C-terminal end and aminoacid Lys48 in HMG-CoA lyase are involved in substrate binding and enzyme activity.
3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase adopts a (betaalpha)(8) TIM barrel structure with an additional beta9, alpha11 and alpha12 helices. Location of HMG part of the substrate has been suggested but the binding mode for the CoA moiety remains to be resolved. As mutation F305 fs(-2), which involves the last 21 residues of the protein, and mutation K48N caused 3-hydroxy-3-methylglutaric aciduria in two patients, we examined the role of the C-terminal end and Lys(48) in enzyme activity. Expression studies of various C-terminal-end-deleted and K48N-mutated proteins revealed that residues 311-313 (localized in the loop between alpha11 and alpha12 helices) and Lys(48) are essential for enzyme activity. An in silico docking model locating HMG-CoA on the surface of the enzyme implicates Asn(311) and Lys(313) in substrate binding by establishing multiple polar contacts with phosphate and ribose groups of adenosine, and Lys(48) by contacting the carboxyl group of the panthotenic acid moiety.
Carrasco
Patricia
P
Department of Biochemistry and Molecular Biology, School of Health Sciences, Universitat Internacional de Catalunya, E-08195 Sant Cugat, Barcelona, Spain.
Menao
Sebastian
S
López-Viñas
Eduardo
E
Santpere
Gabriel
G
Clotet
Josep
J
Sierra
Adriana Y
AY
Gratacós
Esther
E
Puisac
Beatriz
B
Gómez-Puertas
Paulino
P
Hegardt
Fausto G
FG
Pie
Juan
J
Casals
Núria
N
D000214:Acyl Coenzyme A / Q000737:chemistry*; D000595:Amino Acid Sequence; D017124:Conserved Sequence; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D007652:Oxo-Acid-Lyases / Q000096:biosynthesis / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D000214:Acyl Coenzyme A; D011994:Recombinant Proteins; C008047:3-hydroxy-3-methylglutaryl-coenzyme A; D007652:Oxo-Acid-Lyases; C021410:3-hydroxy-3-methylglutaryl-coenzyme A lyase