The flexible C-terminal region of Aspergillus terreus blasticidin S deaminase: identification of its functional roles with deletion enzymes.
Although the four polypeptides of blasticidin S (BS) deaminase (BSD) are packed rather tightly coordinated to the "structural and catalytic" zinc atom of each subunit, the C-terminal region of the enzyme was suggested to be somewhat molten and flexible [M. Kimura, S. Sekido, Y. Isogai, and I. Yamaguchi (2000) J. Biochem. 127, 955-963]. To understand roles of this flexible region, we constructed five C-terminal deletion variants of BSD (each successively deleted from the C-terminal end up to five residues) and analyzed their biochemical properties focusing on the structure and activity of the enzyme. BSD and all of the deletion mutants showed the unique rigid conformation (e.g., characterized by their stabilities in SDS solution) and high levels of resistance against protease digestions. Furthermore, both the wild-type and deletion apoenzymes exhibited similar physical properties in thermodynamic refolding into the stable tetramer conformation. However, these small C-terminal deletions exerted deleterious effects on the catalytic efficiency of the enzyme as indicated by their strongly reduced k(cat)/K(m) value. Judging from the altered kinetic parameters and unaltered structural properties of the deletion variants, these C-terminal residues appear to be directly involved in enzyme-substrate interaction. In this short flexible region, Tyr-126, Trp-128, and Gly-130 were the key residues. Most notably, removal of Gly-130 markedly increased K(m) for BS without affecting its k(cat) value. These results indicate that the flexible C-terminal region is important for catalytic function and that a single Gly residue at the C-terminal end of BSD contributes significantly in facilitating access of a substrate to the active site.
Kimura
Makoto
M
Laboratory for Remediation Research, Plant Science Center, and Microbial Toxicology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. mkimura@postman.riken.go.jp
Furuichi
Makio
M
Yamamoto
Masaki
M
Kumasaka
Takashi
T
Mizuno
Hiroshi
H
Miyano
Masashi
M
Yamaguchi
Isamu
I
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology*; D002384:Catalysis; D020134:Catalytic Domain; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D005998:Glycine / Q000737:chemistry; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009705:Nucleosides / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D009705:Nucleosides; D014443:Tyrosine; C004500:blasticidin S; D014364:Tryptophan; D015032:Zinc; D005998:Glycine