Molecular cloning and characterization of a malic enzyme gene from the oleaginous yeast Lipomyces starkeyi.
The malic enzyme-encoding cDNA (GQ372891) from the oleaginous yeast Lipomyces starkeyi AS 2.1560 was isolated, which has an 1719-bp open reading frame flanked by a 290-bp 5' untranslated sequence and a 92-bp 3' untranslated sequence. The proposed gene, LsME1, encoded a protein with 572 amino acid residues. The protein presented 58% sequence identity with the malic enzymes from Yarrowia lipolytica CLIB122 and Aspergillus fumigatus Af293. The LsME1 gene was cloned into the vector pMAL-p4x to express a fusion protein (MBP-LsME1) in Escherichia coli TB1. The fusion protein was purified and then cleaved by Factor Xa to give the recombinant LsME1. This purified enzyme took either NAD(+) or NADP(+) as the coenzyme but preferred NAD(+). The K (m) values for malic acid, NAD(+) and NADP(+) were 0.85 +/- 0.05 mM, 0.34 +/- 0.08 mM, and 7.4 +/- 0.32 mM, respectively, at pH 7.3.
Tang
Wei
W
Dalian Institute of Chemical Physics, CAS, Dalian 116023, People's Republic of China.
Zhang
Sufang
S
Tan
Haidong
H
Zhao
Zongbao K
ZK
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D055160:Lipomyces / Q000201:enzymology* / Q000235:genetics*; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008293:Malates / Q000378:metabolism; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D005656:Fungal Proteins; D008293:Malates; D011994:Recombinant Proteins; D009243:NAD; C030298:malic acid; D008291:Malate Dehydrogenase