Characterization of gut microbiome composition in Iranian patients with nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.

Gut microbiota dysbiosis is intimately associated with development of non-alcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Nevertheless, the gut microbial community during the course of NAFLD and NASH is yet to be comprehensively profiled. This study evaluated alterations in fecal microbiota composition in Iranian patients with NAFLD and NASH compared with healthy individuals. This cross-sectional study enrolled 15 NAFLD, 15 NASH patients, and 20 healthy controls, and their clinical parameters were examined. The taxonomic composition of the fecal microbiota was determined by sequencing the V3-V4 region of 16S rRNA genes of stool samples. Compared to the healthy controls, NAFLD and NASH patients presented reduced bacterial diversity and richness. We noticed a reduction in the relative abundance of Bacteroidota and a promotion in the relative abundance of Proteobacteria in NAFLD and NASH patients. L-histidine degradation I pathway, pyridoxal 5'-phosphate biosynthesis I pathway, and superpathway of pyridoxal 5'-phosphate biosynthesis and salvage were more abundant in NAFLD patients than in healthy individuals. This study examined fecal microbiota dysbiosis in NAFLD and NASH patients and presented consistent results to European countries. These condition- and ethnicity-specific data could provide different diagnostic signatures and therapeutic targets.

Functional and structural properties of pyridoxal reductase (PdxI) from Escherichia coli: a pivotal enzyme in the vitamin B6 salvage pathway.

Pyridoxine 4-dehydrogenase (PdxI), a NADPH-dependent pyridoxal reductase, is one of the key players in the Escherichia coli pyridoxal 5'-phosphate (PLP) salvage pathway. This enzyme, which catalyses the reduction of pyridoxal into pyridoxine, causes pyridoxal to be converted into PLP via the formation of pyridoxine and pyridoxine phosphate. The structural and functional properties of PdxI were hitherto unknown, preventing a rational explanation of how and why this longer, detoured pathway occurs, given that, in E. coli, two pyridoxal kinases (PdxK and PdxY) exist that could convert pyridoxal directly into PLP. Here, we report a detailed characterisation of E. coli PdxI that explains this behaviour. The enzyme efficiently catalyses the reversible transformation of pyridoxal into pyridoxine, although the reduction direction is thermodynamically strongly favoured, following a compulsory-order ternary-complex mechanism. In vitro, the enzyme is also able to catalyse PLP reduction and use NADH as an electron donor, although with lower efficiency. As with all members of the aldo-keto reductase (AKR) superfamily, the enzyme has a TIM barrel fold; however, it shows some specific features, the most important of which is the presence of an Arg residue that replaces the catalytic tetrad His residue that is present in all AKRs and appears to be involved in substrate specificity. The above results, in conjunction with kinetic and static measurements of vitamins B6 in cell extracts of E. coli wild-type and knockout strains, shed light on the role of PdxI and both kinases in determining the pathway followed by pyridoxal in its conversion to PLP, which has a precise regulatory function.

Chemical Modifications of Pyridoxine for Biological Applications: An Overview.

Pyridoxine and its derivatives, pyridoxamine, and pyridoxal have been recognized for more than 70 years and are known for regulating cellular biology and metabolism. During the past few decades, the anti-oxidant and anti-inflammatory properties of pyridoxine and its vitamers were explored. However, an interesting turnabout was observed in pyridoxine chemical modification in the last two decades. The various important pathophysiological aspects of pyridoxine and its derivatives on several cellular systems have been discovered by researchers. Recent findings have shown that many diseases, like cancer, diabetes, hypertension, tuberculosis, epilepsy, and neurodegenerative diseases are linked to the alteration of pyridoxine. Herein, our main focus is to review the importance of pyridoxine and its derivatives obtained by various chemical modifications, in various disease areas and to recognize important directions for future research.

Agmatine production by Escherichia coli cells expressing SpeA on the extracellular surface.

A plasmid was constructed to express the CapA-SpeA fusion protein from the capA gene, which encodes one of the subunits of capsular poly-γ-glutamate synthetase of Bacillus subtilis subsp. natto, and the speA gene, encoding biosynthetic arginine decarboxylase (EC 4.1.1.19) of Escherichia coli, under the control of the T5 promoter. The expression of SpeA on the extracellular surface of cells was confirmed by confocal microscopy with the anti-SpeAE. coli antibody and anti-rabbit IgG L & H conjugated with Alexa Fluor 488. The constructed strain SH2290 produced 200 mM agmatine from 200 mM arginine, 20 mM MgSO4, 0.9 % NaCl, and 0.02 mg/mL pyridoxal 5'-phosphate (initial pH 5.3) by adjusting pH of the reaction mixture to 6.8 with HCl after each sampling during the reaction. The addition of pyridoxal 5'-phosphate to the reaction mixture was required for the maximum agmatine production. The present results demonstrate that the expression of enzymes on the extracellular surface of cells is a very powerful method for enzymatic conversion.

Microbiota-host crosstalk in the newborn and adult rumen at single-cell resolution.

BACKGROUND: The rumen is the hallmark organ of ruminants, playing a vital role in their nutrition and providing products for humans. In newborn suckling ruminants milk bypasses the rumen, while in adults this first chamber of the forestomach has developed to become the principal site of microbial fermentation of plant fibers. With the advent of single-cell transcriptomics, it is now possible to study the underlying cell composition of rumen tissues and investigate how this relates the development of mutualistic symbiosis between the rumen and its epithelium-attached microbes. RESULTS: We constructed a comprehensive cell landscape of the rumen epithelium, based on single-cell RNA sequencing of 49,689 high-quality single cells from newborn and adult rumen tissues. Our single-cell analysis identified six immune cell subtypes and seventeen non-immune cell subtypes of the rumen. On performing cross-species analysis of orthologous genes expressed in epithelial cells of cattle rumen and the human stomach and skin, we observed that the species difference overrides any cross-species cell-type similarity. Comparing adult with newborn cattle samples, we found fewer epithelial cell subtypes and more abundant immune cells, dominated by T helper type 17 cells in the rumen tissue of adult cattle. In newborns, there were more fibroblasts and myofibroblasts, an IGFBP3+ epithelial cell subtype not seen in adults, while dendritic cells were the most prevalent immune cell subtype. Metabolism-related functions and the oxidation-reduction process were significantly upregulated in adult rumen epithelial cells. Using 16S rDNA sequencing, fluorescence in situ hybridization, and absolute quantitative real-time PCR, we found that epithelial Desulfovibrio was significantly enriched in the adult cattle. Integrating the microbiome and metabolome analysis of rumen tissues revealed a high co-occurrence probability of Desulfovibrio with pyridoxal in the adult cattle compared with newborn ones while the scRNA-seq data indicated a stronger ability of pyroxidal binding in the adult rumen epithelial cell subtypes. These findings indicate that Desulfovibrio and pyridoxal likely play important roles in maintaining redox balance in the adult rumen. CONCLUSIONS: Our integrated multi-omics analysis provides novel insights into rumen development and function and may facilitate the future precision improvement of rumen function and milk/meat production in cattle.

Role of the conserved pyridoxal 5'-phosphate-binding protein YggS/PLPBP in vitamin B6 and amino acid homeostasis.

The YggS/PLPBP protein (also called COG0325 or PLPHP) is a conserved pyridoxal 5'-phosphate (PLP)-binding protein present in all 3 domains of life. Recent studies have demonstrated that disruption or mutation of this protein has multifaceted effects in various organisms, including vitamin B6-dependent epilepsy in humans. In Escherichia coli, disruption of this protein-encoded by yggS-perturbs Thr-Ile/Val metabolism, one-carbon metabolism, coenzyme A synthesis, and vitamin B6 homeostasis. This protein is critical for maintaining low levels of pyridoxine 5'-phosphate (PNP) in various organisms. In the yggS-deficient E. coli strain, inhibition of PLP-dependent enzymes, such as the glycine cleavage system by PNP, is the root cause of metabolic perturbation. Our data suggest that the YggS/PLPBP protein may be involved in the balancing of B6 vitamers by mediating efficient turnover of protein-bound B6 vitamers. This paper reviews recent findings on the function of the YggS/PLPBP protein.

Pyridoxal and α-Ketoglutarate Independently Improve Function of Saccharomyces cerevisiae Thi5 in the Metabolic Network of Salmonella enterica.

Microbial metabolism is often considered modular, but metabolic engineering studies have shown that transferring pathways, or modules, between organisms is not always straightforward. The Thi5-dependent pathway(s) for synthesis of the pyrimidine moiety of thiamine from Saccharomyces cerevisiae and Legionella pneumophila functioned differently when incorporated into the metabolic network of Salmonella enterica. Function of Thi5 from Saccharomyces cerevisiae (ScThi5) required modification of the underlying metabolic network, while LpThi5 functioned with the native network. Here we probe the metabolic requirements for heterologous function of ScThi5 and report strong genetic and physiological evidence for a connection between alpha-ketoglutarate (αKG) levels and ScThi5 function. The connection was built with two classes of genetic suppressors linked to metabolic flux or metabolite pool changes. Further, direct modulation of nitrogen assimilation through nutritional or genetic modification implicated αKG levels in Thi5 function. Exogenous pyridoxal similarly improved ScThi5 function in S. enterica. Finally, directly increasing αKG and PLP with supplementation improved function of both ScThi5 and relevant variants of Thi5 from Legionella pneumophila (LpThi5). The data herein suggest structural differences between ScThi5 and LpThi5 impact their level of function in vivo and implicate αKG in supporting function of the Thi5 pathway when placed in the heterologous metabolic network of S. enterica. IMPORTANCE Thiamine biosynthesis is a model metabolic node that has been used to extend our understanding of metabolic network structure and individual enzyme function. The requirements for in vivo function of the Thi5-dependent pathway found in Legionella and yeast are poorly characterized. Here we suggest that αKG modulates function of the Thi5 pathway in S. enterica and provide evidence that structural variation between ScThi5 and LpThi5 contributes to their functional differences in a Salmonella enterica host.

Pyridoxal Isonicotinoyl Hydrazone Improves Neurological Recovery by Attenuating Ferroptosis and Inflammation in Cerebral Hemorrhagic Mice.

Ferroptosis and inflammation induced by cerebral hemorrhage result in an excessive inflammatory response and irreversible neuronal injury. Alleviating ferroptosis might be an effective way to prevent neuroinflammatory injury and promote neural functional recovery. Pyridoxal isonicotinoyl hydrazine (PIH), a lipophilic iron-chelating agent, has been reported to reduce excess iron-induced cytotoxicity. However, whether PIH could ameliorate the effects of hemorrhagic stroke is not completely understood. In the present study, the preventive effects of PIH in an intracerebral hemorrhage (ICH) mouse model were investigated. Neurological score, rotarod test, and immunofluorescence around the hematoma were assessed to evaluate the effects of PIH on hemorrhagic injury. The involvement of ferroptosis and inflammation was also examined in vitro to explore the underlying mechanism. Results showed that administration of PIH prevented neuronal cell death and reduced lipid peroxidation in Erastin-treated PC-12 cells. In vivo, mice treated with PIH after ICH attenuated neurological deficit scores. Additionally, we found PIH reduced ROS production, iron accumulation, and lipid peroxidation around the hematoma peripheral tissue. Meanwhile, ICH mice treated with PIH showed an upregulation of the key ferroptosis enzyme, glutathione peroxidase 4, and downregulation of cyclooxygenase-2. Moreover, PIH administration inhibited proinflammatory polarization and reduced interleukin-1 beta and tumor necrosis factor alpha in ICH mice. Collectively, these results demonstrated that PIH protects mice against hemorrhage stroke, which was associated with mitigation of inflammation and ferroptosis.

Pyridoxal isonicotinoyl hydrazone inhibition of FXR is involved in the pathogenesis of isoniazid-induced liver injury.

Isoniazid (INH)-induced liver injury may be associated with inhibition of the liver farnesoid X receptor (FXR). However, the relationship between FXR and INH-induced liver injury remained unclear. The present study was performed to clarify the role of inhibition of FXR in the pathogenesis of INH-induced liver injury and to further identify potential inhibitors of FXR from INH and its metabolites. HepaRG cells were treated with INH (10 mM) plus mixed bile acids (BA) and rats were treated with INH (60-600 mg/kg p.o.) or INH plus obeticholic acid (OCA, 10 mg/kg), a potent FXR agonist, for seven days. INH can cause BA-dependent toxicity and apoptosis with elevated intracellular bile acids in vitro; indeed, in these studies, liver bile acids and mRNA levels for Cyp7a1, an FXR target gene were increased, while mRNA levels for FXR and Shp were significantly decreased, and these changes could be prevented by co-treatment with the FXR agonist OCA. In silico molecular docking studies showed that INH, acetyl isoniazid, isonicotinic acid and PIH may be potential FXR inhibitors, and a TR-FRET FXR-coactivator assay confirmed that PIH is a strong antagonist of FXR (IC50 = 52 nM). To further determine if PIH also inhibits FXR activity in vivo, rats were treated with PIH directly (5 mg/kg). Liver total bile acids were significantly increased while FXR expression was not changed, but Shp mRNA levels were significantly decreased and Cyp7a1 mRNA was significantly increased, consistent with PIH acting as an FXR antagonist. In summary, PIH inhibition of liver FXR function leading to bile acid accumulation in hepatocytes may be an early pathogenesis event in INH-induced liver injury.

Skin advanced glycation end products as biomarkers of photosensitivity in schizophrenia.

OBJECTIVES: Photosensitivity to ultraviolet A (UVA) radiation from sunlight is an important side effect of treatment with antipsychotic agents. However, the pathophysiology of drug-induced photosensitivity remains unclear. Recent studies demonstrated the accumulation of advanced glycation end products (AGEs), annotated as carbonyl stress, to be associated with the pathophysiology of schizophrenia. In this study, we investigated the relationship among skin AGE levels, minimal response dose (MRD) with UVA for photosensitivity, and the daily dose of antipsychotic agents in patients with schizophrenia and healthy controls. METHODS: We enrolled 14 patients with schizophrenia and 14 healthy controls. Measurement of skin AGE levels was conducted with AGE scanner, a fluorometric method for assaying skin AGE levels. Measurement of MRD was conducted with UV irradiation device. RESULTS: Skin AGE levels and MRD at 24, 48, and 72 hr in patients with schizophrenia showed a higher tendency for photosensitivity than in the controls, but the difference was statistically insignificant. Multiple linear regression analysis using skin AGE levels failed to show any influence of independent variables. MRD did not affect skin AGE levels. CONCLUSIONS: Photosensitivity to UVA in patients with schizophrenia receiving treatment with antipsychotic agents might not be affected by skin AGE levels.

Bioconversion of pyridoxine to pyridoxamine through pyridoxal using a Rhodococcus expression system.

Pyridoxamine, which is a form of vitamin B6, is a promising candidate for a prophylactic and/or remedy for diabetic complications. Pyridoxamine is chemically synthesized by an oxidative method in manufacturing. However, pyridoxamine production by bioconversion, which is generally preferable for environmental and energetic aspects, has been little investigated. Therefore, I aimed to produce pyridoxamine from pyridoxine, which is a readily and economically available starting material, by bioconversion using a Rhodococcus expression system. I found in the bioconversion of pyridoxine to pyridoxal, approximately 450 mM pyridoxal was produced from 500 mM pyridoxine using recombinant Rhodococcus erythropolis expressing the pyridoxine 4-oxidase gene derived from Mesorhizobium loti. Next, in the bioconversion of pyridoxal to pyridoxamine using recombinant R. erythropolis expressing the pyridoxamine-pyruvate aminotransferase gene derived from M. loti, the bioconversion rate was approximately 80% under the same conditions as pyridoxal production. Finally, in the bioconversion of pyridoxine to pyridoxamine through pyridoxal using recombinant R. erythropolis coexpressing the genes for pyridoxine 4-oxidase and pyridoxamine-pyruvate aminotransferase, the bioconversion rate was approximately 75%. Based on these findings, pyridoxamine production by bioconversion using a Rhodococcus expression system may be of interest for future industrial applications.

Identification of YbhA as the pyridoxal 5'-phosphate (PLP) phosphatase in Escherichia coli: Importance of PLP homeostasis on the bacterial growth.

The gene ybhA of Escherichia coli encodes a phosphatase that has an in vitro specificity to dephosphorylate pyridoxal 5'-phosphate (PLP or vitamin B6), a co-factor for aminotransferases and other enzymes. In this study, we found that excess pyridoxal (PL) in a minimal medium resulted in excess PLP in vivo and growth inhibition, which was alleviated by YbhA overproduction. Conversely, the YbhA overproduction resulted in PLP shortage in vivo and the correlated reduction in growth rate, which was significantly negated by PL in the medium. In addition, the overproduction of a PL kinase, PdxK or PdxY, was inhibitory to cell growth only in the absence of the functional ybhA gene, and the growth defects were alleviated by casamino acids in the medium, which suggested that both the shortage of, and excess, PLP resulted in the disturbance of amino acid metabolism and cell growth, as revealed by a metabolome analysis.

Role of catechins on ET-1-induced stimulation of PLD and NADPH oxidase activities in pulmonary smooth muscle cells: determination of the probable mechanism by molecular docking studies.

The treatment of human pulmonary artery smooth muscle cells with ET-1 stimulates the activity of PLD and NADPH oxidase, but this stimulation is inhibited by pretreatment with bosentan (ET-1 receptor antagonist), FIPI (PLD inhibitor), apocynin (NADPH oxidase inhibitor), and EGCG and ECG (catechins having a galloyl group), but not EGC and EC (catechins devoid of a galloyl group). Herein, using molecular docking analyses based on our biochemical studies, we determined the probable mechanism by which the catechins containing a galloyl group inhibit the stimulation of PLD activity induced by ET-1. The ET-1-induced stimulation of PLD activity was inhibited by SecinH3 (inhibitor of cytohesin). Arf6 and cytohesin-1 are associated in the cell membrane, which is not inhibited by the catechins during ET-1 treatment of the cells. However, EGCG and ECG inhibited the binding of GTPγS with Arf6, even in the presence of cytohesin-1. The molecular docking analyses revealed that the catechins containing a galloyl group (EGCG and ECG) with cytohesin-1-Arf6GDP, but not the catechins without a galloyl group (EGC and EC), prevent GDP-GTP exchange in Arf6, which seems to be an important mechanism for inhibiting the activation of PLD induced by ET-1, and subsequently increases the activity of NADPH oxidase.

Biotransformation of pyridoxal 5'-phosphate from pyridoxal by pyridoxal kinase (pdxY) to support cadaverine production in Escherichia coli.

Cadaverine, a five-carbon diamine (1,5-diaminopentane), can be made by fermentation or direct bioconversion and plays an important role as a building block of polyamides. Lysine decarboxylase (CadA) transforms L-lysine to cadaverine and pyridoxal 5'-phosphate (PLP) can increases conversion rate and yield as a cofactor. Biotransformation of cadaverine using whole Escherichia coli cells that overexpress the lysine decarboxylase has many merits, such as the rapid conversion of l-lysine to cadaverine, possible application of high concentration reactions up to the molar level, production of less byproduct and potential reuse of the enzyme by immobilization. However, the supply of PLP, which is a cofactor of lysine decarboxylase, is the major bottleneck in this system. Therefore, we initiated our study on PLP precursors and PLP-related enzymes and discovered that pyridoxal (PL) can be a viable alternative to supply PLP. Among various PLP systems examined, pyridoxal kinase (PdxY) showed the highest conversion of PL to PLP, resulting in more than 60% conversion of l-lysine to cadaverine with lysine decarboxylase. When the reaction with 0.4M l-lysine, 0.2mM PL and more whole cells was performed, it resulted in an 80% conversion yield. Furthermore, when barium-alginate immobilization was applied, it showed a 90% conversion yield in 1h with PL, suggesting that it is compatible with developed whole-cell systems without a direct supply of exogenous PLP.

Pyridoxal oxime derivative potency to reactivate cholinesterases inhibited by organophosphorus compounds.

Organophosphorus (OP) nerve agents (sarin, tabun VX and soman) inhibit the enzyme acetylcholinesterase (AChE, EC 3.1.1.7) by binding to its active site while preventing neurotransmission in the cholinergic synapses. The protection and treatment of this kind of poisoning are still a challenge as we are yet to discover an antidote that would be effective in all cases of poisoning. To aid the search for more efficient antidotes, we evaluated the ability of nine pyridoxal oxime derivatives, prepared by a novel synthetic pathway, to reactivate recombinant human AChE and the related purified human plasma butyrylcholinesterase (BChE, EC 3.1.1.8) inhibited by VX, tabun and paraoxon. Oximes are derivatives of vitamin B6 bearing a phenacyl moiety attached to the quaternary nitrogen atom and having various substituents on the phenyl ring. As the results have shown, the tested oximes were in general more efficient in the reactivation of OP-inhibited BChE than AChE. The highest observed rate was in the case of VX-inhibited BChE reactivation, where kobs was 0.0087min-1 and the reactivation maximum of 90% was achieved within 5h. The cholinesterases displayed a binding affinity for these derivatives in a µmolar range no matter the substituent on their rings which was in accordance with the molecular modelling results showing a similar binding pattern for all oximes within the active site of both AChE and BChE. Such a positioning reveals also that hydroxy and a metoxy substituents at the vicinity of the oxime moiety present a possible steric hindrance explaining the reactivation results.

Iron(III) complexes of a pyridoxal Schiff base for enhanced cellular uptake with selectivity and remarkable photocytotoxicity.

Iron(III) complexes of pyridoxal (vitamin B6, VB6) or salicylaldehyde Schiff bases and modified dipicolylamines, namely, [Fe(B)(L)](NO3) (1-5), where B is phenyl-N,N-bis((pyridin-2-yl)methyl)methanamine (phbpa in 1), (anthracen-9-yl)-N,N-bis((pyridin-2-yl)methyl)methanamine (anbpa in 2, 4) and (pyren-1-yl)-N,N-bis((pyridin-2-yl)methyl)methanamine (pybpa in 3, 5) (H2L(1) is 3-hydroxy-5-(hydroxymethyl)-4-(((2-hydroxyphenyl)imino)methyl)-2-methylpyridine (1-3) and H2L(2) is 2-[(2-hydroxyphenyl-imino)methyl]phenol), were prepared and their uptake in cancer cells and photocytotoxicity were studied. Complexes 4 and 5, having a non-pyridoxal Schiff base, were prepared to probe the role of the pyridoxal group in tumor targeting and cellular uptake. The PF6 salt (1a) of complex 1 is structurally characterized. The complexes have a distorted six-coordinate FeN4O2 core where the metal is in the +3 oxidation state with five unpaired electrons. The complexes display a ligand to metal charge transfer band near 520 and 420 nm from phenolate to the iron(III) center. The photophysical properties of the complexes are explained from the time dependent density functional theory calculations. The redox active complexes show a quasi-reversible Fe(III)/Fe(II) response near -0.3 V vs saturated calomel electrode. Complexes 2 and 3 exhibit remarkable photocytotoxicity in various cancer cells with IC50 values ranging from 0.4 to 5 µM with 10-fold lower dark toxicity. The cell death proceeded by the apoptotic pathway due to generation of reactive oxygen species upon light exposure. The nonvitamin complexes 4 and 5 display 3-fold lower photocytotoxicity compared to their VB6 analogues, possibly due to preferential and faster uptake of the vitamin complexes in the cancer cells. Complexes 2 and 3 show significant uptake in the endoplasmic reticulum, while complexes 4 and 5 are distributed throughout the cells without any specific localization pattern.

Enzymatic conversion from pyridoxal to pyridoxine caused by microorganisms within tobacco phyllosphere.

Vitamin B6 (VB6) comprises six interconvertible pyridine compounds (vitamers), among which pyridoxal 5'-phosphate (PLP) is a coenzyme involved in a high diversity of biochemical reactions. In plants, PLP is de novo synthesized, and pyridoxine (PN) is usually maintained as the predominant B6 vitamer. Although the conversion from pyridoxal (PL) to PN catalyzed by PL reductase in plants has been confirmed, the enzyme itself remains largely unknown. We previously found pre-incubation at 35 °C dramatically enhanced PL reductase activity in tobacco leaf homogenate. In this study, we demonstrated that the increase in the reductase activity was a consequence of phyllosphere microbial proliferation. VB6 was detected from tobacco phyllosphere, and PL level was the highest among three non-phosphorylated B6 vitamers. When the sterile tobacco rich in PL were kept in an open, warm and humid environment to promote microorganism proliferation, a significant change from PL to PN was observed. Our results suggest that there may be a plant-microbe interaction in the conversion from PL to PN within tobacco phyllosphere.

An inkjet-printed bioactive paper sensor that reports ATP through odour generation.

We describe an inkjet printed paper-based sensor that reports ATP by the enzyme catalysed hydrolysis of S-methyl-L-cysteine generating an odour (methyl mercaptan) that is easily detectable by the human nose.

Emergence of pyridoxal phosphorylation through a promiscuous ancestor during the evolution of hydroxymethyl pyrimidine kinases.

In the family of ATP-dependent vitamin kinases, several bifunctional enzymes that phosphorylate hydroxymethyl pyrimidine (HMP) and pyridoxal (PL) have been described besides enzymes specific towards HMP. To determine how bifunctionality emerged, we reconstructed the sequence of three ancestors of HMP kinases, experimentally resurrected, and assayed the enzymatic activity of their last common ancestor. The latter has ∼ 8-fold higher specificity for HMP due to a glutamine residue (Gln44) that is a key determinant of the specificity towards HMP, although it is capable of phosphorylating both substrates. These results show how a specific enzyme with catalytic promiscuity gave rise to current bifunctional enzymes.