In situ characterization of amine-forming enzymes shows altered oligomeric state.

Enzyme stability can be measured in a number of ways, including melting temperature, activity retention, and size analysis. However, these measurements are often conducted in an idealized storage buffer and not in the relevant enzymatic reaction media. Particularly for reactions that occur in alkaline, volatile, and high ionic strength media, typical analyses using differential scanning calorimetry, light scattering, and sodium dodecyl-sulfate polyacrylamide gel electrophoresis are not satisfactory to track the stability of these enzymes.

A refined picture of the native amine dehydrogenase family revealed by extensive biodiversity screening.

Native amine dehydrogenases offer sustainable access to chiral amines, so the search for scaffolds capable of converting more diverse carbonyl compounds is required to reach the full potential of this alternative to conventional synthetic reductive aminations. Here we report a multidisciplinary strategy combining bioinformatics, chemoinformatics and biocatalysis to extensively screen billions of sequences in silico and to efficiently find native amine dehydrogenases features using computational approaches. In this way, we achieve a comprehensive overview of the initial native amine dehydrogenase family, extending it from 2,011 to 17,959 sequences, and identify native amine dehydrogenases with non-reported substrate spectra, including hindered carbonyls and ethyl ketones, and accepting methylamine and cyclopropylamine as amine donor.

Structure and Mutation of the Native Amine Dehydrogenase MATOUAmDH2.

Native amine dehydrogenases (nat-AmDHs) have recently emerged as a potentially valuable new reservoir of enzymes for the sustainable and selective synthesis of chiral amines, catalyzing the NAD(P)H-dependent ammoniation of carbonyl compounds with high activity and selectivity. MATOUAmDH2, recently identified from the Marine Atlas of Tara Oceans Unigenes (MATOUv1) database of eukaryotic genes, displays exceptional catalytic performance against its best identified substrate, isobutyraldehyde, as well as having broader substrate scope than other nat-AmDHs. In the interests of providing a platform for the rational engineering of this and other nat-AmDHs, we have determined the structure of MATOUAmDH2 in complex with NADP and also with the cofactor and cyclohexylamine.

Purification and Characterization of Nit , a Robust Thermostable Nitrilase From .

Despite the success of some nitrilases in industrial applications, there is a constant demand to broaden the catalog of these hydrolases, especially robust ones with high operational stability. By using the criteria of thermoresistance to screen a collection of candidate enzymes heterologously expressed in , the enzyme Nit from the mesophilic organism was selected and further characterized. Its quick and efficient purification by heat treatment is of major interest for large-scale applications.

Complete Genome Sequences of Two Species Isolated from Marine Environments of the Pacific Ocean.

YsY11 and T9AD were both isolated from marine environments of the Pacific Ocean. Here, we report the whole-genome sequences of these two organisms. YsY11 consists of a single 4.

XszenFHal, a novel tryptophan 5-halogenase from Xenorhabdus szentirmaii.

Flavin-dependent halogenases (FHals) catalyse the halogenation of electron-rich substrates, mainly aromatics. Halogenated compounds have many applications, as pharmaceutical, agrochemicals or as starting materials for the synthesis of complex molecules. By exploring the sequenced bacterial diversity, we discovered and characterized XszenFHal, a novel FHal from Xenorhabdus szentirmaii, a symbiotic bacterium of entomopathogenic nematode.

Characterization of a thermotolerant ROK-type mannofructokinase from Streptococcus mitis: application to the synthesis of phosphorylated sugars.

Most of the "repressor, open reading frame, kinase" (ROK) proteins already characterized so far, and exhibiting a kinase activity, take restrictedly D-glucose as substrate. By exploring the sequenced bacterial diversity, 61 ATP-dependent kinases belonging to the ROK family have been identified and experimentally assayed for the phosphorylation of hexoses. These kinases were mainly found to be thermotolerant and highly active toward D-mannose and D-fructose with notable activities toward D-tagatose.

Enzymatic Cascade Reactions for the Synthesis of Chiral Amino Alcohols from L-lysine.

Amino alcohols are versatile compounds with a wide range of applications. For instance, they have been used as chiral scaffolds in organic synthesis. Their synthesis by conventional organic chemistry often requires tedious multi-step synthesis processes, with difficult control of the stereochemical outcome.

Parallel evolution of non-homologous isofunctional enzymes in methionine biosynthesis.

Experimental validation of enzyme function is crucial for genome interpretation, but it remains challenging because it cannot be scaled up to accommodate the constant accumulation of genome sequences. We tackled this issue for the MetA and MetX enzyme families, phylogenetically unrelated families of acyl-L-homoserine transferases involved in L-methionine biosynthesis. Members of these families are prone to incorrect annotation because MetX and MetA enzymes are assumed to always use acetyl-CoA and succinyl-CoA, respectively.

Revealing the hidden functional diversity of an enzyme family.

Millions of protein database entries are not assigned reliable functions, preventing the full understanding of chemical diversity in living organisms. Here, we describe an integrated strategy for the discovery of various enzymatic activities catalyzed within protein families of unknown or little known function. This approach relies on the definition of a generic reaction conserved within the family, high-throughput enzymatic screening on representatives, structural and modeling investigations and analysis of genomic and metabolic context.