Regulation of Human Cystathionine β-Synthase by <i>S</i>-Adenosyl-<scp>l</scp>-methionine:  Evidence for Two Catalytically Active Conformations Involving an Autoinhibitory Domain in the C-Terminal Region

Jánošík, Miroslav; Kéry, Vladimír; Gaustadnes, Mette; Maclean, Kenneth N.; Kraus, Jan P.

doi:10.1021/bi010711p

Cited by 158 publications

(228 citation statements)

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“…However, the kinetic values published later for wild type CBS [20] are very different from both our previous [21] and the current study. We attribute the differences in kinetic values to the variations in the N-terminal regions of these recombinant proteins.…”

Section: Activation Of Wild Type Cbs By Adomet and Adomet Analogs-adometcontrasting

confidence: 99%

“…3). This result is very similar to the ones we previously published [20,33]. At the same concentration, neither Sinefungin, nor AdoHcy had any effect on CBS activity (Fig.…”

Section: Nih-pa Author Manuscriptsupporting

confidence: 92%

“…The level of activation is comparable to that induced by partial thermal denaturation [20] or limited proteolysis [21].…”

Section: Activation Of Wild Type Cbs By Adomet and Adomet Analogs-adometmentioning

confidence: 68%

“…activates the mammalian CBS enzyme by as much as 5-fold with an apparent K act value of 15-18 μM [20,33]. The level of activation is comparable to that induced by partial thermal denaturation [20] or limited proteolysis [21].…”

Section: Activation Of Wild Type Cbs By Adomet and Adomet Analogs-adometmentioning

confidence: 92%

“…Subsequently, we demonstrated that N-terminal elongation altered the affinity of the enzyme for Hcy. This enzyme had ~9-fold lower K m for Hcy [20] compared to the WT enzyme [21] and was used to obtain the first CBS structure [22]. Here we describe a method to purify CBS using a GST fusion protein which yields CBS as close to normal sequence as possible.…”

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confidence: 99%

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Purification and characterization of the wild type and truncated human cystathionine β-synthase enzymes expressed in E. coli

Frank

Kent

Meier

et al. 2008

Archives of Biochemistry and Biophysics

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In this paper, we describe the expression and characterization of recombinant human cystathionine β-synthase (CBS) in Escherichia coli. We have used a glutathione-S-transferase (GST) fusion protein vector and incorporated a cleavage site with a long hinge region which allows for the independent folding of CBS and its fusion partner. In addition, our construct has the added benefit of yielding a purified CBS which only contains one extra glycine amino acid residue at the Nterminus. In our two-step purification procedure we are able to obtain a highly pure enzyme in sufficient quantities for crystallography and other physical chemical methods. We have investigated the biochemical and catalytic properties of purified full-length human CBS and of two truncation mutants lacking the Cterminal domain or both the N-terminal heme-binding and the C-terminal regulatory regions. Specifically, we have determined the pH optima of the different CBS forms and their kinetic and spectral properties. The full-length and the C-terminally truncated enzyme had a broad pH 8.5 optimum while the pH optimum of the N-and C-terminally truncated enzyme was sharp and shifted to pH 9. Furthermore, we have shown unequivocally that CBS binds one mole of heme per subunit by determining both the heme and the iron content of the enzyme. The activity of the enzyme was unaffected by the redox status of the heme iron. Finally, we show that CBS is stimulated by S-adenosyl-L-methionine but not its analogs. KeywordsHomocystinuria; S-adenosylmethionine; Pyridoxal-5′ phosphate; Heme Cystathionine β-synthase (CBS) 2 (L-Serine hydrolyase (adding homocysteine), EC 4.2.1.22) is central to sulfur amino acid metabolism in eukaryotes, and complete CBS deficiency is the principal cause of homocystinuria in humans. The CBS-catalyzed condensation of serine and homocysteine generates cystathionine which is then cleaved with cystathionine γ-lyase (CGL) to yield cysteine [1].Cysteine synthase (O-acetylserine sulfhydrase, OASS; CS), the CBS counterpart in prokaryotes, plants and enteric protozoa, catalyzes the formation of cysteine from Oacetylserine and hydrogen sulfide (H 2 S) with the concomitant release of acetic acid. Unlike mammals, these organisms are able to assimilate inorganic sulfur into organic sulfur and are therefore essential players in the sulfur cycle in nature. CS and CBS are affiliated with the large β-family of pyridoxal-5′-phosphate (PLP) dependent enzymes and have a high degree * Corresponding author. Fax: +1 303 724 3838. Jan.Kraus@UCHSC.edu (J.P. Kraus). 1 These authors contributed equally to this work.2 Abbreviations used: CBS, Cystathionine β-synthase; GST, glutathione S-transferase; PLP, pyridoxal 5′-phosphate; AdoMet, Sadenosyl-L-methionine; CS, cysteine synthase; OASS, O-acetylserine sulfhydrase; β-gal, β-galactosidase; OAS, O-acetyl serine; AdoHcy, S-adenosylhomocysteine. CBS is the only PLP dependent enzyme which contains heme [7]. The heme is co-ordinated by Cys 52 and His 65 residues located in the N-terminal region of the enzym...

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Section: Activation Of Wild Type Cbs By Adomet and Adomet Analogs-adometcontrasting

confidence: 99%

“…3). This result is very similar to the ones we previously published [20,33]. At the same concentration, neither Sinefungin, nor AdoHcy had any effect on CBS activity (Fig.…”

Section: Nih-pa Author Manuscriptsupporting

confidence: 92%

“…The level of activation is comparable to that induced by partial thermal denaturation [20] or limited proteolysis [21].…”

Section: Activation Of Wild Type Cbs By Adomet and Adomet Analogs-adometmentioning

confidence: 68%

Section: Activation Of Wild Type Cbs By Adomet and Adomet Analogs-adometmentioning

confidence: 92%

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Purification and characterization of the wild type and truncated human cystathionine β-synthase enzymes expressed in E. coli

Frank

Kent

Meier

et al. 2008

Archives of Biochemistry and Biophysics

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Cystathionine β‐Synthase (CBS) Deficiency: Genetics

Kožich¹,

Kraus²

2006

Encyclopedia of Life Sciences

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Cystathionine β‐synthase is an enzyme that catalyzes the condensation of homocysteine and serine to cystathionine. Its deficiency, an autosomal recessive trait resembling Marfan syndrome, is caused by more than 120 different mutations that affect ribonucleic acid processing or properties of the enzyme. The most common pathogenic mutation, c.833T > C, arose by a complex evolutionary process as evidenced by its relatedness to the alleles that contain the frequent polymorphism 844ins68.

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Cystathionine β‐synthase (CBS) Deficiency: Genetics

Kožich¹,

Kruger²,

Kraus³

2010

Encyclopedia of Life Sciences

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Cystathionine β‐synthase (CBS) is an enzyme that catalyses condensation of homocysteine and serine to cystathionine. CBS deficiency, an autosomal recessive trait with estimated population frequency of around 1:10 000–1:20 000, resembles in the most severe forms Marfan syndrome with thromboembolism and neurological impairment, whereas milder forms may manifest with only thromboembolism or may remain asymptomatic. Laboratory findings include grossly elevated plasma total homocysteine and in part of patients also elevated blood methionine; the latter feature is utilised in neonatal screening. CBS binds three cofactors: pyridoxal 5′‐phosphate, an allosteric activator S ‐adenosylmethionine and haem with as yet unresolved function. In the CBS gene, more than 150 different mutations have been described to date, almost 90% of them are missense variants. Although some of the mutations affect ribonucleic acid processing and its stability, the majority of mutations lead to enzyme misfolding and misassembly, which may be in part rescued by chemical or molecular chaperones. Key Concepts: Cystathionine β‐synthase catalyses the first step in homocysteine trans ‐sulfuration. Cystathionine β‐synthase is a modular enzyme composed of a haem‐binding N ‐terminal domain, the catalytically active core and the C ‐terminal autoinhibitory domain. More than 150 different pathogenic mutations have been described in the CBS gene, almost 90% of all mutant CBS alleles carry missense mutations. Misfolding and misassembly of mutant CBS subunits is a common pathogenic mechanism leading to CBS deficiency. Mutation topology predicts in part the behaviour of mutant CBS enzymes, solvent accessible mutations have less severe effect than those buried in the enzyme globule. Deficient CBS activity leads to gross elevation of plasma total homocysteine and often to elevated blood methionine levels. Phenotypic consequences of mutations in the CBS gene include thromboembolism and vascular occlusion, which is accompanied in some patients by lens dislocation, marfanoid features and varying degree of neurological involvement. Population frequency of clinically ascertained patients with CBS deficiency is one to two orders of magnitude lower than the frequency calculated from the prevalence of heterozygotes for pathogenic mutations; this observation indicates an ascertainment bias or lack of symptoms in many CBS‐deficient individuals. Murine models of CBS deficiency recapitulate in part the pathophysiology and organ involvement observed in human patients.

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Regulation of Human Cystathionine β-Synthase by S-Adenosyl-l-methionine: Evidence for Two Catalytically Active Conformations Involving an Autoinhibitory Domain in the C-Terminal Region

Cited by 158 publications

References 33 publications

Purification and characterization of the wild type and truncated human cystathionine β-synthase enzymes expressed in E. coli

Purification and characterization of the wild type and truncated human cystathionine β-synthase enzymes expressed in E. coli

Cystathionine β‐Synthase (CBS) Deficiency: Genetics

Cystathionine β‐synthase (CBS) Deficiency: Genetics

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