• Title/Summary/Keyword: Pyrophosphorylase

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Cloning and characterization of phosphomannose isomerase from sphingomonas chungbukensis DJ77

  • Tran, Sinh Thi;Le, Dung Tien;Kim, Young-Chang;Shin, Malshik;Choi, Jung-Do
    • BMB Reports
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    • v.42 no.8
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    • pp.523-528
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    • 2009
  • Phosphomannose isomerase (PMI) catalyzes the interconversion of fructose-6-phosphate and mannose-6-phosphate in the extracellular polysaccharide (EPS) synthesis pathway. The gene encoding PMI in Sphingomonas chungbukensis DJ77 was cloned and expressed in E. coli. The pmi gene is 1,410 nucleotides long and the deduced amino acid sequence shares high homology with other bifunctional proteins that possess both PMI and GDP-mannose pyrophosphorylase (GMP) activities. The sequence analysis of PMI revealed two domains with three conserved motifs: a GMP domain at the N-terminus and a PMI domain at the C-terminus. Enzyme assays using the PMI protein confirmed its bifunctional activity. Both activities required divalent metal ions such as $Co^{2+}$, $Ca^{2+}$, $Mg^{2+}$, $Ni^{2+}$ or $Zn^{2+}$. Of these ions, $Co^{2+}$ was found to be the most effective activator of PMI. GDP-D-mannose was found to inhibit the PMI activity, suggesting feedback regulation of this pathway.

Comparative analysis of AGPase proteins and conserved domains in sweetpotato (Ipomoea batatas (L.) Lam.) and its two wild relatives

  • Nie, Hualin;Kim, Sujung;Kim, Jongbo;Kwon, Suk-Yoon;Kim, Sun-Hyung
    • Journal of Plant Biotechnology
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    • v.49 no.1
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    • pp.39-45
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    • 2022
  • Conserved domains are defined as recurring units in molecular evolution and are commonly used to interpret the molecular function and biochemical structure of proteins. Herein, the ADP-glucose pyrophosphorylase (AGPase) amino acid sequences of three species of the Ipomoea genus [Ipomoea trifida, I. triloba, and I. batatas (L.) Lam. (sweetpotato)] were identified to investigate their physicochemical and biochemical characteristics. The molecular weight, isoelectric point, instability index, and grand average of hyropathy markedly differed among the three species. The aliphatic index values of sweetpotato AGPase proteins were higher in the small subunit than in the large subunit. The AGPase proteins from sweetpotato were found to contain an LbH_G1P_AT_C domain in the C-terminal region and various domains (NTP_transferase, ADP_Glucose_PP, or Glyco_tranf_GTA) in the N-terminal region. Conversely, most of its two relatives (I. trifida and I. triloba) were found to only contain the NTP_transferase domain in the N-terminal region. These findings suggested that these conserved domains were species-specific and related to the subunit types of AGPase proteins. The study may enable research on the AGPase-related specific characteristics of sweetpotatoes that do not exist in the other two species, such as starch metabolism and tuberization mechanism.

Disaccharide Synthesis using E. coli UDP-glucose regeneration system (대장균의 UDP-glucose regeneration 시스템을 이용한 이당류 합성에 관한 연구)

  • Oh, Jeong-Seok
    • KSBB Journal
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    • v.23 no.6
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    • pp.474-478
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    • 2008
  • UDP-glucose regeneration system using metabolic engineeringis unique and efficient strategy for oligosaccharide synthesis. To exploit the efficient UDP-glucose regeneration system, we introduced four enzymes, which would be important in partitioning the flux of UDP regenerationsuch as UDP-glucose pyrophosphorylase, UDP-Kinase gene, UDP-galactose 4-epimerase, and $\beta$-1, 4-galactasyltrasnsferase, into E. coli AD202. To determine the optimal expression level for UDP-regeneration, LacNAc concentration was compared depending on IPTG concentration. 0.5 mM IPTG induction showed the higher oligosaccharides synthesis. Using metabolic engineering under optimal IPTG induction, LacNAc synthesis of AD202/pQNGLU increased until 16 h and showed the 1.34 mM. This concentration is 10 times higher than that of control strain at same reaction time. Lactose of AD202/pQNGLU showed the maximum synthesis of 0.39 mM at 16 h and showed the 2.6 times higher than that of control strain.

Inhibition of Starch Biosynthesis by Antisense Expression of cDNAs Encoding ADP-Glucose Pyrophosphorylase Small Subunit in Sweetpotato (고구마에서 ADP-Glucose Pyrophosphorylase Small Subunit cDNA의 Antisense 발현에 의한 전분생합성 저해)

  • Min, Sung-Ran;Bae, Jung-Myung;Harn, Chee-Hark;Jeong, Won-Joong;Lee, Young-Bok;Liu, Jang-Ryol
    • Journal of Plant Biotechnology
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    • v.34 no.4
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    • pp.277-283
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    • 2007
  • Embryogenic calluses derived from shoot apical meristem explants of sweetpotato were subjected to particle bombardment to generate transgenic plants for antisense expression of cDNAs encoding two different AGPase small subunit (ibAGP1 and ibAGP2). Plants were generated via somatic embryogenesis. PCR and Southern analysis demonstrated that the incorporation of ibAGP1 and ibAGP2 into the genome in an antisense orientation. Immunoblot analysis confirmed reduced levels of AGPase small subunit in transgenic plant leaves. Plants with both ibAGP1 and ibAGP2 produced a lower level of the protein than plants with ibAGP1 alone. iodine test demonstrated that transgenic plant leaves and storage root accumulated reduced amounts of starch. Iodine staining of leaf tissues indicated that transgenic plants accumulated less amount of starch than control. In accordance with western blot analysis, plants with both ibAGP1 and ibAGP2 accumulated a lower amount of starch than plants with ibAGP1 alone. Both transgenic plants exhibited a severely retarded growth, resulting in bare survival. It is suggested that disrupted expression of the gene encoding AGPase small subunit is lethal to the growth of sweetpotato contrast to other species including potato.

Short-Cut Pathway to Synthesize Cellulose of Encysting Acanthamoeba

  • Moon, Eun-Kyung;Kong, Hyun-Hee
    • Parasites, Hosts and Diseases
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    • v.50 no.4
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    • pp.361-364
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    • 2012
  • The mature cyst of Acanthamoeba is highly resistant to various antibiotics and therapeutic agents. Cyst wall of Acanthamoeba are composed of cellulose, acid-resistant proteins, lipids, and unidentified materials. Because cellulose is one of the primary components of the inner cyst wall, cellulose synthesis is essential to the process of cyst formation in Acanthamoeba. In this study, we hypothesized the key and short-step process in synthesis of cellulose from glycogen in encysting Acanthamoeba castellanii, and confirmed it by comparing the expression pattern of enzymes involving glycogenolysis and cellulose synthesis. The genes of 3 enzymes, glycogen phosphorylase, UDP-glucose pyrophosphorylase, and cellulose synthase, which are involved in the cellulose synthesis, were expressed high at the 1st and 2nd day of encystation. However, the phosphoglucomutase that facilitates the interconversion of glucose 1-phosphate and glucose 6-phosphate expressed low during encystation. This report identified the short-cut pathway of cellulose synthesis required for construction of the cyst wall during the encystation process in Acanthamoeba. This study provides important information to understand cyst wall formation in encysting Acanthamoeba.

Current status on carbon metabolic engineering in plants (식물의 탄소대사공학 연구동향)

  • Kim, Dong-Hern;Lee, Si-Myung;Park, Jong-Suk;Kim, Soo-Jin;Kim, Beom-Ki;Yun, In-Sun;Kim, Dul-I;Byun, Myung-Ok
    • Journal of Plant Biotechnology
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    • v.37 no.2
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    • pp.205-211
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    • 2010
  • Yield productivity of staple crops must be increased at least 50% by 2050, in order to feed the world population which is expected to reach 90 billions. Photosynthetic carbon assimilation and carbohydrate metabolism leading to the production of starch would be the final frontier to quest for new sources of technology enabling such a drastic increase of crop productivity. In this review, attempts to genetically engineer plant photosynthetic carbon reduction cycle and metabolic pathways to increase starch production are introduced.