Expression, purification, and characterization of human malonyl-CoA decarboxylase.
The recombinant human malonyl-CoA decarboxylase (hMCD) was overexpressed in Escherichia coli with and without the first 39 N-terminal amino acids via a cleavable MBP-fusion construct. Proteolytic digestion using genenase I to remove the MBP-fusion tag was optimized for both the full length and truncated hMCD. The apo-hMCD enzymes were solubilized and purified to homogeneity. Steady-state kinetic characterization showed similar kinetic parameters for the MBP-fused and apo-hMCD enzymes with an apparent Km value of approximately 330-520 microM and a turnover rate kcat of 13-28s(-1). For the apo-hMCD enzymes, the N-terminal truncated hMCD was well tolerated over a broad pH range (pH 4-10); whereas the full-length hMCD appeared to be stable only at pH >/= 8.5. Our results showed that the N-terminal region of hMCD has no effect on the catalytic activity of the enzyme but plays a role in the folding process and conformation stability of hMCD.
Zhou
Demin
D
Department of Discovery Biology, Chugai Pharma USA, LLC, 6275 Nancy Ridge Drive, San Diego, CA 92121, USA.
Yuen
Phoebe
P
Chu
Donald
D
Thon
Vicki
V
McConnell
Steve
S
Brown
Steven
S
Tsang
Andria
A
Pena
Michael
M
Russell
Anna
A
Cheng
Jie-Fei
JF
Nadzan
Alex M
AM
Barbosa
Miguel S
MS
Dyck
Jason R B
JR
Lopaschuk
Gary D
GD
Yang
Guang
G
D000595:Amino Acid Sequence; D002262:Carboxy-Lyases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017385:Sequence Homology; D013379:Substrate Specificity
D011994:Recombinant Proteins; D002262:Carboxy-Lyases; C021532:malonyl-CoA decarboxylase