Tài liệu Báo cáo khoa học: Two novel variants of human medium chain acyl-CoA dehydrogenase (MCAD)

Two novel variants of human medium chain acyl-CoA

dehydrogenase (MCAD)

K364R, a folding mutation, and R256T, a catalytic-site mutation

resulting in a well-folded but totally inactive protein

Linda P. O’Reilly1,*, Brage S. Andresen2 and Paul C. Engel1

1 Department of Biochemistry and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin,

Ireland

2 Research Unit for Molecular Medicine, University Hospital, Skejby Sygehus, Aarhus, and Institute of Human Genetics, Aarhus University,

Denmark

Keywords

active site; enzyme deficiency; medium

chain acyl-CoA dehydrogenase (MCAD);

point mutations; protein folding

Correspondence

P. C. Engel, Department of Biochemistry,

Conway Institute, University College Dublin,

Belfield, Dublin 4, Ireland

Fax: +353 12837211

Tel: +353 17166764

E-mail: [email protected]

Website: http://www.ucd.ie/biochem/

Enzymes

Medium chain acyl-CoA dehydrogenase

(MCAD; EC 1.3.99.3); long chain acyl-CoA

dehydrogenase (LCAD; EC 1.3.99.13); short

chain acyl-CoA dehydrogenase (SCAD;

EC 1.3.99.2); glutaryl-CoA dehydrogenase

(GCD; EC 1.3.99.7); isovaleryl-CoA dehydro￾genase (IVD; EC 1.3.99.10); electron trans￾ferring protein (ETF; EC 1.5.5.1).

*Current address

Department of Molecular Genetics and

Biochemistry, University of Pittsburgh, 200

Lothrop Street, Pittsburgh, PA 15261, USA

(Received 13 January 2005, revised 24 June

2005, accepted 25 July 2005)

doi:10.1111/j.1742-4658.2005.04878.x

Two novel rare mutations, MCAD
842GfiC (R256T) and MCAD

1166AfiG (K364R), have been investigated to assess how far the bio￾chemical properties of the mutant proteins correlate with the clinical

phenotype of medium chain acyl-CoA dehydrogenase (MCAD) deficiency.

When the gene for K364R was overexpressed in Escherichia coli, the syn￾thesized mutant protein only exhibited activity when the gene for chapero￾nin GroELS was co-overexpressed. Levels of activity correlated with the

amounts of native MCAD protein visible in western blots. The R256T

mutant, by contrast, displayed no activity either with or without chapero￾nin, but in this case a strong MCAD protein band was seen in the western

blots throughout. The proteins were also purified, and the enzyme function

and thermostability investigated. The K364R protein showed only moder￾ate kinetic impairment, whereas the R256T protein was again totally inac￾tive. Neither mutant showed marked depletion of FAD. The pure K364R

protein was considerably less thermostable than wild-type MCAD. Western

blots indicated that, although the R256T mutant protein is less thermo￾stable than normal MCAD, it is much more stable than K364R. Though

clinically asymptomatic thus far, both mutations have a severe impact on

the biochemical phenotype of the protein. K364R, like several previously

described MCAD mutant proteins, appears to be defective in folding.

R256T, by contrast, is a well-folded protein that is nevertheless devoid of

catalytic activity. How the mutations specifically affect the catalytic activity

and the folding is further discussed.

Abbreviations

ACAD, acyl-CoA dehydrogenase; BCIP, 5-bromo-4-chloro indol-3-yl phosphate; DCPIP, 2,6-dichlorophenolindophenol; ETF, electron￾transferring flavoprotein; GCD, glutaryl-CoA dehydrogenase; INT, 2-(4-iodophenyl) 3-(4-nitrophenyl) 5-phenyl-tetrazolium chloride; IVD,

isovaleryl-CoA dehydrogenase; NBT, 2,2¢-di-p-nitrophenyl 5,5¢-diphenyl 3,3¢-(3,3¢-dimethoxy-4,4¢-diphenylene) ditetrazolium chloride; PES,

phenazine ethosulphate; SCAD, short-chain acyl-CoA dehydrogenase; VLCAD, very long chain acyl-CoA dehydrogenase.

FEBS Journal 272 (2005) 4549–4557 ª 2005 FEBS 4549