Cystathionine Beta-lyase Research Articles

Engineering of methionine-auxotroph Escherichia coli via parallel evolution of two enzymes from Corynebacterium glutamicum's direct-sulfurylation pathway enables its recovery in minimal medium

Methionine biosynthesis relies on the sequential catalysis of multiple enzymes. Escherichia coli, the main bacteria used in research and industry for protein production and engineering, utilizes the three-step trans-sulfurylation pathway catalyzed by L-homoserine O-succinyl transferase, cystathionine gamma synthase and cystathionine beta lyase to convert L-homoserine to L-homocysteine. However, most bacteria employ the two-step direct-sulfurylation pathway involving L-homoserine O-acetyltransferases and O-acetyl homoserine sulfhydrylase. We previously showed that a methionine-auxotroph Escherichiacoli strain (MG1655) with deletion of metA, encoding for L-homoserine O-succinyl transferase, and metB, encoding for cystathionine gamma synthase, could be complemented by introducing the genes metX, encoding for L-homoserine O-acetyltransferases and metY, encoding for O-acetyl homoserine sulfhydrylase, from various sources, thus altering the Escherichia coli methionine biosynthesis metabolic pathway to direct-sulfurylation. However, introducing metX and metY from Corynebacterium glutamicum failed to complement methionine auxotrophy. Herein, we generated a randomized genetic library based on the metX and metY of Corynebacterium glutamicum and transformed it into a methionine-auxotrophic Escherichia coli strain lacking the metA and metB genes. Through multiple enrichment cycles, we successfully isolated active clones capable of growing in M9 minimal media. The dominant metX mutations in the evolved methionine-autotrophs Escherichia coli were L315P and H46R. Interestingly, we found that a metY gene encoding only the N-terminus 106 out of 438 amino acids of the wild-type MetY enzyme is functional and supports the growth of the methionine auxotroph. Recloning the new genes into the original plasmid and transforming them to methionine auxotroph Escherichia coli validated their functionality. These results show that directed enzyme-evolution enables fast and simultaneous engineering of new active variants within the Escherichia coli methionine direct-sulfurylation pathway, leading to efficient complementation.

D-amino acid auxotrophic Escherichia coli strain for invivo functional cloning of novel d-amino acid synthetic enzyme.

Various d-amino acids have been found in a wide range of organisms, including mammals. Although the physiological functions of various d-amino acids have been reported or suggested, the molecular basis of these biological functions has been elucidated in only a few cases. The identification of a d-amino acid biosynthetic enzyme is a critical step in understanding the mechanism of the physiological functions of d-amino acids. While invivo functional screening can be a powerful tool for identifying novel metabolic enzymes, none of the existing organisms exhibit growth dependent on d-amino acid other than d-Ala and d-Glu. Here, we report the first organism that exhibits non-canonical d-amino acid auxotrophy. We found that an Escherichia coli strain lacking the major d-Ala and d-Glu biosynthetic enzymes, alr, dadX, and murI, and expressing the mutated d-amino acid transaminase (DAAT) gene from Bacillus sp. YM-1 (MB3000/mdaat+ ) grew well when supplemented with certain d-amino acid. A multicopy suppression study with plasmids encoding one of the 51 PLP-dependent enzymes of E.coli showed that MB3000/mdaat+ could detect weak and moonlighting racemase activity, such from cystathionine β-lyase (MetC) and a negative regulator of MalT activity/cystathionine β-lyase (MalY)-these exhibit only a few tenths to a few thousandths of the racemization activity of canonical amino acid racemases. We believe that this unique platform will contribute to further research in this field by identifying novel d-amino acid-metabolizing enzymes.

Disruption of the metC Gene Affects Methionine Biosynthesis in Pectobacterium carotovorum subsp. carotovorum Pcc21 and Reduces Soft-Rot Disease.

Plant pathogenic Pectobacterium species cause severe soft rot/blackleg diseases in many economically important crops worldwide. Pectobacterium utilizes plant cell wall degrading enzymes (PCWDEs) as the main virulence determinants for its pathogenicity. In this study, we screened a random mutant, M29 is a transposon insertion mutation in the metC gene encoding cystathionine β-lyase that catalyzes cystathionine to homocysteine at the penultimate step in methionine biosynthesis. M29 became a methionine auxotroph and resulted in growth defects in methionine-limited conditions. Impaired growth was restored with exogenous methionine or homocysteine rather than cystathionine. The mutant exhibited reduced soft rot symptoms in Chinese cabbages and potato tubers, maintaining activities of PCWDEs and swimming motility. The mutant was unable to proliferate in both Chinese cabbages and potato tubers. The reduced virulence was partially restored by a complemented strain or 100 µM of methionine, whereas it was fully restored by the extremely high concentration (1 mM). Our transcriptomic analysis showed that genes involved in methionine biosynthesis or transporter were downregulated in the mutant. Our results demonstrate that MetC is important for methionine biosynthesis and transporter and influences its virulence through Pcc21 multiplication in plant hosts.

Plant homocysteine, a methionine precursor and plant's hallmark of metabolic disorders.

Homocysteine (Hcy) is a sulfur-containing non-proteinogenic amino acid, which arises from redox-sensitive methionine metabolism. In plants, Hcy synthesis involves both cystathionine β-lyase and S-adenosylhomocysteine hydrolase activities. Thus, Hcy itself is crucial for de novo methionine synthesis and S-adenosylmethionine recycling, influencing the formation of ethylene, polyamines, and nicotianamine. Research on mammalian cells has shown biotoxicity of this amino acid, as Hcy accumulation triggers oxidative stress and the associated lipid peroxidation process. In addition, the presence of highly reactive groups induces Hcy and Hcy derivatives to modify proteins by changing their structure and function. Currently, Hcy is recognized as a critical, independent hallmark of many degenerative metabolic diseases. Research results indicate that an enhanced Hcy level is also toxic to yeast and bacteria cells. In contrast, in the case of plants the metabolic status of Hcy remains poorly examined and understood. However, the presence of the toxic Hcy metabolites and Hcy over-accumulation during the development of an infectious disease seem to suggest harmful effects of this amino acid also in plant cells. The review highlights potential implications of Hcy metabolism in plant physiological disorders caused by environmental stresses. Moreover, recent research advances emphasize that recognizing the Hcy mode of action in various plant systems facilitates verification of the potential status of Hcy metabolites as bioindicators of metabolism disorders and thus may constitute an element of broadly understood biomonitoring.

Characterization of human cystathionine γ-lyase enzyme activities toward d-amino acids.

Various d-amino acids play important physiological roles in mammals, but the pathways of their production remain unknown except for d-serine, which is generated by serine racemase. Previously, we found that Escherichia coli cystathionine β-lyase possesses amino acid racemase activity in addition to β-lyase activity. In the present work, we evaluated the enzymatic activities of human cystathionine γ-lyase, which shares a relatively high amino acid sequence identity with cystathionine β-lyase. The enzyme did not show racemase activity toward various amino acids including alanine and lyase and dehydratase activities were highest toward l-cystathionine and l-homoserine, respectively. The enzyme also showed weak activity toward l-cysteine and l-serine but no activity toward d-amino acids. Intriguingly, the pH and temperature profiles of lyase activity were distinct from those of dehydratase activity. Catalytic efficiency was higher for lyase activity than for dehydratase activity.

Identification and functional characterization of a cystathionine β-lyase (CBL) enzyme for H2S production in Arabidopsis thaliana

Sulfide or sulfur metabolism plays an important role in the growth and development of plants. Cystathionine β-lyase (CBL) is an important enzyme in methionine synthesis, but a comprehensive understanding of CBL functions is limited. As the third gasotransmitter, hydrogen sulfide (H2S) plays important physiological roles in plants. In this study, we found that the endogenous H2S content in Arabidopsis thaliana cbl mutants was lower than that in the wild type. Under PEG-based osmotic stress conditions, the H2S contents of CBL-overexpression (OE-CBL) plants increased significantly compared with the wild type. Additionally, the OE-CBL plants increased their tolerance to osmotic stress by increasing the transcription levels of drought-related genes and their relative water-loss rates. Compared with cbl and wild type, OE-CBL plants resisted drought stress by significantly closing their stomata, resulting in improved survival rates. Root tip-bending experiments showed that CBL overexpression relieved osmotic, heavy metal and cold stresses in Arabidopsis. The recombinant CBL activity in vitro revealed that CBL produced H2S using L-cysteine as a substrate. Thus, CBL had a very strong cysteine desulfhydrase activity that could produce endogenous H2S using L-cysteine as a substrate, and it played an important role in plant abiotic stress resistance.

Global Survey, Expressions and Association Analysis of CBLL Genes in Peanut.

Cystathionine γ-synthase (CGS), methionine γ-lyase (MGL), cystathionine β-lyase (CBL) and cystathionine γ-lyase (CGL) share the Cys_Met_Meta_PP domain and play important roles in plant stress response and development. In this study, we defined the genes containing the Cys_Met_Meta_PP domain (PF01053.20) as CBL-like genes (CBLL). Twenty-nine CBLL genes were identified in the peanut genome, including 12 from cultivated peanut and 17 from wild species. These genes were distributed unevenly at the ends of different chromosomes. Evolution, gene structure, and motif analysis revealed that CBLL proteins were composed of five different evolutionary branches. Chromosome distribution pattern and synteny analysis strongly indicated that whole-genome duplication (allopolyploidization) contributed to the expansion of CBLL genes. Comparative genomics analysis showed that there were three common collinear CBLL gene pairs among peanut, Arabidopsis, grape, and soybean, but no collinear CBLL gene pairs between peanut and rice. The prediction results of cis-acting elements showed that AhCBLLs, AdCBLLs, and AiCBLLs contained different proportions of plant growth, abiotic stress, plant hormones, and light response elements. Spatial expression profiles revealed that almost all AhCBLLs had significantly higher expression in pods and seeds. All AhCBLLs could respond to heat stress, and some of them could be rapidly induced by cold, salt, submergence, heat and drought stress. Furthermore, one polymorphic site in AiCBLL7 was identified by association analysis which was closely associated with pod length (PL), pod width (PW), hundred pod weight (HPW) and hundred seed weight (HSW). The results of this study provide a foundation for further research on the function of the CBLL gene family in peanut.

Citrobacter freundii Methionine γ-Lyase: The Role of Serine 339 in the Catalysis of γ- and β-Elimination Reactions.

Serine 339 of the active site of Citrobacter freundii methionine γ-lyase (MGL) is a conserved amino acid in most pyridoxal 5’-phosphate-dependent enzymes of the cystathionine β-lyase subclass, to which MGL belongs. The reaction mechanism of the MGL-catalyzed γ-elimination reaction is poorly explored. We replaced serine 339 with alanine using site-directed mutagenesis. The replacement of serine 339 with alanine led to a significant (by two orders of magnitude) decrease in efficiency in the catalysis of the γ- and β-elimination reactions by the mutant form of the enzyme. The exchange rates of the C-α- and C-β-protons in the amino acids in complexes consisting of the enzyme and competitive inhibitors decreased by one-two orders of magnitude. The spectral characteristics of the mutant form indicated that the replacement did not lead to significant changes in the conformation and tautomerism of MGL internal aldimine. We crystallized the holoenzyme and determined its spatial structure at 1.7 E resolution. The replacement of serine 339 with alanine did not affect the overall course of the polypeptide chain of the MGL subunit and the tetrameric enzyme structure. An analysis of the obtained kinetic and spectral data, as well as the known spatial structures of C. freundii MGL, indicates that serine 339 is necessary for efficient catalysis of γ- and β-elimination reactions at the stage of C-α-proton abstraction from the external aldimine, the γ-elimination reaction at the stages of coenzyme C4’-atom protonation, and C-β-proton abstraction from a ketimine intermediate.

Profiling Cystathionine β/γ-Lyase in Complex Biosamples Using Novel Activatable Fluorogens.

Cystathionine lyase, the key enzyme in transsulfuration and reverse transsulfuration pathways, is involved in a wide array of physiological and pathophysiological processes in both mammals and nonmammals. Though the biological significance of the hydrogen sulfide/cystathionine lyase system in disease states is extensively discussed, the absence of molecular methods for direct monitoring of cystathionine lyase in complex biosamples renders the result unreliable and perplexing. Here, we present the first attempt at designing and developing effective activatable fluorescent probes for cystathionine lyase based on the naphthylamide scaffold. CBLP and CSEP were designed based on the catalytic preference of cystathionine β-lyase (CBL) and cystathionine γ-lyase (CSE). Briefly, incorporation of cysteine/homocysteine as the recognition moiety and a carbamate ethyl sulfide group as a self-immolated linker proved to be an effective strategy for cystathionine lyase fluorescence reporting. CBLP exhibits high selectivity and sensitivity in vitro in semiquantifying CBL levels in roots of wild-type Arabidopsis thaliana and cbl mutants (cbl knockout: SALK_014740C, overexpressed: OE-CBL). Meanwhile, CSEP successfully detected CSE levels in HCC-LM3 cells, zebrafish models, and upregulated CSE in frozen section slides from the liver tissue of cecal ligation and puncture (CLP)-induced septic rats, which was also validated by Western blotting and immunohistochemical analysis. In summary, the practical design strategy facilitates profiling of cystathionine lyase activity in biological processes. It may pave the way for the development of accurate and efficient methods for the direct estimation of cystathionine lyase.

Ability of Exogenous or Wort Endogenous Enzymes to Release Free Thiols from Hop Cysteinylated and Glutathionylated S-Conjugates

Following identification of the substantial pool of aromas represented by cysteinylated and glutathionylated precursors in hop, many studies have focused on developing methods for releasing volatile thiols based on chemical hydrolysis or yeast activity. The present work aimed to evaluate the release activity of either commercially available exogenous enzymes or endogenous wort enzymes (issued from hop or malt) on synthetic Cys- or G-precursors of 3SHol, 3SPol, 3SHptol, 3S4MPol and 4S4M2Pone. In a model medium at pH 7.7, both apotryptophanase and cystathionine β-lyase appeared very efficient on Cys-adducts (up to 77.9% release). Interestingly, a slight proportion of this release (up to 0.9%) appeared to result from chemical hydrolysis. In polyphenols rich media such as hop and beer, total release was much lower (under 0.2%). For release from G-adducts, a combination of γGT and apotryptophanase proved to be effective, highlighting for the first time the ability of β-lyase activity to release free PFTs also from CysGly-adducts. The best release rates (above 2% for all thiols) were obtained with a two-step incubation: 75 units γGT with synthetic glutathionylated precursors for 16 h at 37 °C in the presence of alanine followed by apotryptophanase for 7 h at room temperature. The alternative combination of γGT with S. cerevisiae was found to release free thiols more efficiently than yeast activity alone. Compared to commercially available enzymes, endogenous enzymes extracted from hop or malt displayed lower efficiency (below 0.3% for γGT activity and below 0.002% for β-lyase).

Transcription of Cystathionine β-Lyase (MetC) Is Repressed by HeuR in Campylobacter jejuni, and Methionine Biosynthesis Facilitates Colonocyte Invasion.

A previously identified transcriptional regulator in Campylobacter jejuni, termed HeuR, was found to positively regulate heme utilization. Additionally, transcriptomic work demonstrated that the putative operons CJJ81176_1390 to CJJ81176_1394 (CJJ81176_1390-1394) and CJJ81176_1214-1217 were upregulated in a HeuR mutant, suggesting that HeuR negatively regulates expression of these genes. Because genes within these clusters include a cystathionine β-lyase (metC) and a methionine synthase (metE), it appeared HeuR negatively regulates C. jejuni methionine biosynthesis. To address this, we confirmed mutation of HeuR reproducibly results in metC overexpression under nutrient-replete conditions but did not affect expression of metE, while metC expression in the wild type increased to heuR mutant levels during iron limitation. We subsequently determined that both gene clusters are operonic and demonstrated the direct interaction of HeuR with the predicted promoter regions of these operons. Using DNase footprinting assays, we were able to show that HeuR specifically binds within the predicted -35 region of the CJJ81176_1390-1394 operon. As predicted based on transcriptional results, the HeuR mutant was able to grow and remain viable in a defined medium with and without methionine, but we identified significant impacts on growth and viability in metC and metE mutants. Additionally, we observed decreased adherence, invasion, and persistence of metC and metE mutants when incubated with human colonocytes, while the heuR mutant exhibited increased invasion. Taken together, these results suggest that HeuR regulates methionine biosynthesis in an iron-responsive manner and that the ability to produce methionine is an important factor for adhering to and invading the gastrointestinal tract of a susceptible host. IMPORTANCE As the leading cause of bacterium-derived gastroenteritis worldwide, Campylobacter jejuni has a significant impact on human health. Investigating colonization factors that allow C. jejuni to successfully infect a host furthers our understanding of genes and regulatory elements necessary for virulence. In this study, we have begun to characterize the role of the transcriptional regulatory protein, HeuR, on methionine biosynthesis in C. jejuni. When the ability to synthesize methionine is impaired, detrimental impacts on growth and viability are observed during growth in limited media lacking methionine and/or iron. Additionally, mutations in the methionine biosynthetic pathway result in decreased adhesion, invasion, and intracellular survival of C. jejuni when incubated with human colonocytes, indicating the importance of regulating methionine biosynthesis.

Identification and Characterization of a Novel Genomic Island Harboring Cadmium and Arsenic Resistance Genes in Listeria welshimeri.

Listeria monocytogenes, the bacterial foodborne pathogen responsible for the severe disease listeriosis, frequently exhibits heavy metal resistance. Concurrent resistance to cadmium and arsenic in L. monocytogenes is strongly associated with the 35-kb chromosomal island LGI2. LGI2 has been encountered repeatedly among L. monocytogenes serotype 4b hypervirulent clones but, surprisingly, not among non-pathogenic Listeria spp. Here we describe a novel LGI2 variant, LGI2-3, in two L. welshimeri strains from an urban aquatic environment. Whole genome sequence analysis revealed that the genomes were closely related except for one prophage region and confirmed a chromosomally integrated LGI2-3. It harbored a cystathionine beta-lyase gene previously only encountered in LGI2-1 of L. monocytogenes clonal complex 1 but was otherwise most closely related to LGI2. LGI2-3 harbored a novel cadAC cassette (cadA7C7) that, like LGI2′s cadA4C4, was associated with lower-level tolerance to cadmium (MIC 50 μg/mL) than other cadAC cassettes (MIC ≥ 140 μg/mL). CadA sequence analysis identified two amino acids that may be important for mediating different levels of cadmium tolerance. Our findings clearly demonstrated the potential for LGI2-like islands to be harbored by non-pathogenic Listeria spp. and generate intriguing hypotheses on the genetic diversity mediated by this island and its transfer among Listeria spp.

Gene Expression in 1-Methylcyclopropene (1-MCP) Treated Tomatoes during Pre-Climacteric Ripening Suggests Shared Regulation of Methionine Biosynthesis, Ethylene Production and Respiration

The physiology of fruit ripening is defined as either ‘climacteric’ or ‘non-climacteric’. In climacteric fruit respiration during ripening increases until it reaches a peak, which is accompanied by an increase in autocatalytic ethylene production, whereas the respiration of non-climacteric fruit does not increase and they have no requirement for ethylene to complete their ripening. In an attempt to gain further insight into the involvement of autocatalytic ethylene production with the climacteric rise in respiration, tomato fruit were harvested at three defined stages of maturity prior to the climacteric peak (mature green, breaker, and early orange) and immediately exposed to the gaseous molecule 1-methylcyclopropene (1-MCP). The gene expression profile at each of these stages was monitored after 24 h, using an Affymetrix tomato microarray chip. This approach enabled us to identify ethylene responsive genes that are commonly regulated at early stages of ripening, as well as new candidate genes. In addition, 1-MCP treatment affected the levels of metabolites related to methionine biosynthesis. Methionine feeds climacteric ethylene production and we found that promotors of the genes of enzymes that catalyze the production of homoserine and homocysteine (aspartokinase/homoserine dehydrogenases and cystathionine beta lyase, respectively), precursors in the methionine pathway, contain the AtSR1 binding motif. This binding motif is recognized by ethylene activated transcription factors, hence indicating a role for ethylene in methionine synthesis during early ripening, explaining the autocatalytic ethylene production during subsequent ripening stages.

S0267 Functional and Transcriptional Consequences of Aspirin Use on the Human Fecal Microbiome

INTRODUCTION: Aspirin has been consistently observed to have chemoprotective effects on colorectal neoplasia through multiple interrelated biomechanisms, including prostaglandin synthesis, prostaglandin catabolism, and chronic inflammation via MIC-1. However, it is essential to understand the role human-extrinsic factors, such as the enteric microbiome, have in mediating these effects. It remains unclear how these bacteria may mechanistically influence carcinogenesis, or interfere with cancer prevention, and how regular aspirin use influences the microbiome. METHODS: We have analyzed aspirin use, and other clinical metadata, from a subset of men selected from the Health Professionals Follow-up Study (n = 51,529). Our subset, the “Men’s Lifestyle Validation Study and Microbiome Correlation”, randomly selected for men without history of coronary heart disease, stroke, cancer, or major neurological disease. Stool samples were collected amongst 308 of the participants. Sequencing of the stool samples provided metagenomic and metatranscriptomic data to be analyzed in conjunction with clinical metadata utilizing SAS, R, and the Biobakery pipeline (MaAsLin2, MetaPhlAn2, HUMAnN2), with appropriate multiple-testing hypothesis correction. RESULTS: Metagenomically derived taxonomic and functional profiles of the gut microbiota demonstrated that regular aspirin use significantly shifted community structures. Specifically, Bacteroides caccae and Hungatella hathewayi were enriched with regular aspirin use, whereas Eubacteriumsp. 3_1_31, Odoribacter unclassifised, and Dorea longicatena were depleted. Although similarly influenced by aspirin, they did not appear to highly correlate with one another. Additionally, nine bacterial enzymes, primarily transferases, were depleted by regular aspirin use. Metatranscriptomic analysis found inverse correlations between regular aspirin use and several enzymes: cystathionine beta-lyase, endopeptidase, glucan 1,4-alpha-glucosidase, CoA-disulfide reductase, and an arylsulfatase. This suggests aspirin may influence inhibition of potentially pathogenic bacterial functions.Figure 1.: Species data from metagenomic sequencing was analyzed against different time points of regular aspirin use, with multiple testing control via multivariate analysis, to identify species where regular aspirin use significantly associated with increased, or decreased, abundance.CONCLUSION: Regular aspirin use elicits significant, and consistent, changes in microbial enteric communities, with additional shifts in functional and transcriptional gene expression. These demonstrate means by which aspirin may mediate its chemoprotective effects. By further characterizing these shifts we can identify pathways that could provide targets for potential future therapeutics.Figure 2.: Multivariate analysis, to adjust for potential confounders, led to the identification of species significantly associated with aspirin use from those above. Bacteroides caccae and Hungatella hathewayi were enriched by regular aspirin use. Eubacterium sp. 3_1_31, Odoribacter unclassified, and Dorea longicatena were depleted. When correlated against each other, these species retain relative independence from one another.Figure 3.: Metatranscriptomic data, analyzed based on different time points of regular aspirin use with multiple testing control via multivariate analysis, identified directly and indirectly associated functional genes.

Molecular Modeling and <i>In Vitro</i> Study of Malodor Inhibition Using Thai Herbal Products

Eighty-one microbial odors were isolated and identified based on phenotypic characterization and 16S rRNA gene sequence similarity. The dominant microbial odors (Group C, 27 isolates) were classified as Staphylococcus hominis subsp. hominis DSM 20328T. The genome annotation of all representative microbial odors revealed that they consisted of malodor biosynthesis pathways; short-chain volatile fatty acids (VFAs) and thioalcohol (3-methyl-3-sulfanyl-hexan-1-ol, 3M3SH). Among them, 3M3SH was the most important malodor compound and its key enzyme was cystathionine beta-lyase. To screen the cystathionine beta-lyase inhibitors by docking with PyRx, three bioactive compounds from natural products [gallic acid (CID 370), 1-heneicosanol (CID 85014) and 2,6-dimethylheptadecane (CID 545603)] were predicted to be effective in binding with the target enzyme close to the synthetic inhibitor [N-(2-Hydrazinyl-2-oxoethyl)-3-(trifluoromethyl)benzamide (CID 16109340)]. The water extract of Terminalia catappa L. revealed the highest inhibitory effect against the growth of all microbial odor isolates. Hence, our study concludes that the bioactive compounds of T. catappa L. may be used as an appropriate natural source to develop the natural sport deodorant spray in the future.

Molecular characterization of mimosinase and cystathionine β-lyase in the Mimosoideae subfamily member Mimosa pudica.

Mimosinase degrades the non-protein amino acid mimosine and is thought to have evolved from cystathionine β-lyase (CBL) via gene duplication. However, no study has, to date, compared the molecular characteristics of mimosinase and CBL. We therefore cloned mimosinase and CBL from the Mimosoideae subfamily member Mimosa pudica (Mp) and explored the molecular relationship between mimosinase and CBL for the first time. The recombinant Mp mimosinase degraded both mimosine and cystathionine with a much higher turnover number (kcat) for mimosine compared with cystathionine, and Mp CBL utilized only cystathionine as a substrate. The critical residues implicated in the substrate binding of Arabidopsis thaliana CBL (Tyr-127, Arg-129, Tyr-181, and Arg-440) were highly conserved in both Mp mimosinase and CBL. However, homology modeling and molecular simulation of these enzymes predicted variations in the residues that interact with substrates. A mutation experiment on Mp mimosinase revealed that the disruption of a disulfide bond in the vicinity of the pyridoxal-5'-phosphate domain increased the enzyme's preference toward cystathionine. Treatment of Mp mimosinase with a disulfide-cleavage agent also decreased mimosinase activity. Furthermore, mutation near the conserved binding residue altered the substrate preference between mimosine and cystathionine. Molecular dynamics simulations of Mp mimosinase suggested a closer coordination of the residues that interact with mimosine at the active site compared with cystathionine, indicating a more compact pocket size for mimosine degradation. This study thus may provide new insights into the molecular diversification of CBL, a C-S lyase, into the C-N lyase mimosinase in the Mimosoideae subfamily.

Cystathionine beta-lyase is crucial for embryo patterning and the maintenance of root stem cell niche in Arabidopsis.

The growth and development of roots in plants depends on the specification and maintenance of the root apical meristem. Here, we report the identification of CBL, a gene required for embryo and root development in Arabidopsis, and encodes cystathionine beta-lyase (CBL), which catalyzes the penultimate step in methionine (Met) biosynthesis, and which also led to the discovery of a previous unknown, but crucial, metabolic contribution by the Met biosynthesis pathway. CBL is expressed in embryos and shows quiescent center (QC)-enriched expression pattern in the root. cbl mutant has impaired embryo patterning, defective root stem cell niche, stunted root growth, and reduces accumulation of the root master regulators PLETHORA1 (PLT1) and PLT2. Furthermore, mutation in CBL severely decreases abundance of several PIN-FORMED (PIN) proteins and impairs auxin-responsive gene expression in the root tip. cbl seedlings also exhibit global reduction in histone H3 Lys-4 trimethylation (H3K4me3) and DNA methylation. Importantly, mutation in CBL reduces the abundance of H3K4me3 modification in PLT1/2 genes and downregulates their expression. Overexpression of PLT2 partially rescues cbl root meristem defect, suggesting that CBL acts in part through PLT1/2. Moreover, exogenous supplementation of Metalso restores the impaired QC activity and the root growth defects of cbl. Taken together, our results highlight the unique role of CBL to maintain the root stem cell niche by cooperative actions between Met biosynthesis and epigenetic modification of key developmental regulators.

Elucidating the Heme Domain's Regulatory Role on Human Cystathionine β‐Synthase (hCBS) Product Formation

Human cystathionine β‐synthase (hCBS) is a vital enzyme found within the human transulfuration pathway and is responsible for the biotransformation of serine and homocysteine into cystathionine. hCBS at high cysteine concentrations has been shown to release hydrogen sulfide as a byproduct via an alternate reaction, suggesting hCBS to be closely involved with regulating metabolic sulfur. Catalysis in both the primary and alternate reactions proceeds via a PLP dependent β‐replacement ping‐pong mechanism, with the last step before product dissociation from the internal aldimine (Schiff base) intermediate being the condensation of H2O or H2S, respectively. Consequently, mis‐regulation of substrate levels or loss of CBS activity has been causally linked to hyperhomocysteinemia, a biological condition signifying abnormally high levels of homocysteine within the circulatory system; this condition being one of many causes leading to cardiovascular disease. A highly conserved PLP catalytic and SAM regulatory domain can be seen when comparing yeast analogue (yCBS) to mammalian variants. However, the mammalian variants, specifically the human analog hCBS, differs by containing a unique heme binding domain not found in yCBS. Given that the heme prosthetic group is not required for catalytic activity, the role that this additional domain plays in regulating cystathionine production in hCBS is not yet clearly understood. Current methods for measuring CBS catalytic activity involve using radioactively labeled substrates and typically have focused only on yeast and truncated hCBS variants. We have adapted a previously established colorimetric coupled enzyme assay to measure hCBS kinetics, coupling CBS with cystathionine β‐lyase (CBL), and lactate dehydrogenase (LDH); where the stoichiometric oxidation of NADH, monitored spectrophotometrically at 340 nm, is correlated directly to the formation of cystathionine. Preliminary results suggest this coupled assay is a viable method for measuring product formation kinetics of full length wild type hCBS. Current and future work aims to 1) asses catalytic rate activity for wild type hCBS and 2) asses the activity of single point mutant variants with mutations localized within the heme binding domain. Through these experiments, we hope to elucidate a deeper understanding of how the heme binding domain affects cystathionine product formation.Support or Funding InformationNIH MARC: T34‐GM008574This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cystathionine β-lyase is involved in d-amino acid metabolism.

Non-canonical d-amino acids play important roles in bacteria including control of peptidoglycan metabolism and biofilm disassembly. Bacteria appear to produce non-canonical d-amino acids to adapt to various environmental changes, and understanding the biosynthetic pathways is important. We identified novel amino acid racemases possessing the ability to produce non-canonical d-amino acids in Escherichia coli and Bacillus subtilis in our previous study, whereas the biosynthetic pathways of these d-amino acids still remain unclear. In the present study, we demonstrated that two cystathionine β-lyases (MetC and MalY) from E. coli produce non-canonical d-amino acids including non-proteinogenic amino acids. Furthermore, MetC displayed d- and l-serine (Ser) dehydratase activity. We characterised amino acid racemase, Ser dehydratase and cysteine lyase activities, and all were higher for MetC. Interestingly, all three activities were at a comparable level for MetC, although optimal conditions for each reaction were distinct. These results indicate that MetC and MalY are multifunctional enzymes involved in l-methionine metabolism and the production of d-amino acids, as well as d- and l-Ser metabolism. To our knowledge, this is the first evidence that cystathionine β-lyase is a multifunctional enzyme with three different activities.

Increased Activity of Cystathionine β-Lyase Suppresses 2-Aminoacrylate Stress in Salmonella enterica.

Reactive enamine stress caused by intracellular 2-aminoacrylate accumulation leads to pleiotropic growth defects in a variety of organisms. Members of the well-conserved RidA/YER057c/UK114 protein family prevent enamine stress by enhancing the breakdown of 2-aminoacrylate to pyruvate. In Salmonella enterica, disruption of RidA allows 2-aminoacrylate to accumulate and to inactivate a variety of pyridoxal 5'-phosphate-dependent enzymes by generating covalent bonds with the enzyme and/or cofactor. This study was initiated to identify mechanisms that can overcome 2-aminoacrylate stress in the absence of RidA. Multicopy suppressor analysis revealed that overproduction of the methionine biosynthesis enzyme cystathionine β-lyase (MetC) (EC 4.4.1.8) alleviated the pleiotropic consequences of 2-aminoacrylate stress in a ridA mutant strain. Degradation of cystathionine by MetC was not required for suppression of ridA phenotypes. The data support a model in which MetC acts on a noncystathionine substrate to generate a metabolite that reduces 2-aminoacrylate levels, representing a nonenzymatic mechanism of 2-aminoacrylate depletion.IMPORTANCE RidA proteins are broadly conserved and have been demonstrated to deaminate 2-aminoacrylate and other enamines. 2-Aminoacrylate is generated as an obligatory intermediate in several pyridoxal 5'-phosphate-dependent reactions; if it accumulates, it damages cellular enzymes. This study identified a novel mechanism to eliminate 2-aminoacrylate stress that required the overproduction, but not the canonical activity, of cystathionine β-lyase. The data suggest that a metabolite-metabolite interaction is responsible for quenching 2-aminoacrylate, and they emphasize the need for emerging technologies to probe metabolism in vivo.