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PMID:15003260

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Citation

Zhou, D, Yuen, P, Chu, D, Thon, V, McConnell, S, Brown, S, Tsang, A, Pena, M, Russell, A, Cheng, JF, Nadzan, AM, Barbosa, MS, Dyck, JR, Lopaschuk, GD and Yang, G (2004) Expression, purification, and characterization of human malonyl-CoA decarboxylase. Protein Expr. Purif. 34:261-9

Abstract

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.

Links

PubMed Online version:10.1016/j.pep.2003.11.023

Keywords

Amino Acid Sequence; Carboxy-Lyases/genetics; Carboxy-Lyases/isolation & purification; Carboxy-Lyases/metabolism; Cloning, Molecular; Escherichia coli/genetics; Humans; Hydrogen-Ion Concentration; Molecular Sequence Data; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Sequence Homology; Substrate Specificity

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

HUMAN:DCMC

involved_in

GO:0006633: fatty acid biosynthetic process

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:DCMC

enables

GO:0050080: malonyl-CoA decarboxylase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:DCMC

involved_in

GO:2001294: malonyl-CoA catabolic process

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

See also

References

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