Experiment-based computational method for proper annotation of the molecular function of enzymes

The rate of protein functional elucidation lags far behind the rate of gene and protein sequence discovery, leading to an accumulation of proteins with no known function. Millions of protein database entries are not assigned reliable functions, preventing the full understanding of chemical diver­sity in living organisms. Pfam contains over 16,712 families, among which more than 3,919 families are of unknown function (DUF families). An additional difficulty, often underestimated, is that only a tiny fraction of enzymes have experimentally established functions and in most cases, function is extrapolated from a small number of characterized proteins to all members of a family leading to over-annotation1,2. Here, two examples of an integrated strategy for the discovery of various enzymatic activities catalyzed within protein families will be presented. This approach relies with a high-throughput enzymatic screening on representatives, structural and modeling investigations, analysis of genomic and metabolic context. The structural analysis is in both cases based on the Active Site Clustering Method3 developed at Genoscope. We investigated the protein family with no known function, DUF849 Pfam family, and unearthed 14 potential new enzymatic activities, leading to the designation of these proteins as -keto acid cleavage enzymes4. In addition, we propose an in vivo role for four enzymatic activities and suggest key residues for guiding further functional annotation. The second study will illustrate that proteins with high sequence similarity might not have the same function. We determined the enzymatic activities of 100 O-acyl-L-homoserine transferases representative of the biodiversity of the two unrelated families, MetX and MetA, involved in the first step of the methionine biosynthesis and assumed to always use acetyl-CoA and succinyl-CoA, respectively. We interpreted the results by structural classification of active sites based on protein structure modeling. We identified the specific determining positions responsible for acyl-CoA specificity in the active sites of MetX and MetA enzymes, actually iso-functional for both activities. We then predict that \u3e60% of the 10,000 sequences from these families currently in databases are incorrectly annotated. Finally, we uncovered a divergent subgroup of MetX enzymes in fungi that participate only in L-cysteine biosynthesis as O-succinyl-L-serine transferases5. 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 and the chemical capabilities of biodiversity. References: 1 de Crecy-Lagard, V. Quality Annotations, a Key Frontier in the Microbial Sciences. Microbe 11, 303-310 (2016). 2 Schnoes, A. M., Brown, S. D., Dodevski, I. & Babbitt, P. C. Annotation error in public databases: misannotation of molecular function in enzyme superfamilies. PLoS Comput Biol 5, e1000605 (2009). 3 de Melo-Minardi, R. C., Bastard, K. & Artiguenave, F. Identification of subfamily-specific sites based on active sites modeling and clustering. Bioinformatics 26, 3075-3082, doi:10.1093/bioinformatics/btq595 (2010). 4 Bastard, K. et al. Revealing the hidden functional diversity of an enzyme family. Nature chemical biology 10, 42-49, doi:10.1038/nchembio.1387 (2014). 5 Bastard, K. et al. Parallel evolution of non-homologous isofunctional enzymes in methionine biosynthesis. Nature chemical biology june (2017)

Iterative reconstruction of a global metabolic model of Acinetobacter baylyi ADP1 using high-throughput growth phenotype and gene essentiality data

<p>Abstract</p> <p>Background</p> <p>Genome-scale metabolic models are powerful tools to study global properties of metabolic networks. They provide a way to integrate various types of biological information in a single framework, providing a structured representation of available knowledge on the metabolism of the respective species.</p> <p>Results</p> <p>We reconstructed a constraint-based metabolic model of <it>Acinetobacter baylyi </it>ADP1, a soil bacterium of interest for environmental and biotechnological applications with large-spectrum biodegradation capabilities. Following initial reconstruction from genome annotation and the literature, we iteratively refined the model by comparing its predictions with the results of large-scale experiments: (1) high-throughput growth phenotypes of the wild-type strain on 190 distinct environments, (2) genome-wide gene essentialities from a knockout mutant library, and (3) large-scale growth phenotypes of all mutant strains on 8 minimal media. Out of 1412 predictions, 1262 were initially consistent with our experimental observations. Inconsistencies were systematically examined, leading in 65 cases to model corrections. The predictions of the final version of the model, which included three rounds of refinements, are consistent with the experimental results for (1) 91% of the wild-type growth phenotypes, (2) 94% of the gene essentiality results, and (3) 94% of the mutant growth phenotypes. To facilitate the exploitation of the metabolic model, we provide a web interface allowing online predictions and visualization of results on metabolic maps.</p> <p>Conclusion</p> <p>The iterative reconstruction procedure led to significant model improvements, showing that genome-wide mutant phenotypes on several media can significantly facilitate the transition from genome annotation to a high-quality model.</p

Photobiocatalytic Oxyfunctionalization with High Reaction Rate using a Baeyer-Villiger Monooxygenase from Burkholderia xenovorans in Metabolically Engineered Cyanobacteria

Baeyer-Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to lactones under very mild reaction conditions. This enzymatic route is hindered by the requirement of a stoichiometric supply of auxiliary substrates for cofactor recycling and difficulties with supplying the necessary oxygen. The recombinant production of BVMO in cyanobacteria allows the substitution of auxiliary organic cosubstrates with water as an electron donor and the utilization of oxygen generated by photosynthetic water splitting. Herein, we report the identification of a BVMO from Burkholderia xenovorans (BVMOXeno) that exhibits higher reaction rates in comparison to currently identified BVMOs. We report a 10-fold increase in specific activity in comparison to cyclohexanone monooxygenase (CHMOAcineto) in Synechocystis sp. PCC 6803 (25 vs 2.3 U g(DCW)(-1) at an optical density of OD750 = 10) and an initial rate of 3.7 +/- 0.2 mM h(-1). While the cells containing CHMOAcineto showed a considerable reduction of cyclohexanone to cyclohexanol, this unwanted side reaction was almost completely suppressed for BVMOXeno, which was attributed to the much faster lactone formation and a 10-fold lower KM value of BVMOXeno toward cyclohexanone. Furthermore, the whole-cell catalyst showed outstanding stereoselectivity. These results show that, despite the self-shading of the cells, high specific activities can be obtained at elevated cell densities and even further increased through manipulation of the photosynthetic electron transport chain (PETC). The obtained rates of up to 3.7 mM h-1 underline the usefulness of oxygenic cyanobacteria as a chassis for enzymatic oxidation reactions. The photosynthetic oxygen evolution can contribute to alleviating the highly problematic oxygen mass-transfer limitation of oxygendependent enzymatic processes

A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1

We have constructed a collection of single-gene deletion mutants for all dispensable genes of the soil bacterium Acinetobacter baylyi ADP1. A total of 2594 deletion mutants were obtained, whereas 499 (16%) were not, and are therefore candidate essential genes for life on minimal medium. This essentiality data set is 88% consistent with the Escherichia coli data set inferred from the Keio mutant collection profiled for growth on minimal medium, while 80% of the orthologous genes described as essential in Pseudomonas aeruginosa are also essential in ADP1. Several strategies were undertaken to investigate ADP1 metabolism by (1) searching for discrepancies between our essentiality data and current metabolic knowledge, (2) comparing this essentiality data set to those from other organisms, (3) systematic phenotyping of the mutant collection on a variety of carbon sources (quinate, 2-3 butanediol, glucose, etc.). This collection provides a new resource for the study of gene function by forward and reverse genetic approaches and constitutes a robust experimental data source for systems biology approaches

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Evolution study of the Baeyer-Villiger monooxygenases enzyme family: functional importance of the highly conserved residues.

International audienceBaeyer-Villiger monooxygenases (BVMOs) catalyze the transformation of linear and cyclic ketones into their corresponding esters and lactones by introducing an oxygen atom into a C-C bond. This bioreaction has numerous advantages compared to its chemical version; it does not induce the use of potentially harmful reagents (i.e., green chemistry) and displays significant better enantio- and regio-selectivity. New potential BVMOs were searched using sequence homology for type I BVMO proteins. 116 new sequences were identified as new putative BVMOs respecting the defined selection criteria. Multiple sequence alignments were carried out on the selected sequences to study the conservation of structurally and/or functionally important amino acids during evolution. Type I BVMO signature motif was found to be conserved in 94.8% of the sequences. We noticed also the highly conserved - but previously unnoticed - Threonine 167 (93.1%), located in the signature motif; this position could be added in the pattern used to characterize specific Type I enzymes. Amino acids at the vicinity of the FAD and NADPH cofactors were found also to be highly conserved and the details of the interactions were emphasized. Interestingly, residues at the enzyme binding site were found less conserved in terms of sequence evolution, leading sometimes to some important amino acid changes. These behaviors could explain the enzyme selectivity and specificity for different ligands

PROCÉDÉ DE PRÉPARATION D'AMINES À PARTIR D'ALDÉHYDES ET DE CÉTONES PAR BIOCATALYSE

The present invention relates to the preparation of amines from aldehydes and ketones by reductive amination with enzymes having a reductive aminase activity on aldehydes and ketones devoid of any carboxyl group gamma of the carbonyl group. The invention also relates to the enzymes per se and their uses in biocatalysis. The enzymes are derived from Mycobacterium smegmatis and vaccae, Cystobacter fuscus, Microbacterium sp. and Aminomonas paucivorans

PROCÉDÉ DE PRÉPARATION D'AMINES À PARTIR D'ALDÉHYDES ET DE CÉTONES PAR BIOCATALYSE

The present invention relates to the preparation of amines from aldehydes and ketones by reductive amination with enzymes having a reductive aminase activity on aldehydes and ketones devoid of any carboxyl group gamma of the carbonyl group. The invention also relates to the enzymes per se and their uses in biocatalysis. The enzymes are derived from Mycobacterium smegmatis and vaccae, Cystobacter fuscus, Microbacterium sp. and Aminomonas paucivorans