Aline Mariage

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. As a proof of principle, we investigated the DUF849 Pfam family and unearthed 14 potential new enzymatic activities, leading to the designation of these proteins as β-keto acid cleavage enzymes. We propose an in vivo role for four enzymatic activities and suggest key residues for guiding further functional annotation. Our results show that the functional diversity within a family may be largely underestimated. The extension of this strategy to other families will improve our knowledge of the enzymatic landscape.

Genome Mining for Innovative Biocatalysts: New Dihydroxyacetone Aldolases for the Chemist’s Toolbox

Stereoselective carboligating enzymes were discovered by a genome mining approach to extend the biocatalysis toolbox. Seven hundred enzymes were selected by sequence comparison from diverse prokaryotic species as representatives of the aldolase (FSA) family diversity. The aldol reaction tested involved dihydroxyacetone (DHA) and glyceraldehyde‐3‐phosphate. The hexose‐6‐phosphate formation was monitored by mass spectrometry. Eighteen enzymes annotated either as transaldolases or aldolases were found to exhibit a DHA aldolase activity. Remarkably, six of them proven as aldolases, and not transaldolases, shared very limited similarities with those currently described. Multiple sequence alignment performed on all enzymes revealed a Tyr in the new DHA aldolases as found in FSAcoli instead of a Phe usually found in transaldolases. Four of these DHA aldolases were biochemically characterised in comparison with FSAcoli. In particular, an aldolase from Listeria monocytogenes exhibited interesting catalytic properties.

Broadening the scope of Baeyer–Villiger monooxygenase activities toward α,β-unsaturated ketones: a promising route to chiral enol-lactones and ene-lactones

Three regiodivergent Baeyer-Villiger mono-oxygenases (enantioselectively) oxidized a series of cyclic α,β-unsaturated ketones into (chiral) either enol-lactones or ene-lactones. An easy-to-use and efficient biocatalytic process based on a host-microorganism deprived of unwanted activities (knock-out mutant) was developed to enable the exclusive synthesis of unsaturated lactones.

Synthesis of Mono‐ and Dihydroxylated Amino Acids with New α‐Ketoglutarate‐Dependent Dioxygenases: Biocatalytic Oxidation of CH Bonds

Iron(II)/α‐ketoacid‐dependent oxygenases (αKAOs) are enzymes that mainly catalyse hydroxylation reaction. By using a genomic approach combining sequence comparison and protein‐domain sharing, a set of 131 αKAO enzymes was prepared. The screening of various substrates revealed five new αKAOs. Four αKAOs were found to be active towards L‐lysine, L‐ornithine and L‐arginine with total regio‐ and stereoselectivities and yielding the corresponding 3‐ or 4‐hydroxyamino acids. The enzymatic cascade reaction with two stereoselective regiodivergent αKAOs enabled the synthesis of 3,4‐dihydroxy‐L‐lysine.

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. We determined the enzymatic activities of 100 enzymes from diverse species, and interpreted the results by structural classification of active sites based on protein structure modeling. We predict that >60% of the 10,000 sequences from these families currently present in databases are incorrectly annotated, and suggest that acetyl-CoA was originally the sole substrate of these isofunctional enzymes, which evolved to use exclusively succinyl-CoA in the most recent bacteria. We also uncovered a divergent subgroup of MetX enzymes in fungi that participate only in L-cysteine biosynthesis as O-succinyl-L-serine transferases.

Expanding the reaction space of aldolases using hydroxypyruvate as a nucleophilic substrate

Aldolases are key biocatalysts for stereoselective C–C bond formation allowing access to polyoxygenated chiral units through direct, efficient, and sustainable synthetic processes. The aldol reaction involving unprotected hydroxypyruvate and an aldehyde offers access to valuable polyhydroxy-α-keto acids. However, this undescribed aldolisation is highly challenging, especially regarding stereoselectivity. This reaction was explored using, as biocatalysts, a collection of aldolases selected from biodiversity. Several enzymes that belong to the same pyruvate aldolase Pfam family (PF03328) were found to produce the desired hexulosonic acids from hydroxypyruvate and D-glyceraldehyde with complementary stereoselectivities. One of them was selected for the proof of concept as a biocatalytic tool to prepare five (3S,4S) aldol adducts through an eco-friendly process.

Asymmetric reductive amination by a wild-type amine dehydrogenase from the thermophilic bacteria Petrotoga mobilis

The biocatalytic reductive amination of ketone to chiral amine is one of the most challenging reactions. Using a genome-mining approach, we found proteins catalyzing the reductive amination of ketones without a carboxylic function in the α or β position. The synthesis of (4S)-4-aminopentanoic acid (ee ≥99.5%) was achieved with the thermoactive amine dehydrogenase (AmDH) AmDH4 from Petrotoga mobilis in 88% yield. The high stability and substrate tolerance make AmDH4 a very good starting point for further discovery of reductive amination biocatalysts with an enlarged substrate range. This is the first report of wild-type enzymes with related genes having proper NAD(P)H–AmDH activity.

Synthesis of Branched-Chain Sugars with a DHAP-Dependent Aldolase: Ketones are Electrophile Substrates of Rhamnulose-1-phosphate Aldolases

Dihydroxyacetone phosphate (DHAP)-dependent rhamnulose aldolases display an unprecedented versatility for ketones as electrophile substrates. We selected and characterized a rhamnulose aldolase from Bacteroides thetaiotaomicron (RhuABthet) to provide a proof of concept. DHAP was added as a nucleophile to several α-hydroxylated ketones used as electrophiles. This aldol addition was stereoselective and produced branched-chain monosaccharide adducts with a tertiary alcohol moiety. Several aldols were readily obtained in good to excellent yields (from 76 to 95 %). These results contradict the general view that aldehydes are the only electrophile substrates for DHAP-dependent aldolases and provide a new C-C bond-forming enzyme for stereoselective synthesis of tertiary alcohols.

Corrigendum: Parallel evolution of non-homologous isofunctional enzymes in methionine biosynthesis

Corrigendum: Parallel evolution of non-homologous isofunctional enzymes in methionine biosynthesis