Rescue of Cystathionine β-Synthase (CBS) Mutants with Chemical Chaperones

Majtán, Tomáš; Liu, Lu; Carpenter, John F.; Kraus, Jan P.

doi:10.1074/jbc.m110.107722

Cited by 68 publications

(74 citation statements)

References 39 publications

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“…We have already demonstrated in our previous work, that a gradual thermal denaturation of hCBS resulted in enzyme activation due to irreversible denaturation of the regulatory domains [11], thus mimicking the stimulation by AdoMet [8], [25]. In order to assess the thermal stability of the purified CBS enzymes, we subjected dCBS and yCBS to a similar thermal pre-treatment assay ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Domain Organization, Catalysis and Regulation of Eukaryotic Cystathionine Beta-Synthases

et al. 2014

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Cystathionine beta-synthase (CBS) is a key regulator of sulfur amino acid metabolism diverting homocysteine, a toxic intermediate of the methionine cycle, via the transsulfuration pathway to the biosynthesis of cysteine. Although the pathway itself is well conserved among eukaryotes, properties of eukaryotic CBS enzymes vary greatly. Here we present a side-by-side biochemical and biophysical comparison of human (hCBS), fruit fly (dCBS) and yeast (yCBS) enzymes. Preparation and characterization of the full-length and truncated enzymes, lacking the regulatory domains, suggested that eukaryotic CBS exists in one of at least two significantly different conformations impacting the enzyme’s catalytic activity, oligomeric status and regulation. Truncation of hCBS and yCBS, but not dCBS, resulted in enzyme activation and formation of dimers compared to native tetramers. The dCBS and yCBS are not regulated by the allosteric activator of hCBS, S-adenosylmethionine (AdoMet); however, they have significantly higher specific activities in the canonical as well as alternative reactions compared to hCBS. Unlike yCBS, the heme-containing dCBS and hCBS showed increased thermal stability and retention of the enzyme’s catalytic activity. The mass-spectrometry analysis and isothermal titration calorimetry showed clear presence and binding of AdoMet to yCBS and hCBS, but not dCBS. However, the role of AdoMet binding to yCBS remains unclear, unlike its role in hCBS. This study provides valuable information for understanding the complexity of the domain organization, catalytic specificity and regulation among eukaryotic CBS enzymes.

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Section: Resultsmentioning

confidence: 99%

Domain Organization, Catalysis and Regulation of Eukaryotic Cystathionine Beta-Synthases

et al. 2014

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“…Human CBS (hCBS) was expressed and purified as previously described [9] and buffered exchanged to 20 mM Hepes pH 7.4 using PD-10 columns (GE Healthcare). Protein samples were concentrated to ~200 μM in subunit, flash frozen in liquid nitrogen and stored at −80°C.…”

Section: Methodsmentioning

confidence: 99%

“…Over 160 mutations in the CBS gene have been found to cause classical homocystinuria (HCU) ([5] and http://medschool.ucdenver.edu/krauslab). Missense mutations often lead to protein instability and impaired catalytic activity and regulation [1, 4, 6-9]. Some of the mutations responsible for HCU were modeled based on the structure of the truncated CBS [10, 11], in some cases providing a structural rationale for loss-of-function [12].…”

Section: Introductionmentioning

confidence: 99%

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

Pey

Majtán

Kraus

2014

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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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 is independently regulated by the binding of S-adenosyl-methionine (SAM) to two different type 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.

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“…Thus, most of the data supporting the positive effect of chemical chaperones on mutant CBS folding, assembly and catalytic activity were obtained by studying the overexpressed CBS in crude extracts [15, 17, 21]. We showed that eight CBS mutants, following expression in the presence of a chemical chaperone in the growth medium, were amenable to purification to homogeneity and thus allowed for detailed characterization [16]. We used our established system for protein expression and purification using glutathione-S-transferase (GST)-tagged CBS constructs and a two-column purification procedure including the capturing Glutathione Sepharose affinity and the polishing DEAE Sepharose anion exchange chromatography steps [16, 22, 23].…”

Section: Introductionmentioning

confidence: 99%

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

Majtán

Kraus²

2012

Protein Expression and Purification

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Cystathionine beta-synthase (CBS), a heme-containing 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.

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Rescue of Cystathionine β-Synthase (CBS) Mutants with Chemical Chaperones

Cited by 68 publications

References 39 publications

Domain Organization, Catalysis and Regulation of Eukaryotic Cystathionine Beta-Synthases

Domain Organization, Catalysis and Regulation of Eukaryotic Cystathionine Beta-Synthases

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

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

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