Protein misfolding due to missense mutations is a common pathogenic mechanism in cystathionine β-synthase (CBS) deficiency. In our previous studies, we successfully expressed, purified, and characterized nine CBS mutant enzymes containing the following patient mutations: P49L, P78R, A114V, R125Q, E176K, R266K, P422L, I435T, and S466L. These purified mutants exhibited full heme saturation, normal tetrameric assembly, and high catalytic activity. In this work, we used several spectroscopic and proteolytic techniques to provide a more thorough insight into the conformation of these mutant enzymes. Far-UV circular dichroism, fluorescence, and second-derivative UV spectroscopy revealed that the spatial arrangement of these CBS mutants is similar to that of the wild type, although the microenvironment of the chromophores may be slightly altered. Using proteolysis with thermolysin under native conditions, we found that the majority of the studied mutants is more susceptible to cleavage, suggesting their increased local flexibility or propensity for local unfolding. Interestingly, the presence of the CBS allosteric activator, S-adenosylmethionine (AdoMet), increased the rate of cleavage of the wild type and the AdoMet-responsive mutants, while the proteolytic rate of the AdoMet-unresponsive mutants was not significantly changed. Pulse proteolysis analysis suggested that the protein structure of the R125Q and E176K mutants is significantly less stable than that of the wild type and the other mutants. Taken together, the proteolytic data shows that the conformation of the pathogenic mutants is altered despite retained catalytic activity and normal tetrameric assembly. This study demonstrates that the proteolytic techniques are useful tools for the assessment of the biochemical penalty of missense mutations in CBS.

Charles University Prague Czech Republic

Biochemistry. 2012 Jul 10;51(27):5540. doi: 10.1021/bi300812f. PubMed

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Mudd SH, Levy HL, Kraus JP. Disorders of transsulfuration. In: Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Kinzler K, Vogelstein B, editors. The Metabolic and Molecular Bases of Inherited Disease. 8. McGraw-Hill; New York: 2001.

Gaustadnes M, Ingerslev J, Rutiger N. Prevalence of congenital homocystinuria in Denmark. N Engl J Med. 1999;340:1513. PubMed

Refsum H, Fredriksen A, Meyer K, Ueland PM, Kase BF. Birth prevalence of homocystinuria. J Pediatr. 2004;144:830–832. PubMed

Janosik M, Sokolova J, Janosikova B, Krijt J, Klatovska V, Kozich V. Birth prevalence of homocystinuria in Central Europe: frequency and pathogenicity of mutation c. 1105C>T (p R369C) in the cystathionine beta-synthase gene. J Pediatr. 2009;154:431–437. PubMed PMC

Linnebank M, Homberger A, Kraus JP, Harms E, Kozich V, Koch HG. Haplotyping of wild type and I278T alleles of the human cystathionine beta-synthase gene based on a cluster of novel SNPs in IVS12. Hum Mutat. 2001;17:350–351. PubMed

Miles EW, Kraus JP. Cystathionine beta-synthase: structure, function, regulation, and location of homocystinuria-causing mutations. J Biol Chem. 2004;279:29871–29874. PubMed

Singh S, Madzelan P, Banerjee R. Properties of an unusual heme cofactor in PLP-dependent cystathionine beta-synthase. Nat Prod Rep. 2007;24:631–639. PubMed

Janosik M, Oliveriusova J, Janosikova B, Sokolova J, Kraus E, Kraus JP, Kozich V. Impaired heme binding and aggregation of mutant cystathionine beta-synthase subunits in homocystinuria. Am J Hum Genet. 2001;68:1506–1513. PubMed PMC

Singh LR, Chen X, Kozich V, Kruger WD. Chemical chaperone rescue of mutant human cystathionine beta-synthase. Mol Genet Metab. 2007;91:335–342. PubMed PMC

Singh LR, Kruger WD. Functional rescue of mutant human cystathionine beta-synthase by manipulation of Hsp26 and Hsp70 levels in Saccharomyces cerevisiae. J Biol Chem. 2009;284:4238–4245. PubMed PMC

Kopecka J, Krijt J, Rakova K, Kozich V. Restoring assembly and activity of cystathionine beta-synthase mutants by ligands and chemical chaperones. J Inherit Metab Dis. 2011;34:39–48. PubMed PMC

Majtan T, Liu L, Carpenter JF, Kraus JP. Rescue of cystathionine beta-synthase (CBS) mutants with chemical chaperones: purification and characterization of eight CBS mutant enzymes. J Biol Chem. 2010;285:15866–15873. PubMed PMC

Park C, Marqusee S. Probing the high energy states in proteins by proteolysis. J Mol Biol. 2004;343:1467–1476. PubMed

Park C, Marqusee S. Pulse proteolysis: a simple method for quantitative determination of protein stability and ligand binding. Nat Methods. 2005;2:207–212. PubMed

Young TA, Skordalakes E, Marqusee S. Comparison of proteolytic susceptibility in phosphoglycerate kinases from yeast and E. coli: modulation of conformational ensembles without altering structure or stability. J Mol Biol. 2007;368:1438–1447. PubMed

Hnizda A, Jurga V, Rakova K, Kozich V. Cystathionine beta-synthase mutants exhibit changes in protein unfolding: conformational analysis of misfolded variants in crude cell extracts. J Inherit Metab Dis. 2012;35:469–477. PubMed PMC

Majtan T, Kraus JP. Folding and activity of mutant cystathionine beta-synthase depends on the position and nature of the purification tag: Characterization of the R266K CBS mutant. Protein Expr Purif. 2012;82:317–324. PubMed PMC

Hnizda A, Spiwok V, Jurga V, Kozich V, Kodicek M, Kraus JP. Cross-talk between the catalytic core and the regulatory domain in cystathionine beta-synthase: study by differential covalent labeling and computational modeling. Biochemistry. 2010;49:10526–10534. PubMed PMC

Park C, Marqusee S. Quantitative determination of protein stability and ligand binding by pulse proteolysis. In: Coligan JE, Dunn BM, Speicher DW, Wingfield PT, Ploegh HL, editors. Current protocols in protein science. Wiley; Hoboken: 2006. pp. 20.11.1–20.11.14. PubMed

Hnizda A, Santrucek J, Sanda M, Strohalm M, Kodicek M. Reactivity of histidine and lysine side-chains with diethylpyrocarbonate -- a method to identify surface exposed residues in proteins. J Biochem Biophys Methods. 2008;70:1091–1097. PubMed

Meier M, Janosik M, Kery V, Kraus JP, Burkhard P. Structure of human cystathionine beta-synthase: a unique pyridoxal 5′-phosphate-dependent heme protein. Embo J. 2001;20:3910–3916. PubMed PMC

Meier M, Oliveriusova J, Kraus JP, Burkhard P. Structural insights into mutations of cystathionine beta-synthase. Biochim Biophys Acta. 2003;1647:206–213. PubMed

Willaert K, Engelborghs KW. The quenching of tryptophan fluorescence by protonated and unprotonated imidazole. Eur Biophys J. 1991;20:177–182.

Kery V, Poneleit L, Meyer JD, Manning MC, Kraus JP. Binding of pyridoxal 5′-phosphate to the heme protein human cystathionine beta-synthase. Biochemistry. 1999;38:2716–2724. PubMed

Mach H, Middaugh CR. Simultaneous monitoring of the environment of tryptophan, tyrosine, and phenylalanine residues in proteins by near-ultraviolet second-derivative spectroscopy. Anal Biochem. 1994;222:323–331. PubMed

Prudova A, Bauman Z, Braun A, Vitvitsky V, Lu SC, Banerjee R. S-adenosylmethionine stabilizes cystathionine beta-synthase and modulates redox capacity. Proc Natl Acad Sci U S A. 2006;103:6489–6494. PubMed PMC

Wetlaufer DB. Ultraviolet spectra of proteins and amino acids. Adv Protein Chem. 1962;17:303–390. PubMed

Singh LR, Gupta S, Honig NH, Kraus JP, Kruger WD. Activation of mutant enzyme function in vivo by proteasome inhibitors and treatments that induce Hsp70. PLoS Genet. 2010;6:e1000807. PubMed PMC

Janosik M, Kery V, Gaustadnes M, Maclean KN, Kraus JP. Regulation of human cystathionine beta-synthase by S-adenosyl-L-methionine: evidence for two catalytically active conformations involving an autoinhibitory domain in the C-terminal region. Biochemistry. 2001;40:10625–10633. PubMed

Sen S, Yu J, Yamanishi M, Schellhorn D, Banerjee R. Mapping peptides correlated with transmission of intrasteric inhibition and allosteric activation in human cystathionine beta-synthase. Biochemistry. 2005;44:14210–14216. PubMed

Koutmos M, Kabil O, Smith JL, Banerjee R. Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine {beta}-synthase. Proc Natl Acad Sci U S A. 2010;107:20958–20963. PubMed PMC

Gupta S, Wang L, Hua X, Krijt J, Kozich V, Kruger WD. Cystathionine beta-synthase p. S466L mutation causes hyperhomocysteinemia in mice. Hum Mutat. 2008;29:1048–1054. PubMed PMC

Kozich V, Sokolova J, Klatovska V, Krijt J, Janosik M, Jelinek K, Kraus JP. Cystathionine beta-synthase mutations: effect of mutation topology on folding and activity. Hum Mutat. 2010;31:809–819. PubMed PMC

Sen S, Banerjee R. 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. Biochemistry. 2007;46:4110–4116. PubMed PMC

Waugh DS. Making the most of affinity tags. Trends Biotechnol. 2005;23:316–320. PubMed

A proactive genotype-to-patient-phenotype map for cystathionine beta-synthase