Probing the critical residues for intramolecular fructosyl transfer reaction of a levan fructotransferase.
Levan fructotransferase (LFTase) preferentially catalyzes the transfructosylation reaction in addition to levan hydrolysis, whereas other levan-degrading enzymes hydrolyze levan into a levan-oligosaccharide and fructose. Based on sequence comparisons and enzymatic properties, the fructosyl transfer activity of LFTase is proposed to have evolved from levanase. In order to probe the residues that are critical to the intramolecular fructosyl transfer reaction of the Microbacterium sp. AL-210 LFTase, an error-prone PCR mutagenesis process was carried out, and the mutants that led to a shift in activity from transfructosylation towards hydrolysis of levan were screened by the DNS method. After two rounds of mutagenesis, TLC and HPLC analyses of the reaction products by the selected mutants revealed two major products; one is a di-D-fructose- 2,6':6,2'-dianhydride (DFAIV) and the other is a levanbiose. The newly detected levanbiose corresponds to the reaction product from LFTase lacking transferring activity. Two mutants (2-F8 and 2-G9) showed a high yield of levanbiose (38-40%) compared with the wild-type enzyme, and thus behaved as levanases. Sequence analysis of the individual mutants responsible for the enhanced hydrolytic activity indicated that Asn-85 was highly involved in the transfructosylation activity of LFTase.
Moon
Keum-Ok
KO
Department of Biological Sciences, Pusan National University, Busan, Korea.
Choi
Kyoung-Hwa
KH
Kang
Ho-Young
HY
Oh
Jeong-Il
JI
Jang
Se Bok
SB
Park
Cheon-Seok
CS
Lee
Jong-Hoon
JH
Cha
Jaeho
J
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004187:Disaccharides / Q000096:biosynthesis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005630:Fructans / Q000096:biosynthesis / Q000378:metabolism; D006602:Hexosyltransferases / Q000235:genetics* / Q000378:metabolism*; D006868:Hydrolysis; D016296:Mutagenesis; D016133:Polymerase Chain Reaction
D004187:Disaccharides; D005630:Fructans; C077708:levanbiose; C033869:difructose anhydride IV; C072599:levan; D006602:Hexosyltransferases; C040074:levan fructotransferase