant enzymes, combined with previous structural studies and protein engineering work, we propose a detailed catalytic mechanism for the MAL‐catalyzed reaction, in which the side chains of Gln‐73, Gln‐172, Tyr‐356, Thr‐360, and Leu‐384 provide favorable interactions with the substrate, which are important for substrate binding and activation. This detailed knowledge of the catalytic mechanism of MAL can serve as a guide for future protein engineering experiments.
Drehen für den Wechsel: Eine Phenylalaninaminomutase wurde so mutiert, dass sie die regioselektive Aminierung von Zimtsäurederivaten (siehe Schema, rot) beispielsweise zu β-Aminosäuren katalysiert. Die beobachtete Regioselektivität und die Kristallstrukturen weisen auf zwei Carboxylat-Bindungsarten hin, die sich in der Rotation um die Cβ-Cipso-Bindung unterscheiden, wodurch entschieden wird, ob β- oder (siehe Schema) α-Addition stattfindet.
Non-natural beauty: The enzyme 4-oxalocrotonate tautomerase (4-OT) promiscuously (i.e., with non-natural activity) catalyzes the Michael-type addition of acetaldehyde to nitrostyrene. Catalysis likely proceeds via enamine formation of the amino-terminal proline residue of 4-OT with acetaldehyde (see picture), reminiscent of organocatalysis. High stereoselectivity, low catalyst loading, and water as reaction medium characterize this methodology. Detailed facts of importance to specialist readers are published as "Supporting Information". Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
The Front Cover shows the usefulness of the photocatalyst Ru(bpy)3Cl2 under irradiation with blue light for expanding the catalytic repertoire of a flavin-dependent nitroreductase, using glucose dehydrogenase (GDH) for regeneration of the essential NADH cofactor. In their Research Article, A. Prats Luján, M. F. Bhat et al. report the discovery of a flavoenzyme, BaNTR1, that is able to promote enantio- and chemoselective photoenzymatic reductions of a broad range of ketones to yield the corresponding alcohols with high conversions and outstanding enantiopurity. Most importantly, BaNTR1 was able to promote the photoenzymatic reduction of various α,ß-unsaturated ketones to give the corresponding optically pure alcohols without reducing the C=C or C≡C bond, demonstrating its astoundingly high chemoselectivity. More information can be found in the Research Article by A. Prats Luján, M. F. Bhat et al.
Serendipitous switch: While exploring cis-nitrostyrene as a potential electrophile in Michael-type addition reactions catalysed by the enzyme 4-oxalocrotonate tautomerase (4-OT), it was unexpectedly found that 4-OT catalyses the isomerisation of cis-nitrostyrene to trans-nitrostyrene (kcat/Km= 1.9×103 M−1 s−1).
