Structure of the human sulfhydryl oxidase augmenter of liver regeneration and characterization of a human mutation causing an autosomal recessive myopathy

Vidyadhar N. Daithankar, Stephanie Ramadan, Ming Dong, Brian J. Bahnson, Colin Thorpe

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Abstract

The sulfhydryl oxidase augmenter of liver regeneration (ALR) binds FAD in a helix-rich domain that presents a CxxC disulfide proximal to the isoalloxazine ring of the flavin. Head-to-tail interchain disulfide bonds link subunits within the homodimer of both the short, cytokine-like, form of ALR (sfALR), and a longer form (lfALR) which resides in the mitochondrial intermembrane space (IMS). lfALR has an 80-residue N-terminal extension with an additional CxxC motif required for the reoxidation of reduced Mia40 during oxidative protein folding within the IMS. Recently, Di Fonzo et al. [Di Fonzo, A., Ronchi, D., Lodi, T., Fassone, E., Tigano, M., Lamperti, C., Corti, S., Bordoni, A., Fortunato, F., Nizzardo, M., Napoli, L., Donadoni, C., Salani, S., Saladino, F., Moggio, M., Bresolin, N., Ferrero, I., and Comi, G. P. (2009) Am. J. Hum. Genet. 84, 594-604] described an R194H mutation of human ALR that led to cataract, progressive muscle hypotonia, and hearing loss in three children. The current work presents a structural and enzymological characterization of the human R194H mutant in lf- and sfALR. A crystal structure of human sfALR was determined by molecular replacement using the rat sfALR structure. R194 is located at the subunit interface of sfALR, close to the intersubunit disulfide bridges. The R194 guanidino moiety participates in three H-bonds: two main-chain carbonyl oxygen atoms (from R194 itself and from C95 of the intersubunit disulfide of the other protomer) and with the 2′-OH of the FAD ribose. The R194H mutation has minimal effect on the enzyme activity using model and physiological substrates of short and long ALR forms. However, the mutation adversely affects the stability of both ALR forms: e.g., by decreasing the melting temperature by about 10 °C, by increasing the rate of dissociation of FAD from the holoenzyme by about 45-fold, and by strongly enhancing the susceptibility of sfALR to partial proteolysis and to reduction of its intersubunit disulfide bridges by glutathione. Finally, a comparison of the TROSY-HSQC 2D NMR spectra of wild-type sfALR and its R194H mutant reveals a significant increase in conformational flexibility in the mutant protein. In sum, these in vitro data document the major impact of the seemingly conservative R194H mutation on the stability of dimeric ALR and complement the in vivo observations of Di Fonzo et al.

Original languageEnglish
Pages (from-to)6737-6745
Number of pages9
JournalBiochemistry
Volume49
Issue number31
DOIs
Publication statusPublished - 10 Aug 2010
Externally publishedYes

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Liver Regeneration
Muscular Diseases
Liver
Cytokines
Disulfides
Mutation
Flavin-Adenine Dinucleotide
Proteolysis
Viverridae
Protein folding
Holoenzymes
Muscle Hypotonia
Ribose
Glutathione Disulfide
Protein Folding
Protein Subunits
Enzyme activity
Audition
Mutant Proteins
sulfhydryl oxidase

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structure of the human sulfhydryl oxidase augmenter of liver regeneration and characterization of a human mutation causing an autosomal recessive myopathy. / Daithankar, Vidyadhar N.; Ramadan, Stephanie; Dong, Ming; Bahnson, Brian J.; Thorpe, Colin.

In: Biochemistry, Vol. 49, No. 31, 10.08.2010, p. 6737-6745.

Research output: Contribution to journalArticle

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AU - Thorpe, Colin

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N2 - The sulfhydryl oxidase augmenter of liver regeneration (ALR) binds FAD in a helix-rich domain that presents a CxxC disulfide proximal to the isoalloxazine ring of the flavin. Head-to-tail interchain disulfide bonds link subunits within the homodimer of both the short, cytokine-like, form of ALR (sfALR), and a longer form (lfALR) which resides in the mitochondrial intermembrane space (IMS). lfALR has an 80-residue N-terminal extension with an additional CxxC motif required for the reoxidation of reduced Mia40 during oxidative protein folding within the IMS. Recently, Di Fonzo et al. [Di Fonzo, A., Ronchi, D., Lodi, T., Fassone, E., Tigano, M., Lamperti, C., Corti, S., Bordoni, A., Fortunato, F., Nizzardo, M., Napoli, L., Donadoni, C., Salani, S., Saladino, F., Moggio, M., Bresolin, N., Ferrero, I., and Comi, G. P. (2009) Am. J. Hum. Genet. 84, 594-604] described an R194H mutation of human ALR that led to cataract, progressive muscle hypotonia, and hearing loss in three children. The current work presents a structural and enzymological characterization of the human R194H mutant in lf- and sfALR. A crystal structure of human sfALR was determined by molecular replacement using the rat sfALR structure. R194 is located at the subunit interface of sfALR, close to the intersubunit disulfide bridges. The R194 guanidino moiety participates in three H-bonds: two main-chain carbonyl oxygen atoms (from R194 itself and from C95 of the intersubunit disulfide of the other protomer) and with the 2′-OH of the FAD ribose. The R194H mutation has minimal effect on the enzyme activity using model and physiological substrates of short and long ALR forms. However, the mutation adversely affects the stability of both ALR forms: e.g., by decreasing the melting temperature by about 10 °C, by increasing the rate of dissociation of FAD from the holoenzyme by about 45-fold, and by strongly enhancing the susceptibility of sfALR to partial proteolysis and to reduction of its intersubunit disulfide bridges by glutathione. Finally, a comparison of the TROSY-HSQC 2D NMR spectra of wild-type sfALR and its R194H mutant reveals a significant increase in conformational flexibility in the mutant protein. In sum, these in vitro data document the major impact of the seemingly conservative R194H mutation on the stability of dimeric ALR and complement the in vivo observations of Di Fonzo et al.

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