Orphan Enzyme Research Articles

PTER is a N-acetyltaurine hydrolase that regulates feeding and obesity

Taurine is a conditionally essential micronutrient and one of the most abundant amino acids in humans1–3. In endogenous taurine metabolism, dedicated enzymes are involved in the biosynthesis of taurine from cysteine and in the downstream metabolism of secondary taurine metabolites4,5. One taurine metabolite is N-acetyltaurine6. Levels of N-acetyltaurine are dynamically regulated by stimuli that alter taurine or acetate flux, including endurance exercise7, dietary taurine supplementation8 and alcohol consumption6,9. So far, the identities of the enzymes involved in N-acetyltaurine metabolism, and the potential functions of N-acetyltaurine itself, have remained unknown. Here we show that the body mass index associated orphan enzyme phosphotriesterase-related (PTER)10 is a physiological N-acetyltaurine hydrolase. In vitro, PTER catalyses the hydrolysis of N-acetyltaurine to taurine and acetate. In mice, PTER is expressed in the kidney, liver and brainstem. Genetic ablation of Pter in mice results in complete loss of tissue N-acetyltaurine hydrolysis activity and a systemic increase in N-acetyltaurine levels. After stimuli that increase taurine levels, Pter knockout mice exhibit reduced food intake, resistance to diet-induced obesity and improved glucose homeostasis. Administration of N-acetyltaurine to obese wild-type mice also reduces food intake and body weight in a GFRAL-dependent manner. These data place PTER into a central enzymatic node of secondary taurine metabolism and uncover a role for PTER and N-acetyltaurine in body weight control and energy balance.

Oxamic transcarbamylase of Escherichia coli is encoded by the three genes allFGH (formerly fdrA, ylbE, and ylbF).

Escherichia coli uses allantoin as the sole nitrogen source during anaerobic growth. In the final step of allantoin degradation, oxamic transcarbamylase (OXTCase) converts oxalurate to carbamoyl phosphate (CP) and oxamate. The activity of this enzyme was first measured in Streptococcus allantoicus in the 1960s, but no OXTCase enzyme or the encoding gene(s) have been found in any strain. This study discovered that allFGH (fdrA, ylbE, and ylbF) are the genes that encode the global orphan enzyme OXTCase. The three genes form an operon together with allK (ybcF), encoding catabolic carbamate kinase. The allFGHK operon is located directly downstream of the allECD operon that encodes enzymes for the preceding steps of OXTCase. The OXTCase kinetic parameters were analyzed using the purified protein composed of AllF-AllG-AllH (FdrA-YlbE-YlbF); for the substrate CP, KM and Vmax were 1.3 mM and 15.4 U/mg OXTCase, respectively, and for the substrate oxamate, they were 36.9 mM and 27.0 U/mg OXTCase. In addition, the OXTCase encoded by the three genes is a novel transcarbamylase that shows no similarity with known enzymes of the transcarbamylase family such as aspartate transcarbamylase, ornithine transcarbamylase, and YgeW transcarbamylase. The present study elucidated the anaerobic allantoin degradation pathway of E. coli. Therefore, we suggest that the genes fdrA, ylbE, and ylbF are renamed allF, allG, and allH, respectively.IMPORTANCEThe anaerobic allantoin degradation pathway of Escherichia coli includes a global orphan enzyme, oxamic transcarbamylase (OXTCase), which converts oxalurate to carbamoyl phosphate and oxamate. This study found that the allFGH (fdrA, ylbE, and ylbF) genes encode OXTCase. The OXTCase activity and kinetics were successfully determined with purified recombinant AllF-AllG-AllH (FdrA-YlbE-YlbF). This OXTCase is a novel transcarbamylase that shows no similarity with known enzymes of the transcarbamylase family such as aspartate transcarbamylase (ATCase), ornithine transcarbamylase (OTCase), and YgeW transcarbamylase (YTCase). In addition, OXTCase activity requires three genes, whereas ATCase is encoded by two genes, and OTCase and YTCase are encoded by a single gene. The current study discovered OXTCase, the last unknown step in allantoin degradation, and this enzyme is a new member of the transcarbamylase group that was previously unknown.

An Escherichia coli FdrA Variant Derived from Syntrophic Coculture with a Methanogen Increases Succinate Production Due to Changes in Allantoin Degradation.

ABSTRACTWild-type Escherichia coli was adapted to syntrophic growth with Methanobacterium formicicum for glycerol fermentation over 44 weeks. Succinate production by E. coli started to increase in the early stages of syntrophic growth. Genetic analysis of the cultured E. coli population by pooled sequencing at eight time points suggests that (i) rapid evolution occurred through repeated emergence of mutators that introduced a large number of nucleotide variants and (ii) many mutators increased to high frequencies but remained polymorphic throughout the continuous cultivation. The evolved E. coli populations exhibited gains both in fitness and succinate production, but only for growth under glycerol fermentation with M. formicicum (the condition for this laboratory evolution) and not under other growth conditions. The mutant alleles of the 69 single nucleotide polymorphisms (SNPs) identified in the adapted E. coli populations were constructed individually in the ancestral wild-type E. coli. We analyzed the phenotypic changes caused by 84 variants, including 15 nonsense variants, and found that FdrAD296Y was the most significant variant leading to increased succinate production. Transcription of fdrA was induced under anaerobic allantoin degradation conditions, and FdrA was shown to play a crucial role in oxamate production. The FdrAD296Y variant increased glyoxylate conversion to malate by accelerating oxamate production, which promotes carbon flow through the C4 branch, leading to increased succinate production.IMPORTANCE Here, we demonstrate the ability of E. coli to perform glycerol fermentation in coculture with the methanogen M. formicicum to produce succinate. We found that the production of succinate by E. coli significantly increased during successive cocultivation. Genomic DNA sequencing, evaluation of relative fitness, and construction of SNPs were performed, from which FdrAD296Y was identified as the most significant variant to enable increased succinate production by E. coli. The function of FdrA is uncertain. In this study, experiments with gene expression assays and metabolic analysis showed for the first time that FdrA could be the “orphan enzyme” oxamate:carbamoyltransferase in anaerobic allantoin degradation. Furthermore, we demonstrate that the anaerobic allantoin degradation pathway is linked to succinate production via the glyoxylate pathway during glycerol fermentation.

Optimization of protease production and sequence analysis of the purified enzyme from the cold adapted yeast Rhodotorula mucilaginosa CBMAI 1528

Enzymes from cold-adapted microorganisms are of high interest to industries due to their high activity at low and mild temperatures, which makes them suitable for their use in several processes that either require a supply of exogenous energy or involve the use of heat labile products. In this work, the protease production by the strain Rhodotorula mucilaginosa CBMAI 1528, previously isolated from the Antarctic continent, was optimized, and the purified enzyme analyzed. It was found that protease production was dependent on culture medium composition and growth temperature, being 20 °C and a culture medium containing both glucose and casein peptone (20 and 10 g/L, respectively) the optimal growing conditions in batch as well as in bioreactor. Moreover, mass spectrometry analysis revealed that the enzyme under study has a 100 % sequence identity with the deduced amino acid sequence of a putative aspartic protease from Rhodotorula sp. JG-1b (protein ID: KWU42276.1). This result was confirmed by the decrease of 95 % proteolytic activity by pepstatin A, a specific inhibitor of aspartic proteases. We propose that the enzyme reported here could be Rodothorulapepsin, a protein characterized in 1972 that did not have an associated sequence to date and has been classified as an orphan enzyme.

Implementation of homology based and non-homology based computational methods for the identification and annotation of orphan enzymes: using Mycobacterium tuberculosis H37Rv as a case study

BackgroundHomology based methods are one of the most important and widely used approaches for functional annotation of high-throughput microbial genome data. A major limitation of these methods is the absence of well-characterized sequences for certain functions. The non-homology methods based on the context and the interactions of a protein are very useful for identifying missing metabolic activities and functional annotation in the absence of significant sequence similarity. In the current work, we employ both homology and context-based methods, incrementally, to identify local holes and chokepoints, whose presence in the Mycobacterium tuberculosis genome is indicated based on its interaction with known proteins in a metabolic network context, but have not been annotated. We have developed two computational procedures using network theory to identify orphan enzymes (‘Hole finding protocol’) coupled with the identification of candidate proteins for the predicted orphan enzyme (‘Hole filling protocol’). We propose an integrated interaction score based on scores from the STRING database to identify candidate protein sequences for the orphan enzymes from M. tuberculosis, as a case study, which are most likely to perform the missing function.ResultsThe application of an automated homology-based enzyme identification protocol, ModEnzA, on M. tuberculosis genome yielded 56 novel enzyme predictions. We further predicted 74 putative local holes, 6 choke points, and 3 high confidence local holes in the genome using ‘Hole finding protocol’. The ‘Hole-filling protocol’ was validated on the E. coli genome using artificial in-silico enzyme knockouts where our method showed 25% increased accuracy, compared to other methods, in assigning the correct sequence for the knocked-out enzyme amongst the top 10 ranks. The method was further validated on 8 additional genomes.ConclusionsWe have developed methods that can be generalized to augment homology-based annotation to identify missing enzyme coding genes and to predict a candidate protein for them. For pathogens such as M. tuberculosis, this work holds significance in terms of increasing the protein repertoire and thereby, the potential for identifying novel drug targets.

Utilizing SULT4A1 Mutant Mouse Models to Characterize SULT4A1

Identified by our lab in 2000, sulfotransferase 4A1 (SULT4A1) belongs to the cytosolic sulfotransferase (SULT) gene superfamily of phase II conjugation enzymes based on key sequence and structural similarities. Broadly, SULTs catalyze the transfer of a sulfonate moiety (SO3−) to a diverse set of endogenous and exogenous substrates in tandem with the obligate cofactor 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS). Expression studies have shown SULT4A1 to be localized to neuronal tissues, and SULT4A1 orthologs have been found in all vertebrate species examined. Remarkably, human SULT4A1 shares a high degree of sequence homology with other vertebrate SULT4A1 orthologs, more than any other human SULT. Despite the conserved nature of SULT4A1 among vertebrates, all attempts to demonstrate sulfotransferase activity have failed and the neuronal function of this orphan enzyme remains unclear. To elucidate an understanding of SULT4A1, CRISPR‐Cas9 gene editing technology was performed to disrupt SULT4A1 function in a C57BL/6J mouse background and two mutations of interest were pursued. A 28 bp deletion (Δ28) in exon 1 resulted in a frameshift mutation and a premature stop codon at amino acid (AA) 62. A second mutation of interest was a 12 bp (Δ12) in‐frame deletion in exon 3 of four AA immediately adjacent to the active site histidine. Dynamic modeling studies of the Δ12 mutation suggested a structural disruption to the substrate binding pocket. Interestingly, homozygous mutant pups of both lines displayed a severe and progressive neurological pathology beginning 8–10 days post‐natal, until death at day 21–25. Neurological symptoms included persistent tremor, ataxia, absence seizures, and decreased weight gain. Homozygous mutant mice of both strains were confirmed to lack detectable SULT4A1, while heterozygous mice of both strains showed a modest decrease compared to wild‐type (WT) mice. Since little is known about SULT4A1 function, subcellular fractionation was performed to determine if SULT4A1 localizes to other cellular compartments. Indeed, SULT4A1 was detected in mitochondrial, cytosolic, and microsomal fractions, a previously unreported finding. Due to the early onset of neurological symptoms, SULT4A1 expression was assessed in primary cortical neurons (PCNs) harvested from gestational day 15 mice. PCNs were cultured in vitro to day 12 and robust expression of SULT4A1 was observed in these cells by immunostaining. In summary, SULT4A1 is a critical protein for neurofunction and normal development, as mice lacking functional SULT4A1 displayed a progressively severe and lethal neurological pathology. These mutant mouse models will be instrumental into characterizing the function of SULT4A1.Support or Funding InformationThis work was supported by GM113980 to CNF and NS08695301 to SAA.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Engineering a self-sufficient Mycobacterium tuberculosis CYP130 by gene fusion with the reductase-domain of CYP102A1 from Bacillus megaterium

CYP130 belongs to the subset of cytochrome P450s from Mycobacterium tuberculosis (Mtb) that have been structurally characterized. Despite several efforts for its functional characterization, CYP130 is still considered an orphan enzyme for which no endogenous or exogenous substrate has been identified. In addition, functional redox-partners for CYP130 have not been clearly established yet, hampering the elucidation of its physiological role. In the present study, a catalytically active fusion protein involving CYP130 and the NADPH reductase-domain of CYP102A1 from Bacillus megaterium was created. By screening a panel of known substrates of human P450s, dextromethorphan N-demethylation was identified as a reaction catalyzed by CYP130. The fusion enzyme showed higher catalytic activity, when compared to CYP130 reconstituted with a selection of non-native redox-partners. Molecular dynamics simulation studies based on the crystal structure of CYP130 revealed two primary docking poses of dextromethorphan within the active site consistent with the experimentally observed N-demethylation reaction during the entire molecular dynamics simulation. The dextromethorphan N-demethylation reaction was strongly inhibited by azole-drugs and maybe applied to identify mechanism-based inhibitors of CYP130. Furthermore, the present active CYP130-fusion protein may facilitate the identification of endogenous substrates from Mtb.

Caspase-2: an orphan enzyme out of the shadows.

Caspase-2 has been embodied as an initiator or executioner protease in diverse apoptotic scenarios. However, accumulating evidence is challenging this view, pertaining to its true role. The enzyme's catalytic activity is currently implicated in various functions required for correct cell proliferation, such as counteracting genomic instability, as well as suppressing tumorigenesis. Here, apart from summarizing the latest observations in caspase-2-related research, we make an attempt to reconcile these findings and discuss their implications for future directions.

Fragment Profiling Approach to Inhibitors of the Orphan M. tuberculosis P450 CYP144A1

Similarity between the ligand binding profiles of enzymes may aid functional characterization and be of greater relevance to inhibitor development than sequence similarity or structural homology. Fragment screening is an efficient approach for characterization of the ligand binding profile of an enzyme and has been applied here to study the family of cytochrome P450 enzymes (P450s) expressed by Mycobacterium tuberculosis (Mtb). The Mtb P450s have important roles in bacterial virulence, survival, and pathogenicity. Comparing the fragment profiles of seven of these enzymes revealed that P450s which share a similar biological function have significantly similar fragment profiles, whereas functionally unrelated or orphan P450s exhibit distinct ligand binding properties, despite overall high structural homology. Chemical structures that exhibit promiscuous binding between enzymes have been identified, as have selective fragments that could provide leads for inhibitor development. The similarity between the fragment binding profiles of the orphan enzyme CYP144A1 and CYP121A1, a characterized enzyme that is important for Mtb viability, provides a case study illustrating the subsequent identification of novel CYP144A1 ligands. The different binding modes of these compounds to CYP144A1 provide insight into structural and dynamic aspects of the enzyme, possible biological function, and provide the opportunity to develop inhibitors. Expanding this fragment profiling approach to include a greater number of functionally characterized and orphan proteins may provide a valuable resource for understanding enzyme-ligand interactions.

Progesterone signaling in sperm

Reproductive Biology Cumulus cells surrounding the ovulated egg release the female hormone progesterone, which is necessary for sperm activation in mammals. Miller et al. identified the human sperm progesterone receptor as the orphan enzyme ABHD2. When bound by progesterone, the ABHD2 receptor acts as a lipid hydrolase to deplete an endocannabinoid inhibitor of the plasma membrane's calcium channel. This allows the entry of calcium ions through the calcium channel to activate sperm that are then primed for fertilization. Science , this issue p. [555][1] [1]: /lookup/doi/10.1126/science.aad6887

Unconventional endocannabinoid signaling governs sperm activation via the sex hormone progesterone.

Steroids regulate cell proliferation, tissue development, and cell signaling via two pathways: a nuclear receptor mechanism and genome-independent signaling. Sperm activation, egg maturation, and steroid-induced anesthesia are executed via the latter pathway, the key components of which remain unknown. Here, we present characterization of the human sperm progesterone receptor that is conveyed by the orphan enzyme α/β hydrolase domain-containing protein 2 (ABHD2). We show that ABHD2 is highly expressed in spermatozoa, binds progesterone, and acts as a progesterone-dependent lipid hydrolase by depleting the endocannabinoid 2-arachidonoylglycerol (2AG) from plasma membrane. The 2AG inhibits the sperm calcium channel (CatSper), and its removal leads to calcium influx via CatSper and ensures sperm activation. This study reveals that progesterone-activated endocannabinoid depletion by ABHD2 is a general mechanism by which progesterone exerts its genome-independent action and primes sperm for fertilization.

Physiological roles for butyrylcholinesterase: A BChE-ghrelin axis

Butyrylcholinesterase (BChE) has long been regarded as an “orphan enzyme” with no specific physiological role other than to metabolize exogenous bioactive esters in the diet or in medicines. Human beings with genetic mutations that eliminate all BChE activity appear completely normal, and BChE-knockout mice have been described as “lacking a phenotype” except for faster weight gain on high-fat diets. However, our recent studies with viral gene transfer of BChE in mice reveal that BChE hydrolyzes the so-called “hunger hormone,” ghrelin, at a rate which strongly affects the circulating levels of this peptide hormone. This action has important consequences for weight gain and fat metabolism. Surprisingly, it also impacts emotional behaviors such as aggression. Overexpression of BChE leads to low ghrelin levels in the blood stream and reduces aggression and social stress in mice. Under certain circumstances these combined effects contribute to increased life-span in group-housed animals. These findings may generalize to humans, as recent clinical studies by multiple investigators indicate that, among patients with severe cardiovascular disease, longevity correlates with increasing levels of plasma BChE activity.

Identification of Enzyme Genes Using Chemical Structure Alignments of Substrate-Product Pairs.

Although there are several databases that contain data on many metabolites and reactions in biochemical pathways, there is still a big gap in the numbers between experimentally identified enzymes and metabolites. It is supposed that many catalytic enzyme genes are still unknown. Although there are previous studies that estimate the number of candidate enzyme genes, these studies required some additional information aside from the structures of metabolites such as gene expression and order in the genome. In this study, we developed a novel method to identify a candidate enzyme gene of a reaction using the chemical structures of the substrate-product pair (reactant pair). The proposed method is based on a search for similar reactant pairs in a reference database and offers ortholog groups that possibly mediate the given reaction. We applied the proposed method to two experimentally validated reactions. As a result, we confirmed that the histidine transaminase was correctly identified. Although our method could not directly identify the asparagine oxo-acid transaminase, we successfully found the paralog gene most similar to the correct enzyme gene. We also applied our method to infer candidate enzyme genes in the mesaconate pathway. The advantage of our method lies in the prediction of possible genes for orphan enzyme reactions where any associated gene sequences are not determined yet. We believe that this approach will facilitate experimental identification of genes for orphan enzymes.

Regioselective Oxygenation of Polyunsaturated Fatty Acids by the Thermostable P450 from Thermus thermophilus HB27

Background: CYP175A1 is a thermophilic P450 with high potential to invoke as an industrially viable biocatalyst though very little is known about the natural substrate of this enzyme. The crystal structure of CYP175A1 is similar to its mesophilic analogue CYP102A1, which is a fatty acid metabolizing enzyme (J Biol Chem 1990; 265: 4233-9). We have earlier shown that CYP175A1 catalyzes regioselective mono-oxygenation of different monounsaturated fatty acids (Biochemistry 2012; 51: 7880-90). The present work highlights screening of several important polyunsaturated fatty acids for their potentiality as substrate of this orphan P450 enzyme. Methods: Both molecular modeling/docking as well as spectrophotometric screening of the substrates along with detailed activity assay and mass spectrometric identification of the products have been carried out to determine the reactivity and selectivity of oxygenation of polyunsaturated fatty acids by CYP175A1. Results: Our investigations showed that polyunsaturated fatty acids (arachidonic acid, linoleic acid, α-linolenic acid and - linolenic acid) can be oxygenated by the enzymatic action of CYP175A1 although the enzyme did not show any detectable activity on the corresponding saturated analogues. Results further showed that unlike the monounsaturated fatty acids, the polyunsaturated fatty acids undergo mono- as well as di-oxygenation reactions. Analyses of products also suggested that the monooxygenation of these fatty acids is highly regioselective and is regulated by the number and position of the double bonds in the fatty acids. Conclusion: CYP175A1 may have important roles in lipid metabolism and thus can be used for regioselective oxygenation of different fatty acids. The present study also improves our current understanding on the nature of the enzyme pocket and of the possible natural substrate of this orphan enzyme. Keywords: CYP175A1, epoxidation, hydroxylation, oxygenation, P450, polyunsaturated fatty acids, regioselectivity, thermostability.

A new role for sphingosine: Up-regulation of Fam20C, the genuine casein kinase that phosphorylates secreted proteins

Fam20C is an atypical kinase implicated in bio-mineralization and phosphate homeostasis disorders, and has recently been shown to account for the activity of an orphan enzyme (“genuine casein kinase”, G-CK) previously characterized for its ability to phosphorylate casein and a plethora of secreted proteins at serine residues specified by the S-x-E/pS motif. Fam20C/G-CK activity is only appreciable in the presence of high Mn2+ concentration (>1mM), and is negligible if Mn2+ is replaced by physiological Mg2+ concentrations. Here we show that sphingosine (but not its biological precursor ceramide) not only stimulates several-fold Fam20C activity in the presence of Mn2+, but also confers a comparable activity to Fam20C assayed with Mg2+. Activation by sphingosine is evident using a variety of substrates, and is accounted for by both higher Vmax and decreased Km(ATP), as judged from kinetics run with the β(28–40) substrate peptide and a physiological substrate, BMP-15. Sphingosine also protects Fam20C from thermal inactivation. Consistent with the in vitro results, by treating Fam20C expressing HEK293T cells with myriocin, a potent inhibitor of the sphingosine biosynthetic pathway, the activity of Fam20C released into the conditioned medium is substantially decreased corroborating the concept that sphingosine (or related metabolite(s)) is a co-factor required by Fam20C to optimally display its biological functions. None of the small molecule kinase inhibitors tested so far were able to inhibit Fam20C. Interestingly however fingolimod, an immunosuppressive drug structurally related to sphingosine, used for the treatment of multiple sclerosis, is a powerful activator of Fam20C, both wild type and its pathogenic, loss of function, T268M mutant. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.

Orphan enzyme cuts down on sugar.

The reclusive enzymes that catalyze proteolysis inside the cell membrane are among the most widespread in nature, yet most remain ‘orphans’ whose cellular functions are poorly understood. Now, Voss et al identify Golgi‐resident glycosyltransferases and glycosidases as substrates for the presenilin‐like protease SPPL3. Shedding of these glycan‐modifying enzymes from the membrane down‐regulates global protein N‐glycosylation.

Crystal structures and functional studies clarify substrate selectivity and catalytic residues for the unique orphan enzyme N-acetyl-D-mannosamine dehydrogenase.

NAMDH (N-acetyl-D-mannosamine dehydrogenase), from the soil bacteroidete Flavobacterium sp. 141-8, catalyses a rare NAD+-dependent oxidation of ManNAc (N-acetyl-D-mannosamine) into N-acetylmannosamino-lactone, which spontaneously hydrolyses into N-acetylmannosaminic acid. NAMDH belongs to the SDR (short-chain dehydrogenase/reductase) superfamily and is the only NAMDH characterized to date. Thorough functional, stability, site-directed mutagenesis and crystallographic studies have been carried out to understand better the structural and biochemical aspects of this unique enzyme. NAMDH exhibited a remarkable alkaline pH optimum (pH9.4) with a high thermal stability in glycine buffer (Tm=64°C) and a strict selectivity towards ManNAc and NAD+. Crystal structures of ligand-free and ManNAc- and NAD+-bound enzyme forms revealed a compact homotetramer having point 222 symmetry, formed by subunits presenting the characteristic SDR α3β7α3 sandwich fold. A highly developed C-terminal tail used as a latch connecting nearby subunits stabilizes the tetramer. A dense network of polar interactions with the substrate including the encasement of its acetamido group in a specific binding pocket and the hydrogen binding of the sugar 4OH atom ensure specificity for ManNAc. The NAMDH-substrate complexes and site-directed mutagenesis studies identify the catalytic tetrad and provide useful traits for identifying new NAMDH sequences.

Expression of CYP2W1 in the adrenal gland: relationship with hormone secretion and clinical outcome

Adrenocortical tumors consist of frequent adenomas (ACA) and rare highly malignant carcinomas (ACC). The orphan enzyme cytochrome P450 2W1 (CYP2W1) is selectively expressed in some cancers and represent a promising new drug target in cancer therapy.

Inhibition of SULT4A1 Expression Induces Up-Regulation of Phototransduction Gene Expression in 72-Hour Postfertilization Zebrafish Larvae

Sulfotransferase (SULT) 4A1 is an orphan enzyme that shares distinct structure and sequence similarities with other cytosolic SULTs. SULT4A1 is primarily expressed in neuronal tissue and is also the most conserved SULT, having been identified in every vertebrate investigated to date. Certain haplotypes of the SULT4A1 gene are correlated with higher baseline psychopathology in schizophrenic patients, but no substrate or function for SULT4A1 has yet been identified despite its high level of sequence conservation. In this study, deep RNA sequencing was used to search for alterations in gene expression in 72-hour postfertilization zebrafish larvae following transient SULT4A1 knockdown (KD) utilizing splice blocking morpholino oligonucleotides. This study demonstrates that transient inhibition of SULT4A1 expression in developing zebrafish larvae results in the up-regulation of several genes involved in phototransduction. SULT4A1 KD was verified by immunoblot analysis and quantitative real-time polymerase chain reaction (qPCR). Gene regulation changes identified by deep RNA sequencing were validated by qPCR. This study is the first identification of a cellular process whose regulation appears to be associated with SULT4A1 expression.

Profiling the orphan enzymes

The emergence of Next Generation Sequencing generates an incredible amount of sequence and great potential for new enzyme discovery. Despite this huge amount of data and the profusion of bioinformatic methods for function prediction, a large part of known enzyme activities is still lacking an associated protein sequence. These particular activities are called “orphan enzymes”. The present review proposes an update of previous surveys on orphan enzymes by mining the current content of public databases. While the percentage of orphan enzyme activities has decreased from 38% to 22% in ten years, there are still more than 1,000 orphans among the 5,000 entries of the Enzyme Commission (EC) classification. Taking into account all the reactions present in metabolic databases, this proportion dramatically increases to reach nearly 50% of orphans and many of them are not associated to a known pathway. We extended our survey to “local orphan enzymes” that are activities which have no representative sequence in a given clade, but have at least one in organisms belonging to other clades. We observe an important bias in Archaea and find that in general more than 30% of the EC activities have incomplete sequence information in at least one superkingdom. To estimate if candidate proteins for local orphans could be retrieved by homology search, we applied a simple strategy based on the PRIAM software and noticed that candidates may be proposed for an important fraction of local orphan enzymes. Finally, by studying relation between protein domains and catalyzed activities, it appears that newly discovered enzymes are mostly associated with already known enzyme domains. Thus, the exploration of the promiscuity and the multifunctional aspect of known enzyme families may solve part of the orphan enzyme issue. We conclude this review with a presentation of recent initiatives in finding proteins for orphan enzymes and in extending the enzyme world by the discovery of new activities.ReviewersThis article was reviewed by Michael Galperin, Daniel Haft and Daniel Kahn.