Role of the glutamate 332 residue in the transglycosylation activity of Thermus maltogenic amylase

Tae Jip Kim, Cheon Seok Park, Hee Yeon Cho, Sun Shin Cha, Jeong Sun Kim, Soo-Bok Lee, Tae Wha Moon, Jung Wan Kim, Byung Ha Oh, Kwan Hwa Park

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

A sequence alignment shows that residue 332 is conserved as glutamate in maltogenic amylases (MAases) and in other related enzymes such as cyclodextrinase and neopullulanase, while the corresponding position is conserved as histidine in α-amylases. We analyzed the role of Glu332 in the hydrolysis and the transglycosylation activity of Thermus MAase (ThMA) by site-directed mutagenesis. Replacing Glu332 with histidine reduced transglycosylation activity significantly, but enhanced hydrolysis activity on α-(1,3)-, α-(1,4)-, and α-(1,6)-glycosidic bonds relative to the wild- type (WT) enzyme. The mutant Glu332Asp had catalytic properties similar to those of the WT enzyme, but the mutant Glu332Gln resulted in significantly decreased transglycosylation activity. These results suggest that an acidic side chain at position 332 of MAase plays an important role in the formation and accumulation of transfer products by modulating the relative rates of hydrolysis and transglycosylation. From the structure, we propose that an acidic side chain at position 332, which is located in a pocket, is involved in aligning the acceptor molecule to compete with water molecules in the nucleophilic attack of the glycosyl-enzyme intermediate.

Original languageEnglish
Pages (from-to)6773-6780
Number of pages8
JournalBiochemistry
Volume39
Issue number23
DOIs
Publication statusPublished - 2000 Jun 13

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glucan 1,4-alpha-maltohydrolase
Thermus
Glutamic Acid
Hydrolysis
neopullulanase
cyclomaltodextrinase
Enzymes
Histidine
Mutagenesis
Molecules
Sequence Alignment
Amylases
Site-Directed Mutagenesis
Water

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Kim, T. J., Park, C. S., Cho, H. Y., Cha, S. S., Kim, J. S., Lee, S-B., ... Park, K. H. (2000). Role of the glutamate 332 residue in the transglycosylation activity of Thermus maltogenic amylase. Biochemistry, 39(23), 6773-6780. https://doi.org/10.1021/bi992575i
Kim, Tae Jip ; Park, Cheon Seok ; Cho, Hee Yeon ; Cha, Sun Shin ; Kim, Jeong Sun ; Lee, Soo-Bok ; Moon, Tae Wha ; Kim, Jung Wan ; Oh, Byung Ha ; Park, Kwan Hwa. / Role of the glutamate 332 residue in the transglycosylation activity of Thermus maltogenic amylase. In: Biochemistry. 2000 ; Vol. 39, No. 23. pp. 6773-6780.
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abstract = "A sequence alignment shows that residue 332 is conserved as glutamate in maltogenic amylases (MAases) and in other related enzymes such as cyclodextrinase and neopullulanase, while the corresponding position is conserved as histidine in α-amylases. We analyzed the role of Glu332 in the hydrolysis and the transglycosylation activity of Thermus MAase (ThMA) by site-directed mutagenesis. Replacing Glu332 with histidine reduced transglycosylation activity significantly, but enhanced hydrolysis activity on α-(1,3)-, α-(1,4)-, and α-(1,6)-glycosidic bonds relative to the wild- type (WT) enzyme. The mutant Glu332Asp had catalytic properties similar to those of the WT enzyme, but the mutant Glu332Gln resulted in significantly decreased transglycosylation activity. These results suggest that an acidic side chain at position 332 of MAase plays an important role in the formation and accumulation of transfer products by modulating the relative rates of hydrolysis and transglycosylation. From the structure, we propose that an acidic side chain at position 332, which is located in a pocket, is involved in aligning the acceptor molecule to compete with water molecules in the nucleophilic attack of the glycosyl-enzyme intermediate.",
author = "Kim, {Tae Jip} and Park, {Cheon Seok} and Cho, {Hee Yeon} and Cha, {Sun Shin} and Kim, {Jeong Sun} and Soo-Bok Lee and Moon, {Tae Wha} and Kim, {Jung Wan} and Oh, {Byung Ha} and Park, {Kwan Hwa}",
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Kim, TJ, Park, CS, Cho, HY, Cha, SS, Kim, JS, Lee, S-B, Moon, TW, Kim, JW, Oh, BH & Park, KH 2000, 'Role of the glutamate 332 residue in the transglycosylation activity of Thermus maltogenic amylase', Biochemistry, vol. 39, no. 23, pp. 6773-6780. https://doi.org/10.1021/bi992575i

Role of the glutamate 332 residue in the transglycosylation activity of Thermus maltogenic amylase. / Kim, Tae Jip; Park, Cheon Seok; Cho, Hee Yeon; Cha, Sun Shin; Kim, Jeong Sun; Lee, Soo-Bok; Moon, Tae Wha; Kim, Jung Wan; Oh, Byung Ha; Park, Kwan Hwa.

In: Biochemistry, Vol. 39, No. 23, 13.06.2000, p. 6773-6780.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Role of the glutamate 332 residue in the transglycosylation activity of Thermus maltogenic amylase

AU - Kim, Tae Jip

AU - Park, Cheon Seok

AU - Cho, Hee Yeon

AU - Cha, Sun Shin

AU - Kim, Jeong Sun

AU - Lee, Soo-Bok

AU - Moon, Tae Wha

AU - Kim, Jung Wan

AU - Oh, Byung Ha

AU - Park, Kwan Hwa

PY - 2000/6/13

Y1 - 2000/6/13

N2 - A sequence alignment shows that residue 332 is conserved as glutamate in maltogenic amylases (MAases) and in other related enzymes such as cyclodextrinase and neopullulanase, while the corresponding position is conserved as histidine in α-amylases. We analyzed the role of Glu332 in the hydrolysis and the transglycosylation activity of Thermus MAase (ThMA) by site-directed mutagenesis. Replacing Glu332 with histidine reduced transglycosylation activity significantly, but enhanced hydrolysis activity on α-(1,3)-, α-(1,4)-, and α-(1,6)-glycosidic bonds relative to the wild- type (WT) enzyme. The mutant Glu332Asp had catalytic properties similar to those of the WT enzyme, but the mutant Glu332Gln resulted in significantly decreased transglycosylation activity. These results suggest that an acidic side chain at position 332 of MAase plays an important role in the formation and accumulation of transfer products by modulating the relative rates of hydrolysis and transglycosylation. From the structure, we propose that an acidic side chain at position 332, which is located in a pocket, is involved in aligning the acceptor molecule to compete with water molecules in the nucleophilic attack of the glycosyl-enzyme intermediate.

AB - A sequence alignment shows that residue 332 is conserved as glutamate in maltogenic amylases (MAases) and in other related enzymes such as cyclodextrinase and neopullulanase, while the corresponding position is conserved as histidine in α-amylases. We analyzed the role of Glu332 in the hydrolysis and the transglycosylation activity of Thermus MAase (ThMA) by site-directed mutagenesis. Replacing Glu332 with histidine reduced transglycosylation activity significantly, but enhanced hydrolysis activity on α-(1,3)-, α-(1,4)-, and α-(1,6)-glycosidic bonds relative to the wild- type (WT) enzyme. The mutant Glu332Asp had catalytic properties similar to those of the WT enzyme, but the mutant Glu332Gln resulted in significantly decreased transglycosylation activity. These results suggest that an acidic side chain at position 332 of MAase plays an important role in the formation and accumulation of transfer products by modulating the relative rates of hydrolysis and transglycosylation. From the structure, we propose that an acidic side chain at position 332, which is located in a pocket, is involved in aligning the acceptor molecule to compete with water molecules in the nucleophilic attack of the glycosyl-enzyme intermediate.

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