Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Identification of catalytic acidic residues of levan fructotransferase from Microbacterium sp. AL-210

Microbacterium sp. AL-210이 생산하는 levan fructotransferase의 효소활성에 중요한 아미노산의 동정

  • Sung, Hee-Kyung (Department of Microbiology, College of Natural Sciences, Pusan National University) ;
  • Moon, Keum-Ok (Department of Microbiology, College of Natural Sciences, Pusan National University) ;
  • Choi, Ki-Won (Department of Microbiology, College of Natural Sciences, Pusan National University) ;
  • Choi, Kyung-Hwa (Department of Microbiology, College of Natural Sciences, Pusan National University) ;
  • Hwang, Kyung-Ju (Department of Microbiology, College of Natural Sciences, Pusan National University) ;
  • Kim, Myo-Jung (Food Research Institute and School of Food and Life Science, and Biohealth Products Research Center, Inje University) ;
  • Cha, Jae-Ho (Department of Microbiology, College of Natural Sciences, Pusan National University)
  • Published : 2007.01.29
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Abstract

[ $\beta$ ]-Fructofuranosidases, a family 32 of glycoside hydrolases (GH32), share three conserved domains including the W(L/M)(C/N)DP(Q/N), FRDPK, and ECP(D/G) motifs. The functional role of the conserved acidic residues within three domains of levan fructotransferase, one of the $\beta-fructofuranosidases$, from Microbacterium sp. AL-210 was studied by site-directed mutagenesis. Each mutant was overexpressed in E. coli BL21(DE3) and purified by using Hi-Trap chelating affinity chromatography and fast performance liquid chromatography. Substitution of Asp-63 by Ala, Asp-195 by Asn, and Glu-245 by Ala and Asp decreased the enzyme activity by approximately 100-fold compared to the wild-type enzyme. This result indicates that three acidic residues Asp-63, Asp-195, and Glu-245 play a major role in catalysis. Since the three acidic residues are present in a conserved position in inulinase, levanase, levanfructotransferase, and invertase, they are likely to have a common functional role as nucleophile, transition state stabilizer, and general acid in $\beta-fructofuranosidases$.

당 분해효소의 family 32 (GH32)에 속하는 $\beta-fructofuranosidase$는 3차구조를 근거로 볼 때 W(L/M)(C/N)DP(Q/N), FRDPK, 그리고 ECP(D/G) 부위를 포함하는 세 군데의 보전적인 영역을 가지고 있다. 이러한 $\beta-fructofuranosidase$ family에 속하는 Microbacterium sp. AL-210 유래 levan fructotransferase (LFTase)의 보전적인 산성 아미노산들의 역할이 특정위치 돌연변이법으로 검사되었다. 각각의 돌연변이체는 대장균인 E. coli BL21 (DE3)균주에서 발현되어 대량 생산되었고, 금속 친화 크로마토그래피법과 FPLC법으로 순수 정제되었다. wild-type LFTase의 효소의 활성은 0.74 unit 인 반면 네 개의 돌연변이체인 D63A, D195N, E245A, E245D 각각은 specific activity를 측정해 본 결과 원 균주와 비교해서 약 100배 정도 감소한 효소활성을 보여 주었다. 이로써 아미노산 변형의 target이 되었던 Asp-63, Aps-195, 그리고 Glu-245가 모두 효소 활성 및 기질과의 결합에 상당히 중요한 역할을 하고 있음이 판명되었다. 이러한 세 부위의 산성 아미노산들은 inulinase, levan fructotransferase와 invertase에 모두 보전적으로 위치 하므로 이들은 $\beta-fructofuranosidase$ family내 에서 공통된 역할을 할 것으로 사료된다.

Keywords

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