Unknown Enzyme Research Articles

Naringenin chalcone carbon double-bond reductases mediate dihydrochalcone biosynthesis in apple leaves.

Dihydrochalcones (DHCs) are flavonoids produced as a side branch of the phenylpropanoid pathway. DHCs are found at high concentrations in apples (Malus spp.) but not in pears (Pyrus spp.) or other members of the Rosaceae. Biosynthesis of DHCs in apple has been hypothesized to occur via reduction of p-coumaroyl CoA by a Malus × domestica hydroxycinnamoyl CoA double-bond reductase (MdHCDBR) followed by the action chalcone synthase to produce phloretin or via direct reduction of naringenin chalcone to phloretin via an unknown enzyme. In this study, we report that genetic downregulation of MdHCDBR does not reduce DHC concentrations in apple leaves. We used comparative transcriptome analysis to identify candidate naringenin chalcone reductases (NCRs), designated MdNCR1a-c, expressed in apple leaves but not fruit. These MdNCR1 genes form an expanded gene cluster found exclusively in apple. Transient expression of MdNCR1 genes in Nicotiana benthamiana leaves indicated they produced DHCs at high concentrations in planta. Recombinant MdNCR1 utilized naringenin chalcone to produce phloretin at high efficiency. Downregulation of NCR genes in transgenic apple reduced foliar DHC levels by 85-95%. Reducing DHC production redirected flux to the production of flavonol glycosides. In situ localization indicated that NCR proteins were likely found in the vacuolar membrane. Active site analysis of AlphaFold models indicated that MdNCR1a-c share identical substrate binding pockets, but the pockets differ substantially in related weakly active/inactive NCR proteins. Identifying the missing enzyme required for DHC production provides opportunities to manipulate DHC content in apple and other fruits and has other applications, e.g., in biofermentation and biopharming.

Transformation of 17β-estradiol to estrone by unknown wild-type enzyme in commercial arylsulfatase from Helix pomatia

This work for the first time reported the complete transformation of 17β-estradiol (E2) to estrone (E1) by unknown wild-type enzyme present in the widely used commercial arylsulfatase derived from Helix pomatia. It was found that acetate could effectively inhibit the unknown enzyme with a half inhibitory concentration (IC50) of 140.9 μM, while phosphate and citrate showed no inhibition. Since the buffer solutions with phosphate and citrate have been used in the enzymatic hydrolysis of natural estrogen conjugates for decades, the transformation of E2 to E1 likely occurred during such procedure, inevitably leading to overestimated E1, but underestimated E2. It was further suggested that acetate should be used to prevent this undesirable transformation during the enzymatic hydrolysis of natural estrogen conjugates.

Characterization and heterologous reconstitution of Taxus biosynthetic enzymes leading to baccatin III.

Paclitaxel is a well known anticancer compound. Its biosynthesis involves the formation of a highly functionalized diterpenoid core skeleton (baccatin III) and the subsequent assembly of a phenylisoserinoyl side chain. Despite intensive investigation for half a century, the complete biosynthetic pathway of baccatin III remains unknown. In this work, we identified a bifunctional cytochrome P450 enzyme [taxane oxetanase 1 (TOT1)] in Taxus mairei that catalyzes an oxidative rearrangement in paclitaxel oxetane formation, which represents a previously unknown enzyme mechanism for oxetane ring formation. We created a screening strategy based on the taxusin biosynthesis pathway and uncovered the enzyme responsible for the taxane oxidation of the C9 position (T9αH1). Finally, we artificially reconstituted a biosynthetic pathway for the production of baccatin III in tobacco.

Exploration of DPP-IV Inhibitory Peptide Design Rules Assisted by the Deep Learning Pipeline That Identifies the Restriction Enzyme Cutting Site.

The mining of antidiabetic dipeptidyl peptidase IV (DPP-IV) inhibitory peptides (DPP-IV-IPs) is currently a costly and laborious process. Due to the absence of rational peptide design rules, it relies on cumbersome screening of unknown enzyme hydrolysates. Here, we present an enhanced deep learning model called bidirectional encoder representation (BERT)-DPPIV, specifically designed to classify DPP-IV-IPs and explore their design rules to discover potent candidates. The end-to-end model utilizes a fine-tuned BERT architecture to extract structural/functional information from input peptides and accurately identify DPP-IV-Ips from input peptides. Experimental results in the benchmark data set showed BERT-DPPIV yielded state-of-the-art accuracy and MCC of 0.894 and 0.790, surpassing the 0.797 and 0.594 obtained by the sequence-feature model. Furthermore, we leveraged the attention mechanism to uncover that our model could recognize the restriction enzyme cutting site and specific residues that contribute to the inhibition of DPP-IV. Moreover, guided by BERT-DPPIV, proposed design rules for DPP-IV inhibitory tripeptides and pentapeptides were validated, and they can be used to screen potent DPP-IV-IPs.

In Search of Complementary Extraction Methods for Comprehensive Coverage of the Escherichia coli Metabolome.

Escherichia coli is an invaluable research tool for many fields of biology, in particular for the production of recombinant enzymes. However, the activity of many such recombinant enzymes cannot be determined using standard biochemical assays, as often, the relevant substrates are not known, or the products produced are not detectable. Today, the biochemical footprints of such unknown enzyme activities can be revealed via the analysis of the metabolomes of the recombinant E. coli clones in which they are expressed, using sensitive technologies such as mass spectrometry. However, before any metabolites can be identified, it is necessary to achieve as high a coverage of the potential metabolites present within E. coli as possible. We have therefore analyzed a wide range of different extraction methods against the cell free extracts of various recombinant E. coli clones. The results were analyzed to determine the minimum number of extractions that achieved high recovery and coverage of metabolites. Two methods were selected for further analysis due to their ability to produce not only high numbers of ions, but also wide mass coverage and a high degree of complementarity. One extraction method uses acetonitrile and water, in a 4:1 ratio, which is then dried down and reconstituted in the chromatography running buffer prior to injection onto the chromatography column, and the other extraction method uses a combination of methanol, water and chloroform, in a 3:1:1 ratio, which is injected directly onto the chromatography column. These two extraction methods were shown to be complementary to each other, as regards the respective metabolites extracted, and to cover a large range of metabolites.

Non-canonical d-xylose and l-arabinose metabolism via d-arabitol in the oleaginous yeast Rhodosporidium toruloides

R. toruloides is an oleaginous yeast, with diverse metabolic capacities and high tolerance for inhibitory compounds abundant in plant biomass hydrolysates. While R. toruloides grows on several pentose sugars and alcohols, further engineering of the native pathway is required for efficient conversion of biomass-derived sugars to higher value bioproducts. A previous high-throughput study inferred that R. toruloides possesses a non-canonical l-arabinose and d-xylose metabolism proceeding through d-arabitol and d-ribulose. In this study, we present a combination of genetic and metabolite data that refine and extend that model. Chiral separations definitively illustrate that d-arabitol is the enantiomer that accumulates under pentose metabolism. Deletion of putative d-arabitol-2-dehydrogenase (RTO4_9990) results in > 75% conversion of d-xylose to d-arabitol, and is growth-complemented on pentoses by heterologous xylulose kinase expression. Deletion of putative d-ribulose kinase (RTO4_14368) arrests all growth on any pentose tested. Analysis of several pentose dehydrogenase mutants elucidates a complex pathway with multiple enzymes mediating multiple different reactions in differing combinations, from which we also inferred a putative l-ribulose utilization pathway. Our results suggest that we have identified enzymes responsible for the majority of pathway flux, with additional unknown enzymes providing accessory activity at multiple steps. Further biochemical characterization of the enzymes described here will enable a more complete and quantitative understanding of R. toruloides pentose metabolism. These findings add to a growing understanding of the diversity and complexity of microbial pentose metabolism.

Facile immobilization of pyridoxal 5′-phosphate using p-diazobenzoyl-derivatized Sepharose 4B

Pyridoxal 5′-phosphate (PLP) is a ubiquitous and versatile cofactor utilized by numerous enzymes involved in amino acid biosynthetic pathways. Immobilized PLP is a valuable tool to isolate unknown PLP-dependent enzymes in nature or to perform in vitro selection or directed evolution on existing or de novo PLP-dependent enzymes. The C-6 position is preferred for covalent immobilization of PLP because it maintains all important functional groups in their native, unmodified form. Previously reported diazonium derivatization methods for C-6 immobilization utilized an azide linker compound that is hazardous and not readily available. Here we report a safer and more accessible method to synthesize p-diazobenzoyl-derivatized Sepharose 4B using the N-hydroxysuccinimide (NHS) ester chemistry. The derivative was used to immobilize PLP, and the resulting C-6 immobilized PLP had a loading of ∼2.6 µmol PLP per mL of resin, comparable to commercially available products of other immobilized cofactors.

The History of mARC.

The mitochondrial amidoxime-reducing component (mARC) is the most recently discovered molybdoenzyme in humans after sulfite oxidase, xanthine oxidase and aldehyde oxidase. Here, the timeline of mARC's discovery is briefly described. The story begins with investigations into N-oxidation of pharmaceutical drugs and model compounds. Many compounds are N-oxidized extensively in vitro, but it turned out that a previously unknown enzyme catalyzes the retroreduction of the N-oxygenated products in vivo. After many years, the molybdoenzyme mARC could finally be isolated and identified in 2006. mARC is an important drug-metabolizing enzyme and N-reduction by mARC has been exploited very successfully for prodrug strategies, that allow oral administration of otherwise poorly bioavailable therapeutic drugs. Recently, it was demonstrated that mARC is a key factor in lipid metabolism and likely involved in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). The exact link between mARC and lipid metabolism is not yet fully understood. Regardless, many now consider mARC a potential drug target for the prevention or treatment of liver diseases. This article focusses on discoveries related to mammalian mARC enzymes. mARC homologues have been studied in algae, plants and bacteria. These will not be discussed extensively here.

Core and auxiliary functions of one-carbon metabolism in Pseudomonas putida exposed by a systems-level analysis of transcriptional and physiological responses.

The soil bacterium Pseudomonas putida is a robust biomanufacturing host that assimilates a broad range of substrates while efficiently coping with adverse environmental conditions. P. putida is equipped with functions related to one-carbon (C1) compounds (e.g. methanol, formaldehyde, and formate) oxidation-yet pathways to assimilate these carbon sources are largely absent. In this work, we adopted a systems-level approach to study the genetic and molecular basis of C1 metabolism in P. putida. RNA sequencing identified two oxidoreductases, encoded by PP_0256 and PP_4596, transcriptionally active in the presence of formate. Quantitative physiology of deletion mutants revealed growth defects at high formate concentrations, pointing to an important role of these oxidoreductases in C1 tolerance. Moreover, we describe a concerted detoxification process for methanol and formaldehyde, the C1 intermediates upstream formate. Alcohol oxidation to highly-reactive formaldehyde by PedEH and other broad-substrate-range dehydrogenases underpinned the (apparent) suboptimal methanol tolerance of P. putida. Formaldehyde was mostly processed by a glutathione-dependent mechanism encoded in the frmAC operon, and thiol-independent FdhAB and AldB-II overtook detoxification at high aldehyde concentrations. Deletion strains were constructed and characterized towards unveiling these biochemical mechanisms, underscoring the worth of P. putida for emergent biotechnological applications-e.g. engineering synthetic formatotrophy and methylotrophy. IMPORTANCE C1 substrates continue to attract interest in biotechnology, as their use is both cost-effective and ultimately expected to mitigate the impact of greenhouse gas emissions. However, our current understanding of bacterial C1 metabolism remains relatively limited in species that cannot grow on (i.e., assimilate) these substrates. Pseudomonas putida, a model Gram-negative environmental bacterium, constitutes a prime example of this sort. The biochemical pathways active in response to methanol, formaldehyde, and formate have been largely overlooked-although the ability of P. putida to process C1 molecules has been previously alluded to in the literature. By using a systems-level strategy, this study bridges such knowledge gap through the identification and characterization of mechanisms underlying methanol, formaldehyde, and formate detoxification-including hitherto unknown enzymes that act on these substrates. The results reported herein both expand our understanding of microbial metabolism and lay a solid foundation for engineering efforts toward valorizing C1 feedstocks.

Synthesis of Flavonol-Bearing Probes for Chemoproteomic and Bioinformatic Analyses of Asteraceae Petals in Search of Novel Flavonoid Enzymes.

This study aimed at searching for the enzymes that are responsible for the higher hydroxylation of flavonols serving as UV-honey guides for pollinating insects on the petals of Asteraceae flowers. To achieve this aim, an affinity-based chemical proteomic approach was developed by relying on the use of quercetin-bearing biotinylated probes, which were thus designed and synthesized to selectively and covalently capture relevant flavonoid enzymes. Proteomic and bioinformatic analyses of proteins captured from petal microsomes of two Asteraceae species (Rudbeckia hirta and Tagetes erecta) revealed the presence of two flavonol 6-hydroxylases and several additional not fully characterized proteins as candidates for the identification of novel flavonol 8-hydroxylases, as well as relevant flavonol methyl- and glycosyltransferases. Generally speaking, this substrate-based proteome profiling methodology constitutes a powerful tool for the search for unknown (flavonoid) enzymes in plant protein extracts.

GENETIC VARIATION AND PREDICTED PHENOTYPES IN CYP2C9, CYP2C19, CYP3A4, CYP3A5 AND CYP2D6 GENES AMONG 552 PATIENTS WITH UNCONTROLLED TREATED HYPERTENSION

Objective: Altered drug metabolism may be a contributing factor to uncontrolled hypertension. Beta-blockers, alpha-blockers, calcium antagonists and angiotensin receptor blockers undergo hepatic metabolism by cytochrome P450 (CYP) enzymes. Genetic variation can contribute to interindividual variation, lack of treatment response or side effects with discontinuation of treatment. We aimed to determine common CYP gene variations in patients with treated but uncontrolled hypertension and to compare the distribution with the prevalence in the overall Caucasian population. Design and method: In this national multicenter study we mapped CYP2D6, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 polymorphisms in 552 patients prescribed > = 2 antihypertensive agents and with systolic daytime ambulatory blood pressure (ABPM) > = 135 mmHg. On the (MALDI-TOF) MassARRAY analyzer, single nucleotide variants and small insertions/deletions (indels) were investigated with the VeriDose Core Panel and CYP2D6 copy-number variation with the VeriDose CYP2D6 CNV Panel (Agena Biosciences). Each allele is associated with either normal, decreased, absent, increased or unknown enzyme function based on activity scores defined by the The Clinical Pharmacogenetics Implementation Consortium (CPIC) and describe function and phenotypes for genes (IM = Intermediate metabolizer, PM = poor metabolizer, RM = rapid metabolizer and UM = ultra-rapid metabolizer). Results: 94% of patients were Caucasian. Mean daytime ABPM was 146/84 mmHg (135-190/55-106 mmHg), median age 63 (26-86) years and 35% were female. The distributions of the predicted phenotypes (illustrated in colors in Figure) for each enzyme did not differ from that reported in Caucasian population by The Pharmacogenomics Knowledgebase (PharmGK) (gray). 11% had normal phenotype in all five enzymes whereas 49% had > = 2 enzymes affected by gene variants. Conclusions: The phenotype distribution of five relevant CYP genes in patients with uncontrolled hypertension did not differ from the general Caucasian population. Dosing guidelines based on CYP2D6 genotype currently exist for metoprolol, but for other antihypertensive drugs further investigation is needed. The low proportion of patients with five normal enzymes and the high proportion with > = 2 enzymes affected suggest that pharmacogenetic CYP-testing may be useful for individualizing drug treatment in patients with uncontrolled hypertension.

Development and validation of a proteomic correlation profiling technique to detect and identify enzymes involved in metabolism of drugs of concern.

It is important to be able to detect previously unknown and unsuspected enzymes involved in drug metabolism, in order to predict the variation of pharmacological or toxicological effect caused by pharmacokinetic variance. We investigated the use of proteomic correlation profiling (PCP) as a technique to identify the enzymes involved in metabolism of drugs of concern. By evaluating the metabolic activities of each enzyme (including isoforms of cytochrome P450, uridine 5' diphospho‑glucuronosyltransferase, and hydrolases, plus aldehyde oxidase and carbonyl reductase) on their typical substrates using a panel of human liver samples, we were able to show the validity of PCP for this purpose. Pearson or Spearman's rank correlation coefficient and P values were calculated for the association between the protein abundance profile of each protein and the metabolic rate profile of each typical substrate. For the 18 enzymatic activities examined, 13 of the enzymes reported to be responsible for the reactions had correlation coefficients higher than 0.7, and were ranked first to third. For the remaining five activities, the responsible enzymes had correlation coefficients lower than 0.7 and lower rankings. The reasons for this were diverse, including confounding resulting from low protein abundance ratios, artificially high correlations of other enzymes due to limited sample numbers, the presence of inactive enzyme forms, and genetic polymorphisms. Overall, PCP was able to identify the majority of responsible drug-metabolizing enzymes across several enzyme classes (oxidoreductase, transferase, hydrolase); use of this methodology could allow more timely and accurate identification of unknown drug-metabolizing enzymes. Significance Statement Proteomic correlation profiling using samples from individual human donors was proven to be a useful methodology for the identification of enzymes responsible for drug-metabolism. This methodology could accelerate the identification of unknown drug-metabolizing enzymes in the future.

Rethinking Biosynthesis of Aclacinomycin A.

Aclacinomycin A (ACM-A) is an anthracycline antitumor agent widely used in clinical practice. The current industrial production of ACM-A relies primarily on chemical synthesis and microbial fermentation. However, chemical synthesis involves multiple reactions which give rise to high production costs and environmental pollution. Microbial fermentation is a sustainable strategy, yet the current fermentation yield is too low to satisfy market demand. Hence, strain improvement is highly desirable, and tremendous endeavors have been made to decipher biosynthesis pathways and modify key enzymes. In this review, we comprehensively describe the reported biosynthesis pathways, key enzymes, and, especially, catalytic mechanisms. In addition, we come up with strategies to uncover unknown enzymes and improve the activities of rate-limiting enzymes. Overall, this review aims to provide valuable insights for complete biosynthesis of ACM-A.

Fatty acid acylated peptide therapeutics: discovery of omega-n oxidation of the lipid chain as a novel metabolic pathway in preclinical species

We recently described C18 fatty acid acylated peptides as a new class of potent long-lasting single-chain RXFP1 agonists that displayed relaxin-like activities in vivo. Early pharmacokinetics and toxicological studies of these stearic acid acylated peptides revealed a relevant oxidative metabolism occurring in dog and minipig, and also seen at a lower extent in monkey and rat. Mass spectrometry combined to NMR spectroscopy studies revealed that the oxidation occurred, unexpectedly, on the stearic acid chain at ω-1, ω-2 and ω-3 positions. Structure-metabolism relationship studies on acylated analogues with different fatty acids lengths (C15-C20) showed that the extent of oxidation was higher with longer chains. The oxidized metabolites could be generated in vitro using liver microsomes and engineered bacterial CYPs. These systems were correlating poorly with in vivo metabolism observed across species; however, the results suggest that this biotransformation pathway might be catalyzed by some unknown CYP enzymes.

Terpene synthases and pathways in animals: enzymology and structural evolution in the biosynthesis of volatile infochemicals.

Covering: up to the beginning of 2023Many animals release volatile or semi-volatile terpenes as semiochemicals in intra- and inter-specific interactions. Terpenes are important constituents of pheromones and serve as chemical defenses to ward off predators. Despite the occurrence of terpene specialized metabolites from soft corals to mammals, the biosynthetic origin of these compounds has largely remained obscure. An increasing number of animal genome and transcriptome resources is facilitating the identification of enzymes and pathways that allow animals to produce terpenes independent of their food sources or microbial endosymbionts. Substantial evidence has emerged for the presence of terpene biosynthetic pathways such as in the formation of the iridoid sex pheromone nepetalactone in aphids. In addition, terpene synthase (TPS) enzymes have been discovered that are evolutionary unrelated to canonical plant and microbial TPSs and instead resemble precursor enzymes called isoprenyl diphosphate synthases (IDSs) in central terpene metabolism. Structural modifications of substrate binding motifs in canonical IDS proteins presumably facilitated the transition to TPS function at an early state in insect evolution. Other arthropods such as mites appear to have adopted their TPS genes from microbial sources via horizontal gene transfer. A similar scenario likely occurred in soft corals, where TPS families with closer resemblance to microbial TPSs have been discovered recently. Together, these findings will spur the identification of similar or still unknown enzymes in terpene biosynthesis in other lineages of animals. They will also help develop biotechnological applications for animal derived terpenes of pharmaceutical value or advance sustainable agricultural practices in pest management.

Identification and characterization of proteins that form the inner core Ixodes scapularis tick attachment cement layer.

Ixodes scapularis long-term blood feeding behavior is facilitated by a tick secreted bio adhesive (tick cement) that attaches tick mouthparts to skin tissue and prevents the host from dislodging the attached tick. Understanding tick cement formation is highly sought after as its disruption will prevent tick feeding. This study describes proteins that form the inner core layer of I. scapularis tick cement as disrupting these proteins will likely stop formation of the outer cortical layer. The inner core cement layer completes formation by 24h of tick attachment. Thus, we used laser-capture microdissection to isolate cement from cryosections of 6h and 24h tick attachment sites and to distinguish between early and late inner core cement proteins. LC-MS/MS analysis identified 138 tick cement proteins (TCPs) of which 37 and 35 were unique in cement of 6 and 24h attached ticks respectively. We grouped TCPs in 14 functional categories: cuticular protein (16%), tick specific proteins of unknown function, cytoskeletal proteins, and enzymes (13% each), enzymes (10%), antioxidant, glycine rich, scaffolding, heat shock, histone, histamine binding, proteases and protease inhibitors, and miscellaneous (3-6% each). Gene ontology analysis confirm that TCPs are enriched for bio adhesive properties. Our data offer insights into tick cement bonding patterns and set the foundation for understanding the molecular basis of I. scapularis tick cement formation.

BAHD acyltransferase induced by histone deacetylase inhibitor catalyzes 3-O-hydroxycinnamoylquinic acid formation in bamboo cells.

Suspension-cultured cells of a bamboo species (Bambusa multiplex; Bm) produce 3-O-feruloylquinic acid (3-FQA) and 3-O-p-coumaroylquinic acid (3-pCQA) by treatment with the histone deacetylase inhibitor suberoyl bis-hydroxamic acid (SBHA). Acyltransferases catalyzing the formation of 5-O-hydroxycinnamoylquinic acid esters by transesterification from hydroxycinnamoyl-CoAs to the C-5 hydroxy group of quinic acid (hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferase, HQT) have been identified in the biosynthesis of chlorogenic acids and monolignols; however, an HQT that catalyzes the acylation of the C-3 hydroxy group of quinic acid has not been identified previously. In the present study, we purified a native HQT from SBHA-treated Bm cells. The purified enzyme preferentially accepted feruloyl-/p-coumaroyl-CoAs as acyl-donors and quinic acid as the acyl-acceptor, and the enzyme specifically formed 3-FQA and 3-pCQA but not 5-O-hydroxycinnamoylquinic acid esters or esters with shikimic acid. A cDNA (BmHQT1) encoding this HQT was isolated. Although BmHQT1 is a phylogenetically unique member of the BAHD acyltransferase superfamily that does not cluster with other HQTs, functional characterization of the recombinant enzyme verified that BmHQT1 catalyzes the regiospecific formation of 3-O-hydroxycinnamoylquinic acid esters. Transcript levels of BmHQT1 markedly increased in Bm cells cultured in the presence of SBHA. Moreover, elevated acetylation levels of histone H3 were observed in the coding region of BmHQT1 in the presence of SBHA, indicating that the induced accumulation of 3-FQA/3-pCQA by SBHA is caused by transcriptional activation of BmHQT1 by the action of SBHA as a histone deacetylase inhibitor. The results demonstrate the utility of HDAC inhibitors for discovery of cryptic secondary metabolites and unknown biosynthetic enzymes.

Assigning Functions of Unknown Enzymes by High-Throughput Enzyme Characterization.

The discovery of new enzymes is strongly enabled by the implementation of high-throughput screening methods to detect enzymatic activity in single organisms or clone expression libraries, or to benchmark their performances against known prototypes. In this chapter, a number of methods, applicable at high-throughput scale, are described that allow the screening and characterization of enzymes relevant to biotechnology, particularly, ester-hydrolases (esterases, lipases, phospholipases, and polyester hydrolases).

Halogenation in Fungi: What Do We Know and What Remains to Be Discovered?

In nature, living organisms produce a wide variety of specialized metabolites to perform many biological functions. Among these specialized metabolites, some carry halogen atoms on their structure, which can modify their chemical characteristics. Research into this type of molecule has focused on how organisms incorporate these atoms into specialized metabolites. Several families of enzymes have been described gathering metalloenzymes, flavoproteins, or S-adenosyl-L-methionine (SAM) enzymes that can incorporate these atoms into different types of chemical structures. However, even though the first halogenation enzyme was discovered in a fungus, this clade is still lagging behind other clades such as bacteria, where many enzymes have been discovered. This review will therefore focus on all halogenation enzymes that have been described in fungi and their associated metabolites by searching for proteins available in databases, but also by using all the available fungal genomes. In the second part of the review, the chemical diversity of halogenated molecules found in fungi will be discussed. This will allow the highlighting of halogenation mechanisms that are still unknown today, therefore, highlighting potentially new unknown halogenation enzymes.

Metabolomic approaches for enzyme function and pathway discovery in bacteria.

Most of the chemical diversity present in the natural world derives from the incredible ability of enzymes to act on and control metabolism. Yet, thousands of enzymes have no defined function. The capacity to probe, investigate and assign previously unknown enzyme function with speed and confidence is therefore highly sought-after. Metabolomics is becoming a dominant player in the field of functional genomics and, when coupled with genetic tools and protein biochemistry techniques, has enabled unbiased, de novo annotation of orphan enzymes both in vitro and ex vivo. In this chapter, we describe two distinct experimental and analytical metabolomic methodologies used to reveal enzyme function. Activity-based metabolomic profiling (ABMP) is an in vitro technique that enables tracking of enzyme-induced changes in a complex metabolite extract. Global metabolomic profiling permits the comparison of extracted cellular metabolome of groups of samples (e.g., wild-type versus mutant bacteria). The methods we describe present the advantage of generating cell extracts containing a broad range of metabolites in their native states, which can then be used to identify substrates for orphan enzymes. This chapter aims to provide a guide for the use of these metabolomic techniques by scientists interested in identifying bona fide physiological substrates of orphan enzymes and the metabolic pathways they belong to.