Mutagenesis of Ala290, which modulates substrate subsite affinity at the catalytic interface of dimeric ThMA.
The goal of this study was to develop a maltose-producing enzyme using protein engineering and to clarify the relation between the substrate specificity and the structure of the substrate-binding site of dimeric maltogenic amylase isolated from Thermus (ThMA). Ala290 at the interface of ThMA dimer in the vicinity of the substrate-binding site was substituted with isoleucine, which may cause a structural change due to its bulky side chain. TLC analysis of the action pattern of the mutant ThMA-A290I, using maltooligosaccharides as substrates, revealed that ThMA-A290I used maltotetraose to produce mostly maltose, while wild-type ThMA produced glucose as well as maltose. The wild-type enzyme eventually hydrolyzed the maltose produced from maltotetraose into glucose, but the mutant enzyme did not. For both enzymes, the cleavage frequency of the glycosidic bond of maltooligosaccharides was the highest at the second bond from the reducing end. The mutant ThMA had a much higher Km value for maltose than the wild-type ThMA. The kinetic parameter, kcat/Km) of ThMA-A290I for maltose was 48 times less than that of wild-type ThMA, suggesting that the subsite affinity and hydrolysis mode of ThMA were modulated by the residue located at the interface of ThMA dimer near the active site. The conformational rearrangement in the catalytic interface probably led to the change in the substrate binding affinity of the mutant ThMA. Our results provide basic information for the enzymatic preparation of high-maltose syrup.
Center for Agricultural Biomaterials and Department of Food Science and Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea.
Sung-Hoon
SH
Park
Hyunju
H
Cha
Hee-Kwon
HK
Kang
Jae-Hoon
JH
Shim
Eui-Jeon
EJ
Woo
Jung-Wan
JW
Kim
Kwan-Hwa
KH
Park
D000409:Alanine / Q000235:genetics*; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics*; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007532:Isoleucine / Q000235:genetics; D007700:Kinetics; D008320:Maltose / Q000737:chemistry / Q000378:metabolism; D008757:Methylglucosides / Q000737:chemistry / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity / Q000235:genetics; D013824:Thermus / Q000201:enzymology* / Q000235:genetics
D008757:Methylglucosides; D009844:Oligosaccharides; D011994:Recombinant Proteins; C021705:maltooligosaccharides; D007532:Isoleucine; C027020:methylglucoside; D008320:Maltose; D006026:Glycoside Hydrolases; C120909:glucan 1,4-alpha-maltohydrolase; D005947:Glucose; D000409:Alanine