Purification and characterization of a malic enzyme from the ruminal bacterium Streptococcus bovis ATCC 15352 and cloning and sequencing of its gene.
Malic enzyme (EC 1.1.1.39), which catalyzes L-malate oxidative decarboxylation and pyruvate reductive carboxylation, was purified to homogeneity from Streptococcus bovis ATCC 15352, and properties of this enzyme were determined. The 2.9-kb fragment containing the malic enzyme gene was cloned, and the sequence was determined and analyzed. The enzymatic properties of the S. bovis malic enzyme were almost identical to those of other malic enzymes previously reported. However, we found that the S. bovis malic enzyme catalyzed unknown enzymatic reactions, including reduction of 2-oxoisovalerate, reduction of 2-oxoisocaproate, oxidation of D-2-hydroxyisovalerate, and oxidation of D-2-hydroxyisocaproate. The requirement for cations and the optimum pH of these unique activities were different from the requirement for cations and the optimum pH of the L-malate oxidative decarboxylating activity. A sequence analysis of the cloned fragment revealed the presence of two open reading frames that were 1,299 and 1,170 nucleotides long. The 389-amino-acid polypeptide deduced from the 1,170-nucleotide open reading frame was identified as the malic enzyme; this enzyme exhibited high levels of similarity to malic enzymes of Bacillus stearothermophilus and Haemophilus influenzae and was also similar to other malic enzymes and the malolactic enzyme of Lactococcus lactis.
Kawai
S
S
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Japan.
Suzuki
H
H
Yamamoto
K
K
Inui
M
M
Yukawa
H
H
Kumagai
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D045782:Hemiterpenes; D006863:Hydrogen-Ion Concentration; D007651:Keto Acids / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid; D017386:Sequence Homology, Amino Acid; D016985:Streptococcus bovis / Q000201:enzymology*
D045782:Hemiterpenes; D007651:Keto Acids; D011773:Pyruvates; C001505:alpha-ketoisovalerate; D019289:Pyruvic Acid; D008291:Malate Dehydrogenase; C030574:malate dehydrogenase (decarboxylating)