NO? Binds Human Cystathionine β-Synthase Quickly and Tightly

The hexa-coordinate heme in the H₂S-generating human enzyme cystathionine β-synthase (CBS) acts as a redox-sensitive regulator that impairs CBS activity upon binding of NO^• or CO at the reduced iron. Despite the proposed physiological relevance of this inhibitory mechanism, unlike CO, NO^• was reported to bind at the CBS heme with very low affinity (K_d = 30–281 μm). This discrepancy was herein reconciled by investigating the NO^• reactivity of recombinant human CBS by static and stopped-flow UV-visible absorption spectroscopy. We found that NO^• binds tightly to the ferrous CBS heme, with an apparent K_d ≤0.23 μm. In line with this result, at 25 °C, NO^• binds quickly to CBS (k_on ∼ 8 × 10³ m⁻¹ s⁻¹) and dissociates slowly from the enzyme (k_off ∼ 0.003 s⁻¹). The observed rate constants for NO^• binding were found to be linearly dependent on [NO^•] up to ∼ 800 μm NO^•, and >100-fold higher than those measured for CO, indicating that the reaction is not limited by the slow dissociation of Cys-52 from the heme iron, as reported for CO. For the first time the heme of human CBS is reported to bind NO^• quickly and tightly, providing a mechanistic basis for the in vivo regulation of the enzyme by NO^•. The novel findings reported here shed new light on CBS regulation by NO^• and its possible (patho)physiological relevance, enforcing the growing evidence for an interplay among the gasotransmitters NO^•, CO, and H₂S in cell signaling.     

Nitrite Reductase Activity and Inhibition of H2S Biogenesis by Human Cystathionine ?-Synthase

Nitrite was recognized as a potent vasodilator >130 years and has more recently emerged as an endogenous signaling molecule and modulator of gene expression. Understanding the molecular mechanisms that regulate nitrite metabolism is essential for its use as a potential diagnostic marker as well as therapeutic agent for cardiovascular diseases. In this study, we have identified human cystathionine ß-synthase (CBS) as a new player in nitrite reduction with implications for the nitrite-dependent control of H₂S production. This novel activity of CBS exploits the catalytic property of its unusual heme cofactor to reduce nitrite and generate NO. Evidence for the possible physiological relevance of this reaction is provided by the formation of ferrous-nitrosyl (Fe^II-NO) CBS in the presence of NADPH, the human diflavin methionine synthase reductase (MSR) and nitrite. Formation of Fe^II-NO CBS via its nitrite reductase activity inhibits CBS, providing an avenue for regulating biogenesis of H₂S and cysteine, the limiting reagent for synthesis of glutathione, a major antioxidant. Our results also suggest a possible role for CBS in intracellular NO biogenesis particularly under hypoxic conditions. The participation of a regulatory heme cofactor in CBS in nitrite reduction is unexpected and expands the repertoire of proteins that can liberate NO from the intracellular nitrite pool. Our results reveal a potential molecular mechanism for cross-talk between nitrite, NO and H₂S biology.     

Allosteric Communication between the Pyridoxal 5′-Phosphate (PLP) and Heme Sites in the H2S Generator Human Cystathionine β-Synthase

Human cystathionine β-synthase (CBS) is a unique pyridoxal 5′-phosphate (PLP)-dependent enzyme that has a regulatory heme cofactor. Previous studies have demonstrated the importance of Arg-266, a residue at the heme pocket end of α-helix 8, for communication between the heme and PLP sites. In this study, we have examined the role of the conserved Thr-257 and Thr-260 residues, located at the other end of α-helix 8 on the heme electronic environment and on activity. The mutations at the two positions destabilize PLP binding, leading to lower PLP content and ∼2- to ∼500-fold lower activity compared with the wild-type enzyme. Activity is unresponsive to PLP supplementation, consistent with the pyridoxine-nonresponsive phenotype of the T257M mutation in a homocystinuric patient. The H₂S-producing activities, also impacted by the mutations, show a different pattern of inhibition compared with the canonical transsulfuration reaction. Interestingly, the mutants exhibit contrasting sensitivities to the allosteric effector, S-adenosylmethionine (AdoMet); whereas T257M and T257I are inhibited, the other mutants are hyperactivated by AdoMet. All mutants showed an increased propensity of the ferrous heme to form an inactive species with a 424 nm Soret peak and exhibited significantly reduced enzyme activity in the ferrous and ferrous-CO states. Our results provide the first evidence for bidirectional transmission of information between the cofactor binding sites, suggest the additional involvement of this region in allosteric communication with the regulatory AdoMet-binding domain, and reveal the potential for independent modulation of the canonical transsulfuration versus H₂S-generating reactions catalyzed by CBS.     

Effect of the Disease-Causing R266K Mutation on the Heme and PLP Environments of Human Cystathionine β-Synthase

Cystathionine β-synthase (CBS) is an essential pyridoxal 5'-phosphate (PLP)-dependent enzyme of the transsulfuration pathway that condenses serine with homocysteine to form cystathionine; intriguingly, human CBS also contains a heme b cofactor of unknown function. Herein we describe the enzymatic and spectroscopic properties of a disease-associated R266K hCBS variant, which has an altered hydrogen-bonding environment. The R266K hCBS contains a low-spin, six-coordinate Fe(III) heme bearing a His/Cys ligation motif, like that of WT hCBS; however, there is a geometric distortion that exists at the R266K heme. Using rR spectroscopy, we show that the Fe(III)-Cys(thiolate) bond is longer and weaker in R266K, as evidenced by an 8 cm(-1) downshift in the ν(Fe-S) resonance. Presence of this longer and weaker Fe(III)-Cys(thiolate) bond is correlated with alteration of the fluorescence spectrum of the active PLP ketoenamine tautomer. Activity data demonstrate that, relative to WT, the R266K variant is more impaired in the alternative cysteine-synthesis reaction than in the canonical cystathionine-synthesis reaction. This diminished cysteine synthesis activity and a greater sensitivity to exogenous PLP correlate with the change in PLP environment. Fe-S(Cys) bond weakening causes a nearly 300-fold increase in the rate of ligand switching upon reduction of the R266K heme. Combined, these data demonstrate cross talk between the heme and PLP active sites, consistent with previous proposals, revealing that alteration of the Arg(266)-Cys(52) interaction affects PLP-dependent activity and dramatically destabilizes the ferrous thiolate-ligated heme complex, underscoring the importance of this hydrogen-bonding residue pair.     

Kinetics of Nitrite Reduction and Peroxynitrite Formation by Ferrous Heme in Human Cystathionine β-Synthase

Cystathionine β-synthase (CBS) is a pyridoxal phosphate-dependent enzyme that catalyzes the condensation of homocysteine with serine or with cysteine to form cystathionine and either water or hydrogen sulfide, respectively. Human CBS possesses a noncatalytic heme cofactor with cysteine and histidine as ligands, which in its oxidized state is relatively unreactive. Ferric CBS (Fe(III)-CBS) can be reduced by strong chemical and biochemical reductants to Fe(II)-CBS, which can bind carbon monoxide (CO) or nitric oxide (NO^•), leading to inactive enzyme. Alternatively, Fe(II)-CBS can be reoxidized by O₂ to Fe(III)-CBS, forming superoxide radical anion (O₂^˙̄). In this study, we describe the kinetics of nitrite (NO₂⁻) reduction by Fe(II)-CBS to form Fe(II)NO^•-CBS. The second order rate constant for the reaction of Fe(II)-CBS with nitrite was obtained at low dithionite concentrations. Reoxidation of Fe(II)NO^•-CBS by O₂ showed complex kinetic behavior and led to peroxynitrite (ONOO⁻) formation, which was detected using the fluorescent probe, coumarin boronic acid. Thus, in addition to being a potential source of superoxide radical, CBS constitutes a previously unrecognized source of NO^• and peroxynitrite.     

Modulation of Cystathionine β-Synthase Activity by the Arg-51 and Arg-224 Mutations

Human cystathionine β-synthase (CBS) catalyzes a pyridoxal 5′-phosphate (PLP) dependent β-replacement reaction to synthesize cystathionine from serine and homocysteine. The enzyme is unique in bearing not only a catalytically important PLP but also heme. In order to study a regulatory process mediated by heme, we performed mutagenesis of Arg-51 and Arg-224, which have hydrogen-bonding interactions with propionate side chains of the prosthetic group. It was found that the arginine mutations decrease CBS activity by approximately 50%. The results indicate that structural changes in the heme vicinity are transmitted to PLP existing 20 Å away from heme. A possible explanation of our results is discussed on the basis of CBS structure.     

Enhanced Expression of Cystathionine β-Synthase and Cystathionine γ-Lyase During Acute Cholecystitis-Induced Gallbladder Inflammation

Background

Hydrogen sulfide (H₂S) has recently been shown to play an important role in the digestive system, but the role of endogenous H₂S produced locally in the gallbladder is unknown. The aim of this study was to investigate whether gallbladder possesses the enzymatic machinery to synthesize H₂S, and whether H₂S synthesis is changed in gallbladder inflammation during acute acalculous cholecystitis (AC).

Methods

Adult male guinea pigs underwent either a sham operation or common bile duct ligation (CBDL). One, two, or three days after CBDL, the animals were sacrificed separately. Hematoxylin and eosin-stained slides of gallbladder samples were scored for inflammation. H₂S production rate in gallbladder tissue from each group was determined; immunohistochemistry and western blotting were used to determine expression levels of the H₂S-producing enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in gallbladder.

Results

There was a progressive inflammatory response after CBDL. Immunohistochemistry analysis showed that CBS and CSE were expressed in the gallbladder epithelium, muscular layer, and blood vessels and that the expression increased progressively with increasing inflammation following CBDL. The expression of CBS protein as well as the H₂S-production rate was significantly increased in the animals that underwent CBDL, compared to those that underwent the sham operation.

Conclusions

Both CBS and CSE are expressed in gallbladder tissues. The expression of these enzymes, as well as H₂S synthesis, was up-regulated in the context of inflammation during AC.     

Inter-domain Communication of Human Cystathionine β-Synthase: STRUCTURAL BASIS OF S-ADENOSYL-l-METHIONINE ACTIVATION*

Cystathionine β-synthase (CBS) is a key enzyme in sulfur metabolism, and its inherited deficiency causes homocystinuria. Mammalian CBS is modulated by the binding of S-adenosyl-l-methionine (AdoMet) to its regulatory domain, which activates its catalytic domain. To investigate the underlying mechanism, we performed x-ray crystallography, mutagenesis, and mass spectrometry (MS) on human CBS. The 1.7 Å structure of a AdoMet-bound CBS regulatory domain shows one AdoMet molecule per monomer, at the interface between two constituent modules (CBS-1, CBS-2). AdoMet binding is accompanied by a reorientation between the two modules, relative to the AdoMet-free basal state, to form interactions with AdoMet via residues verified by mutagenesis to be important for AdoMet binding (Phe⁴⁴³, Asp⁴⁴⁴, Gln⁴⁴⁵, and Asp⁵³⁸) and for AdoMet-driven inter-domain communication (Phe⁴⁴³, Asp⁵³⁸). The observed structural change is further supported by ion mobility MS, showing that as-purified CBS exists in two conformational populations, which converged to one in the presence of AdoMet. We therefore propose that AdoMet-induced conformational change alters the interface and arrangement between the catalytic and regulatory domains within the CBS oligomer, thereby increasing the accessibility of the enzyme active site for catalysis.     

Detection of Reaction Intermediates during Human Cystathionine β-Synthase-monitored Turnover and H2S Production

Human cystathionine β-synthase (CBS), a novel heme-containing pyridoxal 5′-phosphate enzyme, catalyzes the condensation of homocysteine and serine or cysteine to produce cystathionine and H₂O or H₂S, respectively. The presence of heme in CBS has limited spectrophotometric characterization of reaction intermediates by masking the absorption of the pyridoxal 5′-phosphate cofactor. In this study, we employed difference stopped-flow spectroscopy to characterize reaction intermediates formed under catalytic turnover conditions. The reactions of l-serine and l-cysteine with CBS resulted in the formation of a common aminoacrylate intermediate (k_obs = 0.96 ± 0.02 and 0.38 ± 0.01 mm⁻¹ s⁻¹, respectively, at 24 °C) with concomitant loss of H₂O and H₂S and without detectable accumulation of the external aldimine or other intermediates. Homocysteine reacted with the aminoacrylate intermediate with k_obs = 40.6 ± 3.8 s⁻¹ and re-formed the internal aldimine. In the reverse direction, CBS reacted with cystathionine, forming the aminoacrylate intermediate with k_obs = 0.38 ± 0.01 mm⁻¹ s⁻¹. This study provides the first insights into the pre-steady-state kinetic mechanism of human CBS and indicates that the reaction is likely to be limited by a conformational change leading to product release.     

Human Polycomb 2 Protein Is a SUMO E3 Ligase and Alleviates Substrate-Induced Inhibition of Cystathionine β-Synthase Sumoylation

Human cystathionine β-synthase (CBS) catalyzes the first irreversible step in the transsulfuration pathway and commits homocysteine to the synthesis of cysteine. Mutations in CBS are the most common cause of severe hereditary hyperhomocysteinemia. A yeast two-hybrid approach to screen for proteins that interact with CBS had previously identified several components of the sumoylation pathway and resulted in the demonstration that CBS is a substrate for sumoylation. In this study, we demonstrate that sumoylation of CBS is enhanced in the presence of human polycomb group protein 2 (hPc2), an interacting partner that was identified in the initial yeast two-hybrid screen. When the substrates for CBS, homocysteine and serine for cystathionine generation and homocysteine and cysteine for H₂S generation, are added to the sumoylation mixture, they inhibit the sumoylation reaction, but only in the absence of hPc2. Similarly, the product of the CBS reaction, cystathionine, inhibits sumoylation in the absence of hPc2. Sumoylation in turn decreases CBS activity by ∼28% in the absence of hPc2 and by 70% in its presence. Based on these results, we conclude that hPc2 serves as a SUMO E3 ligase for CBS, increasing the efficiency of sumoylation. We also demonstrate that γ-cystathionase, the second enzyme in the transsulfuration pathway is a substrate for sumoylation under in vitro conditions. We speculate that the role of this modification may be for nuclear localization of the cysteine-generating pathway under conditions where nuclear glutathione demand is high.     

The Human Cystathionine β-Synthase (CBS) Gene: Complete Sequence, Alternative Splicing, and Polymorphisms

Cystathionine β-synthase [CBS; -serine hydro-lyase (adding homocysteine), EC 4.2.1.22] catalyzes the first committed step of transsulfuration and is the enzyme deficient in classical homocystinuria. In this report, we describe the molecular cloning and the complete nucleotide sequence of the human CBS gene. We report a total of 28,046 nucleotides of sequence, which, in addition to the CBS gene, contains 5 kb of the 5′ flanking region. The human CBS gene contains 23 exons ranging from 42 to 209 bp. The 5′ UTR is formed by 1 of 5 alternatively used exons and 1 invariably present exon, while the 3′ UTR is encoded by exons 16 and 17. We also describe the identification of two alternatively used promoter regions that are GC rich (80%) and contain numerous putative binding sites for Sp1, Ap1, Ap2, and c-myb, but lack the classical TATA box. The CBS locus contains an unusually high number ofAlurepeats, which may predispose this gene to deleterious rearrangements. Additionally, we report on a number of DNA sequence repeats that are polymorphic in North American and European Caucasians.     

Relative Contributions of Cystathionine ��-Synthase and ��-Cystathionase to H2S Biogenesis via Alternative Trans-sulfuration Reactions

In mammals, the two enzymes in the trans-sulfuration pathway, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), are believed to be chiefly responsible for hydrogen sulfide (H₂S) biogenesis. In this study, we report a detailed kinetic analysis of the human and yeast CBS-catalyzed reactions that result in H₂S generation. CBS from both organisms shows a marked preference for H₂S generation by β-replacement of cysteine by homocysteine. The alternative H₂S-generating reactions, i.e. β-elimination of cysteine to generate serine or condensation of 2 mol of cysteine to generate lanthionine, are quantitatively less significant. The kinetic data were employed to simulate the turnover numbers of the various CBS-catalyzed reactions at physiologically relevant substrate concentrations. At equimolar concentrations of CBS and CSE, the simulations predict that H₂S production by CBS would account for ∼25–70% of the total H₂S generated via the trans-sulfuration pathway depending on the extent of allosteric activation of CBS by S-adenosylmethionine. The relative contribution of CBS to H₂S genesis is expected to decrease under hyperhomocysteinemic conditions. CBS is predicted to be virtually the sole source of lanthionine, and CSE, but not CBS, efficiently cleaves lanthionine. The insensitivity of the CBS-catalyzed H₂S-generating reactions to the grade of hyperhomocysteinemia is in stark contrast to the responsiveness of CSE and suggests a previously unrecognized role for CSE in intracellular homocysteine management. Finally, our studies reveal that the profligacy of the trans-sulfuration pathway results not only in a multiplicity of H₂S-yielding reactions but also yields novel thioether metabolites, thus increasing the complexity of the sulfur metabolome.Hydrogen sulfide (H₂S)² elicits an array of physiological effects, including modulation of blood pressure and reduction of ischemia reperfusion injury (1, 2). Other novel effects of H₂S include induction of a state of suspended animation in mouse by decreasing oxygen consumption and drastically reducing the metabolic rate (3) and synchronizing ultradian metabolic oscillation in yeast (4). Under conditions of metabolic cycling in yeast, H₂S production is catalyzed by sulfite reductase in the sulfur assimilation pathway (4). Inhibition of sulfite reductase reduces H₂S production and in turn perturbs metabolic oscillations. H₂S is a specific and potent inhibitor of cytochrome c oxidase in the electron transport chain (3).Although concentrations of H₂S have been reported to range from 50 to 160 μm in brain (5–7) and 30–50 μm in the peripheral system (8), these appear to be grossly overestimated (9). Significantly lower H₂S concentrations of 17 and 14 nm in liver and brain, respectively, have been reported recently (9). The very significant discrepancy between these and the previous estimates of H₂S levels presumably derives from the earlier use of acidic conditions that led to the release of acid-labile sulfur from iron-sulfur centers.In mammals, the primary catalysts for H₂S generation are reported to be the two pyridoxal phosphate (PLP)-dependent enzymes involved in the trans-sulfuration pathway, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) (10, 11). The trans-sulfuration pathway operates in the reverse direction in mammals serving to convert homocysteine to cysteine (Fig. 1), although in yeast and bacteria the pathway is involved in sulfur assimilation from sulfate to cysteine. CBS is widely assumed to be the major contributor to H₂S production in the brain because of its relatively high expression in this organ (10). However, a recent study reported that 3-mercaptopyruvate sulfurtransferase together with cysteine aminotransferase might also generate H₂S in brain (12). The relative contributions of these enzymes and of CSE, which is also present in brain (13, 14), to H₂S production remain to be assessed. Genetic disruption of CSE in mouse leads to cardiac deficits, including pronounced hypertension and reduced endothelium-dependent vasorelaxation, consistent with a major role for CSE in the peripheral system (1). However, brain H₂S levels are reportedly unchanged in CSE^−/− mice.Open in a separate windowFIGURE 1.Diversity of reactions catalyzed by the trans-sulfuration pathway. The turnover numbers (v/[E]) estimated at physiological substrate concentrations, i.e. 10 μm homocysteine, 100 μm cysteine, 560 μm serine, and 5 μm cystathionine, are shown in parentheses for each reaction. The thick arrows highlight reactions that are sensitive to elevated levels of homocysteine. The fold change represents the fold increase in the turnover number of a given reaction under conditions of severe hyperhomocysteinemia (200 μm homocysteine).Despite the growing recognition of the varied physiological effects of H₂S, our understanding of its regulation and mechanism of its biosynthesis is poor. We have recently reported on the complex kinetics of H₂S generation by human CSE (15). The profligacy of the human enzyme affords H₂S generation by a multiplicity of routes involving cysteine and/or homocysteine as substrates. Kinetic simulations predict an increasingly important contribution of homocysteine to H₂S generation with increasing grade of hyperhomocysteinemia, a risk factor for cardiovascular and neurodegenerative diseases (16–18). In addition to H₂S, a variety of products is generated in these reactions, including two novel sulfur metabolites, lanthionine and homolanthionine, which represent the condensation products between 2 mol of cysteine and homocysteine, respectively. Although the steady-state kinetic parameters for H₂S generation from cysteine and homocysteine have been reported for human CBS (hCBS) (19), a comparable detailed kinetic analysis of H₂S generation by CBS by multiple pathways and their sensitivity to the grade of hyperhomocysteinemia is not known. Furthermore, the relative contributions of CBS and CSE to H₂S and lanthionine generation at physiologically relevant concentrations of substrate are not known.Human CBS is a unique heme containing PLP-dependent enzyme (20) that catalyzes the β-replacement of serine by homocysteine to produce cystathionine. The latter is further metabolized by CSE in an α,γ-elimination reaction to produce cysteine. Although yeast and human CBS are highly homologous and catalyze the same chemical reaction with similar kinetic parameters, the yeast enzyme lacks heme and is not allosterically regulated by S-adenosylmethionine (AdoMet) (21).In this study, we have elucidated the kinetics of H₂S biogenesis by yeast and human CBS and used simulations to estimate the relative contributions of CBS and CSE to H₂S production at physiologically relevant concentrations of substrate. We find that CBS and CSE share a common feature, i.e. catalytic promiscuity. However, in contrast to CSE, which is proficient at catalyzing reactions at the β- and γ-carbons of substrates (15), CBS activity is confined to chemical transformations at the β-position. Our studies provide new insights into the existence of alternative trans-sulfuration reactions that can be a source of diverse sulfur metabolites, viz. H₂S, lanthionine, and homolanthionine increasing the diversity of the sulfur metabolome.     

The Regulation of l-Methionine Synthesis and the Properties of Cystathionine γ-Synthase and β-Cystathionase in Corynebacterimn glutamicum

Cystathionine γ-synthase and β-cystathionase activities were found to be present in cell-free extract of Corynebacterium glutamicum. The reactions catalyzed by cystathionine γ-synthase and β-cystathionase were characterized with respect to Michaelis constant, pH optimum, incubation time and optimal enzyme concentration. Cystathionine γ-synthase was sensitive to the inhibition by S-adenosylmethionine. Formation of cystathionine γ-synthase and β-cystathionase was repressed by the addition of methionine to the growth medium although this repression appeared to be non-coordinate.

The regulation of methionine biosynthesis in C. glutamicum was discussed on the basis of these findings.     

Cystathionine β-Synthase Inhibition Is a Potential Therapeutic Approach to Treatment of Ischemic Injury

Hydrogen sulfide (H₂S) has been reported to exacerbate stroke outcome in experimental models. Cystathionine β-synthase (CBS) has been implicated as the predominant H₂S-producing enzyme in central nervous system. When SH-SY5Y cells were transfected to overexpress CBS, these cells were able to synthesize H₂S when exposed to high levels of enzyme substrates but not substrate concentrations that may reflect normal physiological conditions. At the same time, these cells demonstrated exacerbated cell death when subjected to oxygen and glucose deprivation (OGD) together with high substrate concentrations, indicating that H₂S production has a detrimental effect on cell survival. This effect could be abolished by CBS inhibition. The same effect was observed with primary astrocytes exposed to OGD and high substrates or sodium hydrosulfide. In addition, CBS was upregulated and activated by truncation in primary astrocytes subjected to OGD. When rats were subjected to permanent middle cerebral artery occlusion, CBS activation was also observed. These results imply that in acute ischemic conditions, CBS is upregulated and activated by truncation causing an increased production of H₂S, which exacerbate the ischemic injuries. Therefore, CBS inhibition may be a viable approach to stroke treatment.     

Cystathionine β-Synthase (CBS) Domains Confer Multiple Forms of Mg2+-dependent Cooperativity to Family II Pyrophosphatases

Regulated family II pyrophosphatases (CBS-PPases) contain a nucleotide-binding insert comprising a pair of cystathionine β-synthase (CBS) domains, termed a Bateman module. By binding with high affinity to the CBS domains, AMP and ADP usually inhibit the enzyme, whereas ATP activates it. Here, we demonstrate that AMP, ADP, and ATP bind in a positively cooperative manner to CBS-PPases from four bacteria: Desulfitobacterium hafniense, Clostridium novyi, Clostridium perfringens, and Eggerthella lenta. Enzyme interaction with substrate as characterized by the Michaelis constant (K_m) also exhibited positive catalytic cooperativity that decreased in magnitude upon nucleotide binding. The degree of both types of cooperativity increased with increasing concentration of the cofactor Mg²⁺ except for the C. novyi PPase where Mg²⁺ produced the opposite effect on kinetic cooperativity. Further exceptions from these general rules were ADP binding to C. novyi PPase and AMP binding to E. lenta PPase, neither of which had any effect on activity. A genetically engineered deletion variant of D. hafniense PPase lacking the regulatory insert was fully active but differed from the wild-type enzyme in that it was insensitive to nucleotides and bound substrate non-cooperatively and with a smaller K_m value. These results indicate that the regulatory insert acts as an internal inhibitor and confers dual positive cooperativity to CBS domain-containing PPases, making them highly sensitive regulators of the PP_i level in response to the changes in cell energy status that control adenine nucleotide distribution. These regulatory features may be common among other CBS domain-containing proteins.     

Cystathionine β-Synthase in the Brain of the Trout Oncorhynchus mykiss after Unilateral Eye Damage and in Conditions of in vitro Cultivation

Russian Journal of Developmental Biology - Expression of cystathionine β-synthase (CBS) in the brain of adult trout under normal conditions and 1 week after an eye injury was assessed using...     

Characterization of Cystathionine β-Synthase TtCbs1 and Cysteine Synthase TtCsa1 Involved in Cysteine Biosynthesis in Tetrahymena thermophila

Cysteine is implicated in important biological processes. It is synthesized through two different pathways. Cystathionine β-synthase and cystathionine γ-lyase participate in the reverse transsulfuration pathway, while serine acetyltransferase and cysteine synthase function in the de novo pathway. Two evolutionarily related pyridoxal 5′-phosphate-dependent enzymes, cystathionine β-synthase TtCBS1 (TTHERM_00558300) and cysteine synthase TtCSA1 (TTHERM_00239430), were identified from a freshwater protozoan Tetrahymena thermophila. TtCbs1 contained the N-terminal heme binding domain, catalytic domain, and C-terminal regulatory domain, whereas TtCsa1 consisted of two α/β domains. The catalytic core of the two enzymes is similar. TtCBS1 and TtCSA1 showed high expression levels in the vegetative growth stage and decreased during the sexual developmental stage. TtCbs1 and TtCsa1 were localized in the cytoplasm throughout different developmental stages. His-TtCbs1 and His-TtCsa1 were expressed and purified in vitro. TtCbs1 catalyzed the canonical reaction with the highest velocity and possessed serine sulfhydrylase activity. TtCsa1 showed cysteine synthase activity with high K_m for O-acetylserine and low K_m for sulfide and also had serine sulfhydrylase activity toward serine. Both TtCbs1 and TtCsa1 catalyzed hydrogen sulfide producing. TtCBS1 knockdown and TtCSA1 knockout mutants affected cysteine and glutathione synthesis. TtCbs1 and TtCsa1 are involved in cysteine synthesis through two different pathways in T. thermophila.     

Cloning of the cDNA and Genes for the Hamster and Human β2-Adrenergic Receptors

Abstract

The adenylate cyclase-stimulatory β₂-adrenergic receptor has been purified to apparent homogeneity from hamster lung. Partial amino acid sequence obtained from isolated CNBr peptides was used to clone the gene and cDNA for this receptor. The predicted amino acid sequence for the hamster β₂-adrenergic receptor revealed that the protein consists of a single polypeptide chain of 418 aa with consensus N-glycosylation and phosphorylation sites predicted by previous in vitro data. The most striking feature of the receptor protein however, is that it contains seven stretches of hydrophobic residues similar to the proposed seven transmembrane segments of the light receptor rhodopsin. Significant amino acid homology (30-35%) can be found between the hamster β₂-adrenergic receptor and rhodopsin within these putative membrane spanning regions. Using a hamster β₂-adrenergic receptor probe, the gene and cDNA for the human β₂-adrenergic receptor were isolated, revealing a high degree of homology (87%) between the two proteins from different species. Unlike the genes encoding the family of opsin pigments, of which rhodopsin is a member, the genes encoding both hamster and human β₂-adrenergic receptors are devoid of introns in their coding as well as 5′ and 3′ untranslated nucleotide sequences. The cloning of the genes and the elucidation of the aa sequences for these G-protein coupled receptors should help to determine the structure-function as well as the evolutionary relationship of these proteins.