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Query: EC:4.2.1.22 (cystathionine beta-synthase)
 965 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cystathionine beta-synthase is a unique heme protein that catalyzes a pyridoxal phosphate (or PLP)-dependent beta-replacement reaction. The reaction involves the condensation of serine and homocysteine and constitutes one of the two major avenues for detoxification of homocysteine in mammals. The enzyme is allosterically regulated by S-adenosylmethionine (AdoMet). In this study, we have characterized the kinetic, spectroscopic, and ligand binding properties of a truncated catalytic core of cystathionine beta-synthase extending from residues 1 through 408 in which the C-terminal 143 residues have been deleted. This is similar to a natural variant of the protein that has been described in a homocystinuric patient in which the predicted peptide is 419 amino acids in length. Truncation leads to the formation of a dimeric enzyme in contrast to the tetrameric organization of the native enzyme. Some of the kinetic properties of the truncated enzyme are different from the full-length form, most notably, significantly higher K(m)s for the two substrates, and loss of activation by AdoMet. This is paralleled by the absence of AdoMet binding to the truncated form, whereas four AdoMet molecules bind cooperatively to the full-length tetrameric enzyme with a K(d) of 7. 4 microM. Steady-state kinetic analysis indicates that the order of substrate addition is important. Thus, preincubation of the enzyme with homocysteine leads to a 2-fold increase in V(max) relative to preincubation of the enzyme with serine. Since the intracellular concentration of serine is significantly greater than that of homocysteine, the physiological significance of this phenomenon needs to be considered. Based on ligand binding studies and homology searches with protein sequences in the database, we assign residues 68-209 as being important for PLP binding, residues 241-341 for heme binding, and residues 421-469 for AdoMet binding.
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PMID:Assignment of enzymatic functions to specific regions of the PLP-dependent heme protein cystathionine beta-synthase. 1052 87

Homocysteine is a key junction metabolite that can be converted to cystathionine in a reaction catalyzed by the heme and pyridoxal phosphate-dependent cystathionine beta-synthase. The heme has unusual spectroscopic properties and the axial ligands have been assigned as histidine and cysteine, respectively. Its role in the protein is not obvious from the chemistry of the beta-replacement reaction that is catalyzed. We have characterized the binding of the gaseous signaling molecule, NO, to cystathionine beta-synthase and examined its effect on the reactions catalyzed by the truncated dimeric form of the enzyme, W409X, which is a natural variant. Binding of NO appears to result in the formation of a five-coordinate ferrous nitrosyl species in which both endogenous ligands have been lost. This is in contrast to CO binding which is reported to displace the thiolate ligand and form a six-coordinate species. NO binds to the full-length enzyme with a K(d) of 281+/-50 microM and to the truncated enzyme with a K(d) of 350+/-44 microM. Binding of NO to the full-length enzyme inhibits activity with a K(i) of 320+/-60 microM. These studies demonstrate that as with CO, perturbation of the heme environment by NO is communicated to the active site with concomitant inhibition of enzyme activity, and suggests a regulatory role for heme in cystathionine beta-synthase.
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PMID:Characterization of NO binding to human cystathionine beta-synthase: possible implications of the effects of CO and NO binding to the human enzyme. 1174 62

All eukaryotic CLC Cl(-) channel subunits possess a long cytoplasmic carboxy-terminus that contains two so-called CBS (cystathionine beta-synthase) domains. These domains are found in various unrelated proteins from all phylae. The crystal structure of the CBS domains of inosine monophosphate dehydrogenase (IMPDH) is known, but it is not known whether this structure is conserved in CLC channels. Working primarily with ClC-1, we used deletion scanning mutagenesis, coimmunoprecipitation and electrophysiology to demonstrate that its CBS domains interact. The replacement of CBS domains of ClC-1 with the corresponding CBS domains from other CLC channels and even human IMPDH yielded functional channels, indicating a high degree of structural conservation. Based on a homology model of the pair of CBS domains of CLC channels, we identified some residues that, when mutated, affected the common gate which acts on both pores of the dimeric channel. Thus, we propose that the structure of CBS domains from CLC channels is highly conserved and that they play a functional role in the common gate.
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PMID:Functional and structural conservation of CBS domains from CLC chloride channels. 1472 90

Cystathionine beta-synthase (CBS) condenses homocysteine, a toxic metabolite, with serine in a pyridoxal phosphate-dependent reaction. It also contains a heme cofactor to which carbon monoxide (CO) or nitric oxide can bind, resulting in enzyme inhibition. To understand the mechanism of this regulation, we have investigated the equilibria and kinetics of CO binding to the highly active catalytic core of CBS, which is dimeric. CBS exhibits strong anticooperativity in CO binding with successive association constants of 0.24 and 0.02 microm(-1). Stopped flow measurements reveal slow CO association (0.0166 s(-1)) limited by dissociation of the endogenous ligand, Cys-52. Rebinding of CO and of Cys-52 following CO photodissociation were independently monitored via time-resolved resonance Raman spectroscopy. The Cys-52 rebinding rate, 4000 s(-1), is essentially unchanged between pH 7.6 and 10.5, indicating that the pK(a) of Cys-52 is shifted below pH 7.6. This effect is attributed to the nearby Arg-266 residue, which is proposed to form a salt bridge with the dissociated Cys-52, thereby inhibiting its protonation and slowing rebinding to the Fe. This salt bridge suggests a pathway for enzyme inactivation upon CO binding, because Arg-266 is located on a helix that connects the heme and pyridoxal phosphate cofactor domains.
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PMID:Dynamics of carbon monoxide binding to cystathionine beta-synthase. 1650 79

Cystathionine beta-synthase catalyzes the condensation of serine and homocysteine to yield cystathionine and is the single most common locus of mutations associated with homocystinuria. In this study, we have examined the kinetic consequences of a pair of linked patient mutations, P78R/K102N, that are housed in the catalytic core of the protein and compared it to the effects of the corresponding single mutations. The P78R mutation affords purification of a mixture of higher order oligomers, P78R-I, which resembles the mixed quaternary state associated with wild-type enzyme. However, unlike wild-type enzyme, P78R-I converts over time to P78R-II, which exists predominantly as a full-length dimer. The specific activities of the K102N, P78R-I, and P78R-II mutants in the absence of AdoMet are approximately 3-, 9-, and 3-fold lower than of wild-type enzyme and are stimulated 2.9-, 2.5-, and 1.4-fold respectively by AdoMet. However, when linked, the specific activity of the resulting double mutant is comparable to that of wild-type enzyme but it is unresponsive to AdoMet, revealing that interactions between the two sites modulate the phenotype of the enzyme. Steady-state kinetic analysis for the double mutant reveals a sigmoidal dependence on homocysteine that is not observed with wild-type enzyme, which is ascribed to the mutation at the K102 locus and indicates changes in subunit interactions. Hydrogen-deuterium mass spectrometric analysis reveals that, even in the absence of AdoMet, the double mutant is locked in an activated conformation that is observed for wild-type enzyme in the presence of AdoMet, providing a structural rationale for loss of this allosteric regulation. To our knowledge, this is the first example of mutations in the catalytic core of cystathionine beta-synthase that result in failure of AdoMet-dependent regulation. Furthermore, analysis of individual single mutations has permitted, for the first time, partial kinetic characterization of a full-length dimeric form of human cystathionine beta-synthase.
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PMID:A pathogenic linked mutation in the catalytic core of human cystathionine beta-synthase disrupts allosteric regulation and allows kinetic characterization of a full-length dimer. 1735 95

We have identified a novel family of proteins, in which the N-terminal cystathionine beta-synthase (CBS) domain is fused to the C-terminal Zn ribbon domain. Four proteins were overexpressed in Escherichia coli and purified: TA0289 from Thermoplasma acidophilum, TV1335 from Thermoplasma volcanium, PF1953 from Pyrococcus furiosus, and PH0267 from Pyrococcus horikoshii. The purified proteins had a red/purple color in solution and an absorption spectrum typical of rubredoxins (Rds). Metal analysis of purified proteins revealed the presence of several metals, with iron and zinc being the most abundant metals (2-67% of iron and 12-74% of zinc). Crystal structures of both mercury- and iron-bound TA0289 (1.5-2.0 A resolution) revealed a dimeric protein whose intersubunit contacts are formed exclusively by the alpha-helices of two cystathionine beta-synthase subdomains, whereas the C-terminal domain has a classical Zn ribbon planar architecture. All proteins were reversibly reduced by chemical reductants (ascorbate or dithionite) or by the general Rd reductase NorW from E. coli in the presence of NADH. Reduced TA0289 was found to be capable of transferring electrons to cytochrome C from horse heart. Likewise, the purified Zn ribbon protein KTI11 from Saccharomyces cerevisiae had a purple color in solution and an Rd-like absorption spectrum, contained both iron and zinc, and was reduced by the Rd reductase NorW from E. coli. Thus, recombinant Zn ribbon domains from archaea and yeast demonstrate an Rd-like electron carrier activity in vitro. We suggest that, in vivo, some Zn ribbon domains might also bind iron and therefore possess an electron carrier activity, adding another physiological role to this large family of important proteins.
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PMID:Biochemical and structural characterization of a novel family of cystathionine beta-synthase domain proteins fused to a Zn ribbon-like domain. 1802

Cystathionine beta-synthase (CBS) catalyzes the condensation of serine and homocysteine to cystathionine, which represents the committing step in the transsulfuration pathway. CBS is unique in being a pyridoxal phosphate-dependent enzyme that has a heme cofactor. The activity of CBS under in vitro conditions is responsive to the redox state of the heme, which is distant from the active site and has been postulated to play a regulatory role. The heme in CBS is unusual; it is six-coordinate, low spin, and contains cysteine and histidine as axial ligands. In this study, we have assessed the redox behavior of a human CBS dimeric variant lacking the C-terminal regulatory domain. Potentiometric redox titrations showed a reversible response with a reduction potential of -291 +/- 5 mV versus the normal hydrogen electrode, at pH 7.2. Stopped-flow kinetic determinations demonstrated that Fe(II)CBS reacted with dioxygen yielding Fe(III)CBS without detectable formation of an intermediate species. A linear dependence of the apparent rate constant of Fe(II)CBS decay on dioxygen concentration was observed and yielded a second-order rate constant of (1.11 +/- 0.07) x 10 (5) M (-1) s (-1) at pH 7.4 and 25 degrees C for the direct reaction of Fe(II)CBS with dioxygen. A similar reactivity was observed for full-length CBS. Heme oxidation led to superoxide radical generation, which was detected by the superoxide dismutase (SOD)-inhibitable oxidation of epinephrine. Our results show that CBS may represent a previously unrecognized source of cytosolic superoxide radical.
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PMID:Dioxygen reactivity and heme redox potential of truncated human cystathionine beta-synthase. 1827 72

Cysteine plays a major role in the antioxidative defense mechanisms of the human parasite Entameoba histolytica. The major route of cysteine biosynthesis in this parasite is the condensation of O-acetylserine with sulfide by the de novo cysteine biosynthetic pathway involving two key enzymes O-acetyl-L-serine sulfhydrylase (OASS) and serine acetyl transferase (SAT). The crystal structure of native OASS from Entameoba histolytica (EhOASS) has been determined at 1.86 A resolution and in complex with its product cysteine at 2.4 A resolution. In comparison with other known OASS structures, insertion in the N-terminal region and C-terminal helix reveal critical differences, which may influence the protein-protein interactions. In spite of lacking chloride binding site at the dimeric interface, the N-terminal extension compared with other known cysteine synthases, participates in dimeric interactions in an interesting domain swapping manner, enabling it to form a stronger dimer. Sulfate is bound in the active site of the native structure, which is replaced by cysteine in the cysteine bound form causing reorientation of the small N-terminal domain and thus closure of the active site. Ligand binding constants of OAS, Cys, and Met with EhOASS are comparable with other known OASS indicating similar active site arrangement and dynamics. The cysteine complexed structure represents the snapshot of the enzyme just before releasing the final product with a closed active site. The C-terminal helix positioning in the EhOASS may effect its interactions with EhSAT and thus influencing the formation of the cysteine synthase complex in this organism.
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PMID:Crystal structure of native O-acetyl-serine sulfhydrylase from Entamoeba histolytica and its complex with cysteine: structural evidence for cysteine binding and lack of interactions with serine acetyl transferase. 1835 May 70

Cystathionine beta-synthase domains are found in a myriad of proteins from organisms across the tree of life and have been hypothesized to function as regulatory modules that sense the energy charge of cells. Here we characterize the structure and stability of PAE2072, a dimeric tandem cystathionine beta-synthase domain protein from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum. Crystal structures of the protein in unliganded and AMP-bound forms, determined at resolutions of 2.10 and 2.35 A, respectively, reveal remarkable conservation of key functional features seen in the gamma subunit of the eukaryotic AMP-activated protein kinase. The structures also confirm the presence of a suspected intermolecular disulfide bond between the two subunits that is shown to stabilize the protein. Our AMP-bound structure represents a first step in investigating the function of a large class of uncharacterized prokaryotic proteins. In addition, this work extends previous studies that have suggested that, in certain thermophilic microbes, disulfide bonds play a key role in stabilizing intracellular proteins and protein-protein complexes.
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PMID:Structures and functional implications of an AMP-binding cystathionine beta-synthase domain protein from a hyperthermophilic archaeon. 1851 46

A new crystal structure of the dimeric cysteine synthase CysM from Mycobacterium tuberculosis reveals an open and a closed conformation of the enzyme. In the closed conformation the five carboxy-terminal amino acid residues are inserted into the active site cleft. Removal of this segment results in a decreased lifetime of the alpha-aminoacrylate reaction intermediate, an increased sensitivity to oxidants such as hydrogen peroxide, and loss of substrate selectivity with respect to the sulfur carrier thiocarboxylated CysO. These results highlight features of CysM that might be of particular importance for cysteine biosynthesis under oxidative stress in M. tuberculosis.
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PMID:The C-terminal of CysM from Mycobacterium tuberculosis protects the aminoacrylate intermediate and is involved in sulfur donor selectivity. 1910 53


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