Journal of Molecular Medicine

, Volume 90, Issue 1, pp 81–88

A novel molecular mechanism to explain biotin-unresponsive holocarboxylase synthetase deficiency

  • Lungisa Mayende
  • Rachel D. Swift
  • Lisa M. Bailey
  • Tatiana P. Soares da Costa
  • John C. Wallace
  • Grant W. Booker
  • Steven W. Polyak
Original Article


Biotin (vitamins H and B7) is an important micronutrient as defects in its availability, metabolism or adsorption can cause serious illnesses, especially in the young. A key molecule in the biotin cycle is holocarboxylase synthetase (HLCS), which attaches biotin onto the biotin-dependent enzymes. Patients with congenital HLCS deficiency are prescribed oral biotin supplements that, in most cases, reverse the clinical symptoms. However, some patients respond poorly to biotin therapy and have an extremely poor long-term prognosis. Whilst a small number of mutations in the HLCS gene have been implicated, the molecular mechanisms that lead to the biotin-unresponsive phenotype are not understood. To improve our understanding of HLCS, limited proteolysis was performed together with yeast two-hybrid analysis. A structured domain within the N-terminal region that contained two missense mutations was identified in patients who were refractory to biotin therapy, namely p.L216R and p.L237P. Genetic studies demonstrated that the interaction between the enzyme and the protein substrate was disrupted by mutation. Further dissection of the binding mechanism using surface plasmon resonance demonstrated that the mutations reduced affinity for the substrate through a >15-fold increase in dissociation rate. Together, these data provide the first molecular explanation for HLCS-deficient patients that do not respond to biotin therapy.


Protein interaction Biotin protein ligase Holocarboxylase synthetase Multiple carboxylase deficiency Enzyme 

Supplementary material

109_2011_811_MOESM1_ESM.pdf (321 kb)
Supplementary Figure 1(PDF 321 kb)
109_2011_811_MOESM2_ESM.pdf (1.1 mb)
Supplementary Figure 2(PDF 1152 kb)
109_2011_811_MOESM3_ESM.pdf (763 kb)
Supplementary Figure 3(PDF 763 kb)
109_2011_811_MOESM4_ESM.pdf (177 kb)
Supplementary Figure 4(PDF 177 kb)
109_2011_811_MOESM5_ESM.pdf (17 kb)
Supplementary Table 1(PDF 17 kb)


  1. 1.
    Polyak SW, Chapman-Smith A (2004) Biotin. In: Lane MD, Lennarz WJ (eds) Encyclopedia of biological chemistry. Elsevier, Oxford, pp 174–178CrossRefGoogle Scholar
  2. 2.
    Attwood PV, Wallace JC (2002) Chemical and catalytic mechanisms of carboxyl transfer reactions in biotin-dependent enzymes. Acc Chem Res 35:113–120PubMedCrossRefGoogle Scholar
  3. 3.
    Jitrapakdee S, Wallace JC (2003) The biotin enzyme family: conserved structural motifs and domain rearrangements. Curr Protein Pept Sci 4:217–229PubMedCrossRefGoogle Scholar
  4. 4.
    Saunders M, Sweetman L, Robinson B, Roth K, Cohn R, Gravel RA (1979) Biotin-response organicaciduria. Multiple carboxylase defects and complementation studies with propionicacidemia in cultured fibroblasts. J Clin Invest 64:1695–1702PubMedCrossRefGoogle Scholar
  5. 5.
    Wolf B, Feldman GL (1982) The biotin-dependent carboxylase deficiencies. Am J Hum Genet 34:699–716PubMedGoogle Scholar
  6. 6.
    Wolf B (1995) Disorders of biotin metabolism. In: Beavder AL, Sly WS, Scriver CR (eds) The metabolic and molecular basis of inherited diseases. McGraw-Hill, New York, pp 3151–3177Google Scholar
  7. 7.
    Baumgartner ER, Suormala T (1997) Multiple carboxylase deficiency—inherited and acquired disorders of biotin metabolism. Int J Vit Nutr Res 67:377–384Google Scholar
  8. 8.
    Wilson CJ, Myer M, Darlow BA, Stanley T, Thomson G, Baumgartner ER, Kirby DM, Thorburn DR (2005) Severe holocarboxylase synthetase deficiency with incomplete biotin responsiveness resulting in antenatal insult in samoan neonates. J Pediatr 147:115–118PubMedCrossRefGoogle Scholar
  9. 9.
    Pendini NR, Bailey LM, Booker GW, Wilce MC, Wallace JC, Polyak SW (2008) Microbial biotin protein ligases aid in understanding holocarboxylase synthetase deficiency. Biochim Biophys Acta 1784:973–982PubMedGoogle Scholar
  10. 10.
    Suzuki Y, Yang X, Aoki Y, Kure S, Matsubara Y (2005) Mutations in the holocarboxylase synthetase gene HLCS. Hum Mutat 26:285–290PubMedCrossRefGoogle Scholar
  11. 11.
    Zempleni J, Mock DM (1999) Bioavailability of biotin given orally to humans in pharmacologic doses. Am J Clin Nutr 69:504–508PubMedGoogle Scholar
  12. 12.
    Wolf B, Hsia YE, Sweetman L, Feldman G, Boychuk RB, Bart RD, Crowell DH, Di Mauro RM, Nyhan WL (1981) Multiple carboxylase deficiency: clinical and biochemical improvement following neonatal biotin treatment. Pediatrics 68:113–118PubMedGoogle Scholar
  13. 13.
    Yang X, Aoki Y, Li X, Sakamoto O, Hiratsuka M, Kure S, Taheri S, Christensen E, Inui K, Kubota M, Ohira M, Ohki M, Kudoh J, Kawasaki K, Shibuya K, Shintani A, Asakawa S, Minoshima S, Shimizu N, Narisawa K, Matsubara Y, Suzuki Y (2001) Structure of human holocarboxylase synthetase gene and mutation spectrum of holocarboxylase synthetase deficiency. Hum Genet 109:526–534PubMedCrossRefGoogle Scholar
  14. 14.
    Dupuis L, Campeau E, Leclerc D, Gravel RA (1999) Mechanism of biotin responsiveness in biotin-responsive multiple carboxylase deficiency. Mol Gen Metabol 66:80–90CrossRefGoogle Scholar
  15. 15.
    Aoki Y, Li X, Sakamoto O, Hiratsuka M, Akaishi H, Xu LQ, Briones P, Suormala T, Baumgartner ER, Suzuki Y, Narisawa K (1999) Identification and characterization of mutations in patients with holocarboxylase synthetase deficiency. Hum Genet 104:143–148PubMedCrossRefGoogle Scholar
  16. 16.
    Sweetman L, Nyhan WL (1986) Inheritable biotin-treatable disorders and associated phenomena. Annu Rev Nutr 6:317–343PubMedCrossRefGoogle Scholar
  17. 17.
    Aoki Y, Suzuki Y, Sakamoto O, Li X, Takahashi K, Ohtake A, Sakuta R, Ohura T, Miyabayashi S, Narisawa K (1995) Molecular analysis of holocarboxylase synthetase deficiency—a missense mutation and a single base deletion are predominant in Japanese patients. Biochim Biophys Acta Mol Bas Dis 1272:168–174Google Scholar
  18. 18.
    Morrone A, Malvagia S, Donati MA, Funghini S, Ciani F, Pela I, Boneh A, Peters H, Pasquini E, Zammarchi E (2002) Clinical findings and biochemical and molecular analysis of four patients with holocarboxylase synthetase deficiency. Am J Med Genet 111:10–18PubMedCrossRefGoogle Scholar
  19. 19.
    Dupuis L, Leondelrio A, Leclerc D, Campeau E, Sweetman L, Saudubray JM, Herman G, Gibson KM, Gravel RA (1996) Clustering of mutations in the biotin-binding region of holocarboxylase synthetase in biotin-responsive multiple carboxylase deficiency. Hum Mol Gen 5:1011–1016PubMedCrossRefGoogle Scholar
  20. 20.
    Bagautdinov B, Kuroishi C, Sugahara M, Kunishima N (2005) Crystal structures of biotin protein ligase from Pyrococcus horikoshii OT3 and its complexes: structural basis of biotin activation. J Mol Biol 353:322–333PubMedCrossRefGoogle Scholar
  21. 21.
    Wilson KP, Shewchuk LM, Brennan RG, Otsuka AJ, Matthews BW (1992) Escherichia coli biotin holoenzyme synthetase/bio repressor crystal structure delineates the biotin- and DNA-binding domains. Proc Natl Acad Sci USA 89:9257–9261PubMedCrossRefGoogle Scholar
  22. 22.
    Campeau E, Gravel RA (2001) Expression in Escherichia coli of N- and C-terminally deleted human holocarboxylase synthetase. Influence of the N-terminus on biotinylation and identification of a minimum functional protein. J Biol Chem 276:12310–12316PubMedCrossRefGoogle Scholar
  23. 23.
    Hassan YI, Moriyama H, Olsen LJ, Bi X, Zempleni J (2009) N- and C-terminal domains in human holocarboxylase synthetase participate in substrate recognition. Mol Genet Metabol 96:183–188CrossRefGoogle Scholar
  24. 24.
    Ingaramo M, Beckett D (2009) Distinct amino termini of two human HCS isoforms influence biotin acceptor substrate recognition. J Biol Chem 284:30862–30870PubMedCrossRefGoogle Scholar
  25. 25.
    Lee CK, Cheong C, Jeon YH (2010) The N-terminal domain of human holocarboxylase synthetase facilitates biotinylation via direct interaction with the substrate protein. FEBS Lett 584:675–680PubMedCrossRefGoogle Scholar
  26. 26.
    Bailey LM, Ivanov RA, Jitrapakdee S, Wilson CJ, Wallace JC, Polyak SW (2008) Reduced half-life of holocarboxylase synthetase from patients with severe multiple carboxylase deficiency. Hum Mutat 29:E47–57PubMedCrossRefGoogle Scholar
  27. 27.
    Aoki Y, Suzuki Y, Li X, Sakamoto O, Chikaoka H, Takita S, Narisawa K (1997) Characterization of mutant holocarboxylase synthetase (HCS)—a Km for biotin was not elevated in a patient with HCS deficiency. Ped Res 42:849–854CrossRefGoogle Scholar
  28. 28.
    Jitrapakdee S, Walker ME, Wallace JC (1999) Functional expression, purification, and characterization of recombinant human pyruvate carboxylase. Biochem Biophys Res Commun 266:512–517PubMedCrossRefGoogle Scholar
  29. 29.
    Polyak SW, Chapman-Smith A, Brautigan PJ, Wallace JC (1999) Biotin protein ligase from Saccharomyces cerevisiae. The N-terminal domain is required for complete activity. J Biol Chem 274:32847–32854PubMedCrossRefGoogle Scholar
  30. 30.
    Polyak SW, Chapman-Smith A, Mulhern TD, Cronan JE Jr, Wallace JC (2001) Mutational analysis of protein substrate presentation in the post-translational attachment of biotin to biotin domains. J Biol Chem 276:3037–3045PubMedCrossRefGoogle Scholar
  31. 31.
    Kowalski K, Merkel AL, Booker GW (2004) 1H, 13C and 15N resonance assignments of the third spectrin repeat of alpha-actinin-4. J Biomol NMR 29:533–534PubMedCrossRefGoogle Scholar
  32. 32.
    Xu Y, Nenortas E, Beckett D (1995) Evidence for distinct ligand-bound conformational states of the multifunctional Escherichia coli repressor of biotin biosynthesis. Biochemistry 34:16624–16631PubMedCrossRefGoogle Scholar
  33. 33.
    Naganathan S, Beckett D (2007) Nucleation of an allosteric response via ligand-induced loop folding. J Mol Biol 373:96–111PubMedCrossRefGoogle Scholar
  34. 34.
    Streaker ED, Beckett D (2003) Coupling of protein assembly and DNA binding: biotin repressor dimerization precedes biotin operator binding. J Mol Biol 325:937–948PubMedCrossRefGoogle Scholar
  35. 35.
    Bailey LM, Wallace JC, Polyak SW (2010) Holocarboxylase synthetase: correlation of protein localisation with biological function. Arch Biochem Biophys 496:45–52PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Lungisa Mayende
    • 1
  • Rachel D. Swift
    • 2
  • Lisa M. Bailey
    • 3
  • Tatiana P. Soares da Costa
    • 1
  • John C. Wallace
    • 1
  • Grant W. Booker
    • 1
  • Steven W. Polyak
    • 1
  1. 1.School of Molecular and Biomedical ScienceUniversity of AdelaideAdelaideAustralia
  2. 2.Boston Consulting GroupChifley TowerSydneyAustralia
  3. 3.Royal Institution of AustraliaAdelaideAustralia

Personalised recommendations