Enantioselective Reduction of Noncovalent Complexes of Amino Acids with CuII via Resonant Collision-Induced Dissociation: Collision Energy, Activation Duration Effects, and RRKM Modeling.

Formation of noncovalent complexes is one of the approaches to perform chiral analysis with mass spectrometry. Enantiomeric distinction of amino acids (AAs) based on the relative rate constants of competitive fragmentations of quaternary copper complexes is an efficient method for chiral differentiation. Here, we studied the complex [CuII,(Phe,PhG,Pro-H)]+ (m/z 493) under resonant collision-induced dissociation conditions while varying the activation time. The precursor ion can yield two main fragments through the loss of the non-natural AA phenylglycine (PhG): the expected product ion [CuII,(Phe,Pro-H)]+ (m/z 342) and the reduced product ion [CuI,(Phe,Pro)]+ (m/z 343). Enantioselective reduction describes the difference in relative abundance of these ions, which depends on the chirality of the precursor ion: the formation of the reduced ion m/z 343 is favored in homochiral complexes (DDD) compared to heterochiral complexes (such as LDD). Energy-resolved mass spectrometry data show that reduction, which arises from rearrangement, is favored at a low collision energy (CE) and long activation time (ActT), whereas direct cleavage preferentially occurs at a high CE and short ActT. These results were confirmed with kinetic modeling based on RRKM theory. For this modeling, it was necessary to set a pre-exponential factor as a reference, so that the E0 values obtained are relative values. Interestingly, these simulations showed that the critical energy E0 required to form the reduced ion is comparable in both homochiral and heterochiral complexes. However, the formation of product ion m/z 342 through direct cleavage is associated with a lower E0 in heterochiral complexes. Consequently, enantioselectivity would not be caused by enhanced reduction in homochiral complexes but rather by direct cleavage being favored in heterochiral complexes.

Characterization of a novel ß-alanine biosynthetic pathway consisting of promiscuous metabolic enzymes.

Bacteria adapt to utilize the nutrients available in their environment through a sophisticated metabolic system composed of highly specialized enzymes. Although these enzymes can metabolize molecules other than those for which they evolved, their efficiency toward promiscuous substrates is considered too low to be of physiological relevance. Herein, we investigated the possibility that these promiscuous enzymes are actually efficient enough at metabolizing secondary substrates to modify the phenotype of the cell. For example, in the bacterium Acinetobacter baylyi ADP1 (ADP1), panD (coding for l-aspartate decarboxylase) encodes the only protein known to catalyze the synthesis of ß-alanine, an obligate intermediate in CoA synthesis. However, we show that the ADP1 ΔpanD mutant could also form this molecule through an unknown metabolic pathway arising from promiscuous enzymes and grow as efficiently as the wildtype strain. Using metabolomic analyses, we identified 1,3-diaminopropane and 3-aminopropanal as intermediates in this novel pathway. We also conducted activity screening and enzyme kinetics to elucidate candidate enzymes involved in this pathway, including 2,4-diaminobutyrate aminotransferase (Dat) and 2,4-diaminobutyrate decarboxylase (Ddc) and validated this pathway in vivo by analyzing the phenotype of mutant bacterial strains. Finally, we experimentally demonstrate that this novel metabolic route is not restricted to ADP1. We propose that the occurrence of conserved genes in hundreds of genomes across many phyla suggests that this previously undescribed pathway is widespread in prokaryotes.

Pyruvate kinase, a metabolic sensor powering glycolysis, drives the metabolic control of DNA replication.

BACKGROUND: In all living organisms, DNA replication is exquisitely regulated in a wide range of growth conditions to achieve timely and accurate genome duplication prior to cell division. Failures in this regulation cause DNA damage with potentially disastrous consequences for cell viability and human health, including cancer. To cope with these threats, cells tightly control replication initiation using well-known mechanisms. They also couple DNA synthesis to nutrient richness and growth rate through a poorly understood process thought to involve central carbon metabolism. One such process may involve the cross-species conserved pyruvate kinase (PykA) which catalyzes the last reaction of glycolysis. Here we have investigated the role of PykA in regulating DNA replication in the model system Bacillus subtilis. RESULTS: On analysing mutants of the catalytic (Cat) and C-terminal (PEPut) domains of B. subtilis PykA we found replication phenotypes in conditions where PykA is dispensable for growth. These phenotypes are independent from the effect of mutations on PykA catalytic activity and are not associated with significant changes in the metabolome. PEPut operates as a nutrient-dependent inhibitor of initiation while Cat acts as a stimulator of replication fork speed. Disruption of either PEPut or Cat replication function dramatically impacted the cell cycle and replication timing even in cells fully proficient in known replication control functions. In vitro, PykA modulates activities of enzymes essential for replication initiation and elongation via functional interactions. Additional experiments showed that PEPut regulates PykA activity and that Cat and PEPut determinants important for PykA catalytic activity regulation are also important for PykA-driven replication functions. CONCLUSIONS: We infer from our findings that PykA typifies a new family of cross-species replication control regulators that drive the metabolic control of replication through a mechanism involving regulatory determinants of PykA catalytic activity. As disruption of PykA replication functions causes dramatic replication defects, we suggest that dysfunctions in this new family of universal replication regulators may pave the path to genetic instability and carcinogenesis.

Genetic and biocatalytic basis of formate dependent growth of Escherichia coli strains evolved in continuous culture.

The reductive glycine pathway was described as the most energetically favorable synthetic route of aerobic formate assimilation. Here we report the successful implementation of formatotrophy in Escherichia coli by means of a stepwise adaptive evolution strategy. Medium swap and turbidostat regimes of continuous culture were applied to force the channeling of carbon flux through the synthetic pathway to pyruvate establishing growth on formate and CO2 as sole carbon sources. Labeling with 13C-formate proved the assimilation of the C1 substrate via the pathway metabolites. Genetic analysis of intermediate isolates revealed a mutational path followed throughout the adaptation process. Mutations were detected affecting the copy number (gene ftfL) or the coding sequence (genes folD and lpd) of genes which specify enzymes implicated in the three steps forming glycine from formate and CO2, the central metabolite of the synthetic pathway. The mutation R191S present in methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (FolD) abolishes the inhibition of cyclohydrolase activity by the substrate formyl-tetrahydrofolate. The mutation R273H in lipoamide dehydrogenase (Lpd) alters substrate affinities as well as kinetics at physiological substrate concentrations likely favoring a reactional shift towards lipoamide reduction. In addition, genetic reconstructions proved the necessity of all three mutations for formate assimilation by the adapted cells. The largely unpredictable nature of these changes demonstrates the usefulness of the evolutionary approach enabling the selection of adaptive mutations crucial for pathway engineering of biotechnological model organisms.

Change in Cofactor Specificity of Oxidoreductases by Adaptive Evolution of an Escherichia coli NADPH-Auxotrophic Strain.

The nicotinamide cofactor specificity of enzymes plays a key role in regulating metabolic processes and attaining cellular homeostasis. Multiple studies have used enzyme engineering tools or a directed evolution approach to switch the cofactor preference of specific oxidoreductases. However, whole-cell adaptation toward the emergence of novel cofactor regeneration routes has not been previously explored. To address this challenge, we used an Escherichia coli NADPH-auxotrophic strain. We continuously cultivated this strain under selective conditions. After 500 to 1,100 generations of adaptive evolution using different carbon sources, we isolated several strains capable of growing without an external NADPH source. Most isolated strains were found to harbor a mutated NAD+-dependent malic enzyme (MaeA). A single mutation in MaeA was found to switch cofactor specificity while lowering enzyme activity. Most mutated MaeA variants also harbored a second mutation that restored the catalytic efficiency of the enzyme. Remarkably, the best MaeA variants identified this way displayed overall superior kinetics relative to the wild-type variant with NAD+. In other evolved strains, the dihydrolipoamide dehydrogenase (Lpd) was mutated to accept NADP+, thus enabling the pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase complexes to regenerate NADPH. Interestingly, no other central metabolism oxidoreductase seems to evolve toward reducing NADP+, which we attribute to several biochemical constraints, including unfavorable thermodynamics. This study demonstrates the potential and biochemical limits of evolving oxidoreductases within the cellular context toward changing cofactor specificity, further showing that long-term adaptive evolution can optimize enzyme activity beyond what is achievable via rational design or directed evolution using small libraries. IMPORTANCE In the cell, NAD(H) and NADP(H) cofactors have different functions. The former mainly accepts electrons from catabolic reactions and carries them to respiration, while the latter provides reducing power for anabolism. Correspondingly, the ratio of the reduced to the oxidized form differs for NAD+ (low) and NADP+ (high), reflecting their distinct roles. We challenged the flexibility of E. coli's central metabolism in multiple adaptive evolution experiments using an NADPH-auxotrophic strain. We found several mutations in two enzymes, changing the cofactor preference of malic enzyme and dihydrolipoamide dehydrogenase. Upon deletion of their corresponding genes we performed additional evolution experiments which did not lead to the emergence of any additional mutants. We attribute this restricted number of mutational targets to intrinsic thermodynamic barriers; the high ratio of NADPH to NADP+ limits metabolic redox reactions that can regenerate NADPH, mainly by mass action constraints.

First Direct Evidence of Interpartner Hydride/Deuteride Exchanges for Stored Sodiated Arginine/Fructose-6-phosphate Complex Anions within Salt-Solvated Structures.

Mass spectrometric investigations of noncovalent binding between low molecular weight compounds revealed the existence of gas-phase (GP) noncovalent complex (NCC) ions involving zwitterionic structures. ESI MS is used to prove the formation of stable sodiated NCC anions between fructose (F6P) and arginine (R) moieties. Theoretical calculations indicate a folded solvated salt (i.e., sodiated carboxylate interacting with phosphate) rather than a charge-solvated form. Under standard CID conditions, [(F6P+R-H+Na)-H]- competitively forms two major product ions (PIs) through partner splitting [(R-H+Na) loss] and charge-induced cross-ring cleavage while preserving the noncovalent interactions (noncovalent product ions (NCPIs)). MS/MS experiments combined with in-solution proton/deuteron exchanges (HDXs) demonstrated an unexpected labeling of PIs, i.e., a correlated D-enrichment/D-depletion. An increase in activation time up to 3000 ms favors such processes when limited to two H/D exchanges. These results are rationalized by interpartner hydride/deuteride exchanges (⟨HDX⟩) through stepwise isomerization/dissociation of sodiated NCC-d11 anions. In addition, the D-enrichment/D-depletion discrepancy is further explained by back HDX with residual water in LTQ (selective for the isotopologue NCPIs as shown by PI relaxation experiments). Each isotopologue leads to only one back HDX unlike multiple HDXs generally observed in GP. This behavior shows that NCPIs are zwitterions with charges solvated by a single water molecule, thus generating a back HDX through a relay mechanism, which quenches the charges and prevents further back HDX. By estimating back HDX impact on D-depletion, the interpartner ⟨HDX⟩ complementarity was thus illustrated. This is the first description of interpartner ⟨HDX⟩ and selective back HDX validating salt-solvated structures.

Continuous Culture Adaptation of Methylobacterium extorquens AM1 and TK 0001 to Very High Methanol Concentrations.

The bio-economy relies on microbial strains optimized for efficient large scale production of chemicals and fuels from inexpensive and renewable feedstocks under industrial conditions. The reduced one carbon compound methanol, whose production does not involve carbohydrates needed for the feed and food sector, can be used as sole carbon and energy source by methylotrophic bacteria like Methylobacterium extorquens AM1. This strain has already been engineered to produce various commodity and high value chemicals from methanol. The toxic effect of methanol limits its concentration as feedstock to 1% v/v. We obtained M. extorquens chassis strains tolerant to high methanol via adaptive directed evolution using the GM3 technology of automated continuous culture. Turbidostat and conditional medium swap regimes were employed for the parallel evolution of the recently characterized strain TK 0001 and the reference strain AM1 and enabled the isolation of derivatives of both strains capable of stable growth with 10% methanol. The isolates produced more biomass at 1% methanol than the ancestor strains. Genome sequencing identified the gene metY coding for an O-acetyl-L-homoserine sulfhydrylase as common target of mutation. We showed that the wildtype enzyme uses methanol as substrate at elevated concentrations. This side reaction produces methoxine, a toxic homolog of methionine incorporated in polypeptides during translation. All mutated metY alleles isolated from the evolved populations coded for inactive enzymes, designating O-acetyl-L-homoserine sulfhydrylase as a major vector of methanol toxicity. A whole cell transcriptomic analysis revealed that genes coding for chaperones and proteases were upregulated in the evolved cells as compared with the wildtype, suggesting that the cells had to cope with aberrant proteins formed during the adaptation to increasing methanol exposure. In addition, the expression of ribosomal proteins and enzymes related to energy production from methanol like formate dehydrogenases and ATP synthases was boosted in the evolved cells upon a short-term methanol stress. D-lactate production from methanol by adapted cells overexpressing the native D-lactate dehydrogenase was quantified. A significant higher lactate yield was obtained compared with control cells, indicating an enhanced capacity of the cells resistant to high methanol to assimilate this one carbon feedstock more efficiently.

ABC Transporters Required for Hexose Uptake by Clostridium phytofermentans.

The mechanisms by which bacteria uptake solutes across the cell membrane broadly impact their cellular energetics. Here, we use functional genomic, genetic, and biophysical approaches to reveal how Clostridium (Lachnoclostridium) phytofermentans, a model bacterium that ferments lignocellulosic biomass, uptakes plant hexoses using highly specific, nonredundant ATP-binding cassette (ABC) transporters. We analyze the transcription patterns of its 173 annotated sugar transporter genes to find those upregulated on specific carbon sources. Inactivation of these genes reveals that individual ABC transporters are required for uptake of hexoses and hexo-oligosaccharides and that distinct ABC transporters are used for oligosaccharides versus their constituent monomers. The thermodynamics of sugar binding shows that substrate specificity of these transporters is encoded by the extracellular solute-binding subunit. As sugars are not phosphorylated during ABC transport, we identify intracellular hexokinases based on in vitro activities. These mechanisms used by Clostridia to uptake plant hexoses are key to understanding soil and intestinal microbiomes and to engineer strains for industrial transformation of lignocellulose.IMPORTANCE Plant-fermenting Clostridia are anaerobic bacteria that recycle plant matter in soil and promote human health by fermenting dietary fiber in the intestine. Clostridia degrade plant biomass using extracellular enzymes and then uptake the liberated sugars for fermentation. The main sugars in plant biomass are hexoses, and here, we identify how hexoses are taken in to the cell by the model organism Clostridium phytofermentans We show that this bacterium uptakes hexoses using a set of highly specific, nonredundant ABC transporters. Once in the cell, the hexoses are phosphorylated by intracellular hexokinases. This study provides insight into the functioning of abundant members of soil and intestinal microbiomes and identifies gene targets to engineer strains for industrial lignocellulosic fermentation.

De novo structure determination of 3-((3-aminopropyl)amino)-4-hydroxybenzoic acid, a novel and abundant metabolite in Acinetobacter baylyi ADP1.

INTRODUCTION: Metabolite identification remains a major bottleneck in the understanding of metabolism. Many metabolomics studies end up with unknown compounds, leaving a landscape of metabolites and metabolic pathways to be unraveled. Therefore, identifying novel compounds within a metabolome is an entry point into the 'dark side' of metabolism. OBJECTIVES: This work aimed at elucidating the structure of a novel metabolite that was first detected in the soil bacterium Acinetobacter baylyi ADP1 (ADP1). METHODS: We used high resolution multi-stage tandem mass spectrometry for characterizing the metabolite within the metabolome. We purified the molecule for 1D- and 2D-NMR (1H, 13C, 1H-1H-COSY, 1H-13C-HSQC, 1H-13C-HMBC and 1H-15N-HMBC) analyses. Synthetic standards were chemically prepared from MS and NMR data interpretation. RESULTS: We determined the de novo structure of a previously unreported metabolite: 3-((3-aminopropyl)amino)-4-hydroxybenzoic acid. The proposed structure was validated by comparison to a synthetic standard. With a concentration in the millimolar range, this compound appears as a major metabolite in ADP1, which we anticipate to participate to an unsuspected metabolic pathway. This novel metabolite was also detected in another γ-proteobacterium. CONCLUSION: Structure elucidation of this abundant and novel metabolite in ADP1 urges to decipher its biosynthetic pathway and cellular function.

Characterization of l-Carnitine Metabolism in Sinorhizobium meliloti.

l-Carnitine is a trimethylammonium compound mostly known for its contribution to fatty acid transport into mitochondria. In bacteria, it is synthesized from γ-butyrobetaine (GBB) and can be used as a carbon source. l-Carnitine can be formed directly by GBB hydroxylation or synthesized via a biosynthetic route analogous to fatty acid degradation. However, this multistep pathway has not been experimentally characterized. In this work, we identified by gene context analysis a cluster of l-carnitine anabolic genes next to those involved in its catabolism and proceeded to the complete in vitro characterization of l-carnitine biosynthesis and degradation in Sinorhizobium meliloti The five enzymes catalyzing the seven steps that convert GBB to glycine betaine are described. Metabolomic analysis confirmed the multistage synthesis of l-carnitine in GBB-grown cells but also revealed that GBB is synthesized by S. meliloti To our knowledge, this is the first report of aerobic GBB synthesis in bacteria. The conservation of l-carnitine metabolism genes in different bacterial taxonomic classes underscores the role of l-carnitine as a ubiquitous nutrient.IMPORTANCE The experimental characterization of novel metabolic pathways is essential for realizing the value of genome sequences and improving our knowledge of the enzymatic capabilities of the bacterial world. However, 30% to 40% of genes of a typical genome remain unannotated or associated with a putative function. We used enzyme kinetics, liquid chromatography-mass spectroscopy (LC-MS)-based metabolomics, and mutant phenotyping for the characterization of the metabolism of l-carnitine in Sinorhizobium meliloti to provide an accurate annotation of the corresponding genes. The occurrence of conserved gene clusters for carnitine metabolism in soil, plant-associated, and marine bacteria underlines the environmental abundance of carnitine and suggests this molecule might make a significant contribution to ecosystem nitrogen and carbon cycling.

Elucidation of the trigonelline degradation pathway reveals previously undescribed enzymes and metabolites.

Trigonelline (TG; N-methylnicotinate) is a ubiquitous osmolyte. Although it is known that it can be degraded, the enzymes and metabolites have not been described so far. In this work, we challenged the laboratory model soil-borne, gram-negative bacterium Acinetobacter baylyi ADP1 (ADP1) for its ability to grow on TG and we identified a cluster of catabolic, transporter, and regulatory genes. We dissected the pathway to the level of enzymes and metabolites, and proceeded to in vitro reconstruction of the complete pathway by six purified proteins. The four enzymatic steps that lead from TG to methylamine and succinate are described, and the structures of previously undescribed metabolites are provided. Unlike many aromatic compounds that undergo hydroxylation prior to ring cleavage, the first step of TG catabolism proceeds through direct cleavage of the C5-C6 bound, catalyzed by a flavin-dependent, two-component oxygenase, which yields (Z)-2-((N-methylformamido)methylene)-5-hydroxy-butyrolactone (MFMB). MFMB is then oxidized into (E)-2-((N-methylformamido) methylene) succinate (MFMS), which is split up by a hydrolase into carbon dioxide, methylamine, formic acid, and succinate semialdehyde (SSA). SSA eventually fuels up the TCA by means of an SSA dehydrogenase, assisted by a Conserved Hypothetical Protein. The cluster is conserved across marine, soil, and plant-associated bacteria. This emphasizes the role of TG as a ubiquitous nutrient for which an efficient microbial catabolic toolbox is available.

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.

A Synthetic Alternative to Canonical One-Carbon Metabolism.

One-carbon metabolism is an ubiquitous metabolic pathway that encompasses the reactions transferring formyl-, hydroxymethyl- and methyl-groups bound to tetrahydrofolate for the synthesis of purine nucleotides, thymidylate, methionine and dehydropantoate, the precursor of coenzyme A. An alternative cyclic pathway was designed that substitutes 4-hydroxy-2-oxobutanoic acid (HOB), a compound absent from known metabolism, for the amino acids serine and glycine as one-carbon donors. It involves two novel reactions, the transamination of l-homoserine and the transfer of a one-carbon unit from HOB to tetrahydrofolate releasing pyruvate as coproduct. Since canonical reactions regenerate l-homoserine from pyruvate by carboxylation and subsequent reduction, every one-carbon moiety made available for anabolic reactions originates from CO2. The HOB-dependent pathway was established in an Escherichia coli auxotroph selected for prototrophy using long-term cultivation protocols. Genetic, metabolic and biochemical evidence support the emergence of a functional HOB-dependent one-carbon pathway achieved with the recruitment of the two enzymes l-homoserine transaminase and HOB-hydroxymethyltransferase and of HOB as an essential metabolic intermediate. Escherichia coli biochemical reprogramming was achieved by minimally altering canonical metabolism and leveraging on natural selection mechanisms, thereby launching the resulting strain on an evolutionary trajectory diverging from all known extant species.

Meaning and consequence of the coexistence of competitive hydrogen bond/salt forms on the dissociation orientation of non-covalent complexes.

Non-covalent complexes (NCC) between hexose monophosphates (HexP) and arginine (R) were analyzed using ESI MS and MS/MS in negative mode under different (hard, HC and soft, SC) desolvation conditions. High resolution mass spectrometry (HRMS) revealed the presence of different ionic species, namely, homo- and heteromultimers of R and HexP. Deprotonated heterodimers and corresponding sodiated species were enhanced under HC likely due to a decrease in available charge number associated with the reduction of H+/Na+ exchange. The quantum calculations showed that the formation of covalent systems is very little exothermic, therefore, such systems are disfavored. Desolvation dependent CID spectra of deprotonated [(HexP+R)‒H]- complexes demonstrated that they can exist within the hydrogen bond (HB) and salt bridge (SB) forms, yielding either NCC separation or covalent bond cleavages, respectively. Although HB forms are the main species, they cannot survive under HC; therefore, the minor SB forms became detectable. Energy-resolved mass spectrometry (ERMS) experiments revealed diagnostic fragment ions from both SB and HB forms, providing evidence that these isomeric forms are inconvertible. SB formation should result from the ionic interactions of highly acidic group of HexP with strongly basic guanidine group of arginine and thus requires an arginine zwitterion (ZW) form. This was confirmed by quantum calculations. Ion-ion interactions are significantly affected by the presence of sodium cation as demonstrated by the fragmentation patterns of sodiated complex species. Regarding CID data, only SB between protonated amino group of R and deprotonated phosphate group of HexP could be suggested, but the primary amine is not enough basic then, the SB must be fleeting. Nevertheless, the observation of the covalent bond cleavages suggests the presence of structures with a free negative charge able to induce fragmentations. Indeed, according to quantum calculations, solvated salt (SS) systems involving Na+/COO- salt solvated by neutral phosphate and negative charge on sugar ring are preferentially formed.

Novel metabolic features in Acinetobacter baylyi ADP1 revealed by a multiomics approach.

Expansive knowledge of bacterial metabolism has been gained from genome sequencing output, but the high proportion of genes lacking a proper functional annotation in a given genome still impedes the accurate prediction of the metabolism of a cell. To access to a more global view of the functioning of the soil bacterium Acinetobacter baylyi ADP1, we adopted a multi 'omics' approach. Application of RNA-seq transcriptomics and LC/MS-based metabolomics, along with the systematic phenotyping of the complete collection of single-gene deletion mutants of A. baylyi ADP1 made possible to interrogate on the metabolic perturbations encountered by the bacterium upon a biotic change. Shifting the sole carbon source from succinate to quinate elicited in the cell not only a specific transcriptional response, necessary to catabolize the new carbon source, but also a major reorganization of the transcription pattern. Here, the expression of more than 12 % of the total number of genes was affected, most of them being of unknown function. These perturbations were ultimately reflected in the metabolome, in which the concentration of about 50 % of the LC/MS-detected metabolites was impacted. And the differential regulation of many genes of unknown function is probably related to the synthesis of the numerous unidentified compounds that were present exclusively in quinate-grown cells. Together, these data suggest that A. baylyi ADP1 metabolism involves unsuspected enzymatic reactions that await discovery.

Validation of RetroPath, a computer-aided design tool for metabolic pathway engineering.

Metabolic engineering has succeeded in biosynthesis of numerous commodity or high value compounds. However, the choice of pathways and enzymes used for production was many times made ad hoc, or required expert knowledge of the specific biochemical reactions. In order to rationalize the process of engineering producer strains, we developed the computer-aided design (CAD) tool RetroPath that explores and enumerates metabolic pathways connecting the endogenous metabolites of a chassis cell to the target compound. To experimentally validate our tool, we constructed 12 top-ranked enzyme combinations producing the flavonoid pinocembrin, four of which displayed significant yields. Namely, our tool queried the enzymes found in metabolic databases based on their annotated and predicted activities. Next, it ranked pathways based on the predicted efficiency of the available enzymes, the toxicity of the intermediate metabolites and the calculated maximum product flux. To implement the top-ranking pathway, our procedure narrowed down a list of nine million possible enzyme combinations to 12, a number easily assembled and tested. One round of metabolic network optimization based on RetroPath output further increased pinocembrin titers 17-fold. In total, 12 out of the 13 enzymes tested in this work displayed a relative performance that was in accordance with its predicted score. These results validate the ranking function of our CAD tool, and open the way to its utilization in the biosynthesis of novel compounds.

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 ß-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.

Prediction and identification of sequences coding for orphan enzymes using genomic and metagenomic neighbours.

Despite the current wealth of sequencing data, one-third of all biochemically characterized metabolic enzymes lack a corresponding gene or protein sequence, and as such can be considered orphan enzymes. They represent a major gap between our molecular and biochemical knowledge, and consequently are not amenable to modern systemic analyses. As 555 of these orphan enzymes have metabolic pathway neighbours, we developed a global framework that utilizes the pathway and (meta)genomic neighbour information to assign candidate sequences to orphan enzymes. For 131 orphan enzymes (37% of those for which (meta)genomic neighbours are available), we associate sequences to them using scoring parameters with an estimated accuracy of 70%, implying functional annotation of 16,345 gene sequences in numerous (meta)genomes. As a case in point, two of these candidate sequences were experimentally validated to encode the predicted activity. In addition, we augmented the currently available genome-scale metabolic models with these new sequence-function associations and were able to expand the models by on average 8%, with a considerable change in the flux connectivity patterns and improved essentiality prediction.

Microbial urate catabolism: characterization of HpyO, a non-homologous isofunctional isoform of the flavoprotein urate hydroxylase HpxO.

In aerobic cells, urate is oxidized to 5-hydroxyisourate by two distinct enzymes: a coenzyme-independent urate oxidase (EC 1.7.3.3) found in eukaryotes and bacteria like Bacillus subtilis and a prokaryotic flavoprotein urate hydroxylase (HpxO) originally found in some Klebsiella species. More cases of analogous or non-homologous isofunctional enzymes (NISE) for urate catabolism have been hypothesized by inspecting bacterial genomes. Here, we used a functional complementation approach in which a candidate gene for urate oxidation is integrated by homologous recombination in the Acinetobacter baylyi ADP1 genome at the locus of its original hpxO gene. Catabolism of urate was restored in A. baylyi ADP1 expressing a FAD-dependent protein from Xanthomonas campestris, representing a new urate hydroxylase family that we called HpyO. This enzyme was kinetically characterized and compared with other HpxO enzymes. In contrast to the latter, HpyO is a typical Michaelian enzyme. This work provides the first experimental evidences for the function of HpyO in bacterial urate catabolism and establishes it as a NISE of HpxO.