Over-expression of cystathionine γ-synthase in Arabidopsis thaliana leads to increased levels of methionine and S-methylmethionine

Cystathionine γ-synthase (CGS, EC 4.2.99.9), the first committed enzyme in methionine biosynthesis, was over-expressed in Arabidopsis thaliana by introducing in the genome of this plant the coding sequence of the Arabidopsis enzyme under the control of the cauliflower mosaic virus 35S promoter. In order to target the recombinant protein to the chloroplast, the transgene included the sequence encoding the N-terminal transit peptide of Arabidopsis CGS. CGS activity and polypeptide were increased several fold in these plants. There was a markedly increased level of soluble methionine in the leaves of the transformed plants, up to 15-fold, indicating that CGS is a rate-limiting enzyme in this metabolic pathway. In addition, the transformed plants strongly over-accumulated S-methylmethionine, but not S-adenosylmethionine, in agreement with the view that S-methylmethionine corresponds to a storage form of labile methyl groups in plants and/or plays a role in preventing S-adenosylmethionine accumulation. The same strategy was used to increase the level of cystathionine β-lyase (CBL, EC 4.4.1.8), the second committed enzyme in methionine biosynthesis, in transformed A. thaliana. Despite an increase in both CBL activity and polypeptide in transformed Arabidopsis plants over-expressing Arabidopsis CBL, there was very little change in the contents of soluble methionine and S-methylmethionine, suggesting strongly that CBL is not rate limiting in the methionine biosynthetic pathway.     

Expression of cystathionine β-synthase and cystathionine γ-lyase in human pregnant myometrium and their roles in the control of uterine contractility

Background

Human uterus undergoes distinct molecular and functional changes during pregnancy and parturition. Hydrogen sulfide (H₂S) has recently been shown to play a key role in the control of smooth muscle tension. The role of endogenous H₂S produced locally in the control of uterine contractility during labour is unknown.

Methodology/Principal Findings

Human myometrium biopsies were obtained from pregnant women undergoing cesarean section at term. Immunohistochemistry analysis showed that cystathionine-γ-lyase (CSE) and cystathionine-β-synthetase (CBS), the principle enzymes responsible for H₂S generation, were mainly localized to smooth muscle cells of human pregnant myometrium. The mRNA and protein expression of CBS as well as H₂S production rate were down-regulated in labouring tissues compared to nonlabouring tissues. Cumulative administration of L-cysteine (10⁻⁷–10⁻² mol/L), a precursor of H₂S, caused a dose-dependent decrease in the amplitude of spontaneous contractions in nonlabouring and labouring myometrium strips. L-cysteine at high concentration (10⁻³ mol/L) increased the frequency of spontaneous contractions and induced tonic contraction. These effects of L-cysteine were blocked by the inhibitors of CBS and CSE. Pre-treatment of myometrium strips with glibenclamide, an inhibitor of ATP-sensitive potassium (K_ATP) channels, abolished the inhibitory effect of L-cysteine on spontaneous contraction amplitude. The effects of L-cysteine on the amplitude of spontaneous contractions and baseline muscle tone were less potent in labouring tissues than that in nonlabouring strips.

Conclusion/Significance

H₂S generated by CSE and CBS locally exerts dual effects on the contractility of pregnant myometrium. Expression of H₂S synthetic enzymes is down-regulated during labour, suggesting that H₂S is one of the factors involved in the transition of pregnant uterus from quiescence to contractile state after onset of parturition.     

Differential response of methionine metabolism in two grain legumes,soybean and azuki bean,expressing a mutated form of Arabidopsis cystathionine γ-synthase

Methionine (Met) is a sulfur-containing amino acid that is essential in mammals and whose low abundance limits the nutritional value of grain legumes. Cystathionine γ-synthase (CGS) catalyzes the first committed step of Met biosynthesis, and the stability of its mRNA is autoregulated by the cytosolic concentration of S-adenosyl-l-methionine (SAM), a direct metabolite of Met. The mto1-1 mutant of Arabidopsis thaliana harbors a mutation in the AtCGS1 gene that renders the mRNA resistant to SAM-dependent degradation and therefore results in the accumulation of free Met to high levels in young leaves. To manipulate Met biosynthesis in soybean and azuki bean, we introduced the AtCGS1 mto1-1 gene into the two grain legumes under the control of a seed-specific glycinin gene promoter. Transgenic seeds of both species accumulated soluble Met to levels at least twice those apparent in control seeds. However, the increase in free Met did not result in an increase in total Met content of the transgenic seeds. In transgenic azuki bean seeds, the amount of cystathionine, the direct product of CGS, was markedly increased whereas the total content of Met was significantly decreased compared with control seeds. Similar changes were not detected in soybean. Our data suggest that the regulation of Met biosynthesis differs between soybean and azuki bean, and that the expression of AtCGS1 mto1-1 differentially affects the metabolic stability of sulfur amino acids and their metabolites in the two grain legumes.     

Crystal structure of the single cystathionine β-synthase domain-containing protein CBSX1 from Arabidopsis thaliana

The single cystathionine β-synthase (CBS) pair proteins from Arabidopsis thaliana have been identified as being a redox regulator of the thioredoxin (Trx) system. CBSX1 and CBSX2, which are two of the six Arabidopsis cystathione β-synthase domain-containing proteins that contain only a single CBS pair, have close sequence similarity. Recently, the crystal structure of CBSX2 was determined and a significant portion of the internal region was disordered. In this study, crystal structures of full-length CBSX1 and the internal loop deleted (Δloop) form are reported at resolutions of 2.4 and 2.2 Å, respectively. The structures of CBSX1 show that they form anti-parallel dimers along their central twofold axis and have a unique ～155° bend along the side. This is different from the angle of CBSX2, which is suggestive of the flexible nature of the relative angle between the monomers. The biochemical data that were obtained using the deletion as well as point mutants of CBSX1 confirmed the importance of AMP-ligand binding in terms of enhancing Trx activity.     

Cystathionine β-synthase and cystathionine γ-lyase in tissues of the nemertean Cerebratulus marginatus Renier, 1804 (nemertea)

Using immunohistochemistry, we examined the distribution of the hydrogen sulfide (H₂S) synthesizing enzymes cystathionine β-synthase (CBS) and cystathionine Γ-lyase (CSE) in tissues of the nemertean Cerebratulus marginatus Renier, 1804 (Heteronemertea: Lineidae). The expression of both CBS and CSE was found in the body wall and in the receptor cells of the canals of the cerebral organs; the expression of CBS was found in the foregut and intestine; and CSE was found in the brain, lateral nerve cords, and cerebral organs. The peculiarities of the distribution and the possible functional role of H₂S-synthesizing elements in nemerteans are discussed.     

Human cystathionine β-synthase: gene organization and expression of different 5′ alternative splicing

The human cystathionine β-synthase (CBS) gene spans in excess of 30 kb and consists of 19 exons, with three different 5′ untranslated regions including three different exons 1 (exons 1 a, b, and c). Exon la and 1b are 390 bp apart from each other and are linked to exon 2 in cDNA « a » and cDNA « b ». Exon 1c, which linked to exon 5 in cDNA « c », is 7 kb apart from exon 1b. All splice sites conform to the GT/AG rule, including those from exon la or 1b to exon 2 and from exon 1c to exon 5. Upstream of exons la and 1b, we found two putative promoter sequences with high C + G nucleotide content, one CAAT box at —70 nucleotides (for exon lb), no TATA box, several Sp1 binding regulatory consensus sequences, and some other regulatory sequences. Human adult and fetal Northern blots hybridized with total cDNA containing exon 1b, or specific probes from exons 1 (b and c) showed mRNAs of 2.5 kb, 2.7 kb, and 3.7 kb. These results suggest that the mRNAs containing the different exons 1 are under the control of different promoters.     

Cytosolic γ-glutamyl peptidases process glutathione conjugates in the biosynthesis of glucosinolates and camalexin in Arabidopsis

The defense-related plant metabolites known as glucosinolates play important roles in agriculture, ecology, and human health. Despite an advanced biochemical understanding of the glucosinolate pathway, the source of the reduced sulfur atom in the core glucosinolate structure remains unknown. Recent evidence has pointed toward GSH, which would require further involvement of a GSH conjugate processing enzyme. In this article, we show that an Arabidopsis thaliana mutant impaired in the production of the γ-glutamyl peptidases GGP1 and GGP3 has altered glucosinolate levels and accumulates up to 10 related GSH conjugates. We also show that the double mutant is impaired in the production of camalexin and accumulates high amounts of the camalexin intermediate GS-IAN upon induction. In addition, we demonstrate that the cellular and subcellular localization of GGP1 and GGP3 matches that of known glucosinolate and camalexin enzymes. Finally, we show that the purified recombinant GGPs can metabolize at least nine of the 10 glucosinolate-related GSH conjugates as well as GS-IAN. Our results demonstrate that GSH is the sulfur donor in the biosynthesis of glucosinolates and establish an in vivo function for the only known cytosolic plant γ-glutamyl peptidases, namely, the processing of GSH conjugates in the glucosinolate and camalexin pathways.     

Characterization of the S289A,D mutants of yeast cystathionine β-synthase

Cystathionine β-synthase (CBS) catalyzes the pyridoxal 5′-phosphate (PLP)-dependent condensation of l-serine and l-homocysteine to form l-cystathionine in the first step of the reverse transsulfuration pathway. Residue S289 of yeast CBS, predicted to form a hydrogen bond with the pyridine nitrogen of the PLP cofactor, was mutated to alanine and aspartate. The k_cat/K_m^l-Ser of the S289A mutant is reduced by a factor of ～ 800 and the β-replacement activity of the S289D mutant is undetectable. Fluorescence energy transfer between tryptophan residue(s) of the enzyme and the PLP cofactor, observed in the wild-type enzyme and diminished in the S289A mutant, is absent in S289D. These results demonstrate that residue S289 is essential in maintaining the properties and orientation of the pyridine ring of the PLP cofactor. The reduction in activity of ytCBS-S289A suggests that ytCBS catalyzes the α,β-elimination of l-Ser via an E1cB mechanism.     

Cloning and expression of sucrose phosphorylase gene from Bifidobacterium longum in E. coli and characterization of the recombinant enzyme

A DNA fragment, which complemented the growth of E. coli both on M9 medium containing raffinose and on LB medium containing ampicillin, IPTG and 5-bromo-4-chloro-3-indoxyl--d-galactoside, was isolated from the genomic library of Bifidobacterium longum SJ32, which had been digested with EcoRI. In the cloned DNA fragment, a gene encoding a sucrose phosphorylase (splP) and a partially cloned putative sucrose regulator gene (splR) were identified using the deletion analysis and sequence analysis. A 56 kDa protein was synthesized in E. coli and partially purified by DEAE-ion exchange chromatography. The partially purified enzyme did not react with melibiose, melezitoze and raffinose but did with sucrose. It had transglucosylation activity in addition to hydrolytic activity.     

The expression level of threonine synthase and cystathionine-γ-synthase is influenced by the level of both threonine and methionine in <Emphasis Type="Italic">Arabidopsis</Emphasis> plants

The biosynthesis pathways of the essential amino acids methionine and threonine diverge from O-phosphohomoserine, an intermediate metabolite in the aspartate family of amino acids. Thus, the enzymes cystathionine-γ-synthase (CGS) in the methionine pathway and threonine synthase (TS), the last enzyme in the threonine pathway, compete for this common substrate. To study this branching point, we overexpressed TS in sense and antisense orientation in Arabidopsis plants with the aim to study its effect on the level of threonine but more importantly on the methionine content. Positive correlation was found not only between TS expression level and threonine content, but also between TS/threonine and CGS expression level. Plants expressing the sense orientation of TS showed a higher level of threonine, increased expression level of CGS, and a significantly higher level of S-methylmethionine, the transport form of methionine. By contrast, plants expressing the antisense form of TS showed lower levels of threonine and of CGS expression level. In these antisense plants, the methionine level increased up to 47-fold compared to wild-type plants. To study further the effect of threonine on CGS expression level, wild-type plants were irrigated with threonine and control plants were irrigated with methionine or water. While threonine increased the expression level of CGS but reduced that of TS, methionine reduced the expression level of CGS but increased that of TS. This data demonstrate that both methionine and threonine affect the two enzymes at the branching point, thus controlling not only their own level, but also the level of each other. This mechanism probably aids in keeping the levels of these two essential amino acids sufficiently high to support plant growth.     

Involvement of the vacuolar processing enzyme γVPE in response of Arabidopsis thaliana to water stress

Plant vacuoles play several roles in controlling development, pathogen defence, and stress response. γVPE is a vacuolarlocalised cysteine protease with a caspase-1 like activity involved in the activation and maturation of downstream vacuolar hydrolytic enzymes that trigger hypersensitive cell death and tissue senescence. This work provides evidence that γVPE is strongly expressed in Arabidopsis guard cells and is involved in water stress response. The γvpe knock-out mutants showed reduced stomatal opening and an increased resistance to desiccation suggesting a new role of γVPE in control of stomatal movements.     

Exploration of structure–function relationships in Escherichia coli cystathionine γ-synthase and cystathionine β-lyase via chimeric constructs and site-specific substitutions

Cystathionine γ-synthase (CGS) and cystathionine β-lyase (CBL) share a common structure and several active-site residues, but catalyze distinct side-chain rearrangements in the two-step transsulfuration pathway that converts cysteine to homocysteine, the precursor of methionine. A series of 12 chimeric variants of Escherichia coli CGS (eCGS) and CBL (eCBL) was constructed to probe the roles of two structurally distinct, ~ 25-residue segments situated in proximity to the amino and carboxy termini and located at the entrance of the active-site. In vivo complementation of methionine-auxotrophic E. coli strains, lacking the genes encoding eCGS and eCBL, demonstrated that exchange of the targeted regions impairs the activity of the resulting enzymes, but does not produce a corresponding interchange of reaction specificity. In keeping with the in vivo results, the catalytic efficiency of the native reactions is reduced by at least 95-fold, and α,β versus α,γ-elimination specificity is not modified. The midpoint of thermal denaturation monitored by circular dichroism, ranges between 59 and 80 °C, compared to 66 °C for the two wild-type enzymes, indicating that the chimeric enzymes adopt a stable folded structure and that the observed reductions in catalytic efficiency are due to reorganization of the active site. Alanine-substitution variants of residues S32 and S33, as well as K42 of eCBL, situated in proximity to and within, respectively, the targeted amino-terminal region were also investigated to explore their role as determinants of reaction specificity via positioning of key active-site residues. The catalytic efficiency of the S32A, S33A and the K42A site-directed variants of eCBL is reduced by less than 10-fold, demonstrating that, while these residues may participate in positioning S339, which tethers the catalytic base, their role is minor.     

The role of surface electrostatics on the stability,function and regulation of human cystathionine β-synthase,a complex multidomain and oligomeric protein

Human cystathionine β-synthase (hCBS) is a key enzyme of sulfur amino acid metabolism, controlling the commitment of homocysteine to the transsulfuration pathway and antioxidant defense. Mutations in hCBS cause inherited homocystinuria (HCU), a rare inborn error of metabolism characterized by accumulation of toxic homocysteine in blood and urine. hCBS is a complex multidomain and oligomeric protein whose activity and stability are independently regulated by the binding of S-adenosyl-methionine (SAM) to two different types of sites at its C-terminal regulatory domain. Here we study the role of surface electrostatics on the complex regulation and stability of hCBS using biophysical and biochemical procedures. We show that the kinetic stability of the catalytic and regulatory domains is significantly affected by the modulation of surface electrostatics through noticeable structural and energetic changes along their denaturation pathways. We also show that surface electrostatics strongly affect SAM binding properties to those sites responsible for either enzyme activation or kinetic stabilization. Our results provide new insight into the regulation of hCBS activity and stability in vivo with implications for understanding HCU as a conformational disease. We also lend experimental support to the role of electrostatic interactions in the recently proposed binding modes of SAM leading to hCBS activation and kinetic stabilization.     

Increased expression of PS1 is sufficient to elevate the level and activity of γ-secretase in vivo

Increase in the generation and deposition of amyloid-β (Aβ) plays a central role in the development of Alzheimer's Disease (AD). Elevation of the activity of γ-secretase, a key enzyme required for the generation for Aβ, can thus be a potential risk factor in AD. However, it is not known whether γ-secretase can be upregulated in vivo. While in vitro studies showed that expression of all four components of γ-secretase (Nicastrin, Presenilin, Pen-2 and Aph-1) are required for upregulation of γ-secretase, it remains to be established as to whether this is true in vivo. To investigate whether overexpressing a single component of the γ-secretase complex is sufficient to elevate its level and activity in the brain, we analyzed transgenic mice expressing either wild type or familial AD (fAD) associated mutant PS1. In contrast to cell culture studies, overexpression of either wild type or mutant PS1 is sufficient to increase levels of Nicastrin and Pen-2, and elevate the level of active γ-secretase complex, enzymatic activity of γ-secretase and the deposition of Aβ in brains of mice. Importantly, γ-secretase comprised of mutant PS1 is less active than that of wild type PS1-containing γ-secretase; however, γ-secretase comprised of mutant PS1 cleaves at the Aβ42 site of APP-CTFs more efficiently than at the Aβ40 site, resulting in greater accumulation of Aβ deposits in the brain. Our data suggest that whereas fAD-linked PS1 mutants cause early onset disease, upregulation of PS1/γ-secretase activity may be a risk factor for late onset sporadic AD.     

Cloning, analysis and expression of the gene for β-glycosidase of Thermus caldophilus GK24 and properties of the enzyme

The gene (bglT) encoding Thermus caldophilus GK24 -glycosidase (Tca -glycosidase) was cloned and sequenced. The gene contains an open reading frame encoding 431 amino acids with a M _r of 48 658 Da. The bglT gene was expressed under the control of tac promoter on a high-copy plasmid in E. coli. The recombinant Tca -glycosidase was purified 41.5-fold with a 59% yield and a specific activity of 83 U mg^–1 protein.     

Crystal structure of a key enzyme in the agarolytic pathway, α-neoagarobiose hydrolase from Saccharophagus degradans 2-40

In agarolytic microorganisms, α-neoagarobiose hydrolase (NABH) is an essential enzyme to metabolize agar because it converts α-neoagarobiose (O-3,6-anhydro-alpha-l-galactopyranosyl-(1,3)-d-galactose) into fermentable monosaccharides (d-galactose and 3,6-anhydro-l-galactose) in the agarolytic pathway. NABH can be divided into two biological classes by its cellular location. Here, we describe a structure and function of cytosolic NABH from Saccharophagus degradans 2–40 in a native protein and d-galactose complex determined at 2.0 and 1.55 Å, respectively. The overall fold is organized in an N-terminal helical extension and a C-terminal five-bladed β-propeller catalytic domain. The structure of the enzyme–ligand (d-galactose) complex predicts a +1 subsite in the substrate binding pocket. The structural features may provide insights for the evolution and classification of NABH in agarolytic pathways.     

Folding and activity of mutant cystathionine β-synthase depends on the position and nature of the purification tag: characterization of the R266K CBS mutant

Cystathionine β-synthase (CBS), a heme-containing pyridoxal-5-phosphate (PLP)-dependent enzyme, catalyzes the condensation of serine and homocysteine to yield cystathionine. Missense mutations in CBS, the most common cause of homocystinuria, often result in misfolded proteins. Arginine 266, where the pathogenic missense mutation R266K was identified, appears to be involved in the communication between heme and the PLP-containing catalytic center. Here, we assessed the effect of a short affinity tag (6xHis) compared to a bulky fusion partner (glutathione S-transferase - GST) on CBS wild type (WT) and R266K mutant enzyme properties. While WT CBS was successfully expressed either in conjunction with a GST or with a 6xHis tag, the mutant R266K CBS had no activity, did not form native tetramers and did not respond to chemical chaperone treatment when expressed with a GST fusion partner. Interestingly, expression of R266K CBS constructs with a 6xHis tag at either end yielded active enzymes. The purified, predominantly tetrameric, R266K CBS with a C-terminal 6xHis tag had ～82% of the activity of a corresponding WT CBS construct. Results from thermal pre-treatment of the enzyme and the denaturation profile of R266K suggests a lower thermal stability of the mutant enzyme compared to WT, presumably due to a disturbed heme environment.     

δ-Aminolaevulinate dehydratase, the regulatory enzyme of the haem-biosynthetic pathway in Neurospora crassa

The activity of delta-aminolaevulinate dehydratase is very low in the mould Neurospora crassa compared with the activities detected in bacterial and animal systems. The enzyme is inducible in iron-deficient cultures by addition of iron and is repressed by protoporphyrin. The properties of the purified enzyme indicate its allosteric nature and susceptibility to feedback inhibition by coproporphyrinogen III. Neurospora extracts also contain a protein inhibitor of the enzyme and a small-molecule activator, which appears to be associated with the enzyme. The regulatory function of this enzyme in vivo is correlated with the accumulation of delta-aminolaevulinic acid in normal cultures of N. crassa. The decay curve of the iron-induced enzyme in vivo shows a biphasic pattern, with one of the components showing a half-life of 4-5 min.