Affiliation:
1. Laboratory of Applied Microbiology, Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo, Japan
Abstract
ABSTRACT
Acyl coenzyme A carboxylase (acyl-CoA carboxylase) was purified from
Acidianus brierleyi
. The purified enzyme showed a unique subunit structure (three subunits with apparent molecular masses of 62, 59, and 20 kDa) and a molecular mass of approximately 540 kDa, indicating an α
4
β
4
γ
4
subunit structure. The optimum temperature for the enzyme was 60 to 70°C, and the optimum pH was around 6.4 to 6.9. Interestingly, the purified enzyme also had propionyl-CoA carboxylase activity. The apparent
K
m
for acetyl-CoA was 0.17 ± 0.03 mM, with a
V
max
of 43.3 ± 2.8 U mg
−1
, and the
K
m
for propionyl-CoA was 0.10 ± 0.008 mM, with a
V
max
of 40.8 ± 1.0 U mg
−1
. This result showed that
A. brierleyi
acyl-CoA carboxylase is a bifunctional enzyme in the modified 3-hydroxypropionate cycle. Both enzymatic activities were inhibited by malonyl-CoA, methymalonyl-CoA, succinyl-CoA, or CoA but not by palmitoyl-CoA. The gene encoding acyl-CoA carboxylase was cloned and characterized. Homology searches of the deduced amino acid sequences of the 62-, 59-, and 20-kDa subunits indicated the presence of functional domains for carboxyltransferase, biotin carboxylase, and biotin carboxyl carrier protein, respectively. Amino acid sequence alignment of acetyl-CoA carboxylases revealed that archaeal acyl-CoA carboxylases are closer to those of
Bacteria
than to those of
Eucarya
. The substrate-binding motifs of the enzymes are highly conserved among the three domains. The ATP-binding residues were found in the biotin carboxylase subunit, whereas the conserved biotin-binding site was located on the biotin carboxyl carrier protein. The acyl-CoA-binding site and the carboxybiotin-binding site were found in the carboxyltransferase subunit.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
44 articles.
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