• 中国精品科技期刊
  • 中国高校百佳科技期刊
  • 中国中文核心期刊
  • 中国科学引文数据库核心期刊
Advanced Search
LIU Zhangmin, CHAO Jiajia, FENG Yan, LI Qian, CUI Li. Characterization of glutamate dehydrogenase SHJG_7666 from Streptomyces hygroscopicus 5008[J]. Journal of China Pharmaceutical University, 2018, 49(3): 360-368. DOI: 10.11665/j.issn.1000-5048.20180316
Citation: LIU Zhangmin, CHAO Jiajia, FENG Yan, LI Qian, CUI Li. Characterization of glutamate dehydrogenase SHJG_7666 from Streptomyces hygroscopicus 5008[J]. Journal of China Pharmaceutical University, 2018, 49(3): 360-368. DOI: 10.11665/j.issn.1000-5048.20180316

Characterization of glutamate dehydrogenase SHJG_7666 from Streptomyces hygroscopicus 5008

More Information
  • Glutamate dehydrogenase(GDH)a key enzyme in the nitrogen metabolism pathway catalyzes the conversion between α-ketoglutarate and glutamate reversibly using NAD(P)H as a cofactor. Based on genomic studies, it was concluded that SHJG_7666 was a potential GDH in Streptomyces hygroscopicus 5008(S5008), and its expression level in vivo was positively correlated with the biosynthesis of an important aminocyclol compound validamycin. Phylogenetic tree analysis showed that the S5008 SHJG_7666 GDH belonged to the Glu/Leu/Phe/Val dehydrogenase family, with conserved glutamate-α-ketoglutarate binding domain and the classical GXGXXG dinucleotide binding motif. Further homologous modeling and structural comparison revealed that SHJG_7666 contained conserved Lys60, Lys78 and Asp120 catalytic functional sites and ligand binding residues Ser36, Gly38, Gln119and Asp166, Asn300, Ala330. Moreover, recombinant expression of SHJG_7666 in E. coli and in vitro enzyme activity demonstrated that glutamate dehydrogenase can convert ammonium salt to glutamate with pH and temperature being optimal at 7. 5 and 37 °C respectively. Enzyme activity under optimum reaction condition has Km value of(25. 3±9. 1)μmol/L and kcat of(3±0. 8)×10-5 s-1 for the substrate α-ketoglutarate. Results of this study further improved the catalytic activity of SHJG_7666, thus laying the foundation for the ultimate increase of validamycin production.
  • [1]
    Spanaki C,Plaitakis A.The role of glutamate dehydrogenase in mammalian ammonia metabolism[J].Neurotox Res,2012,21(1):117-127.
    [2]
    Miflin BJ,Habash DZ.The role of glutamine synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops[J].J Exp Bot,2002,53(370):979.
    [3]
    Ameziane R,Bernhard K,Lightfoot D,et al.Expression of the bacterial gdhA gene encoding a NADPH glutamate dehydrogenase in tobacco affects plant growth and development[J].Plant Soil,2000,221(1):47-57.
    [4]
    Fonnum F.Glutamate:a neurotransmitter in mammalian brain[J].J Neurochem,1984,42(1):1-11.
    [5]
    Shashidharan P,Plaitakis A.The discovery of human of GLUD2 glutamate dehydrogenase and its implications for cell function in health and disease[J].Neurochem Res,2014,39(3):460-470.
    [6]
    Kanamori K, Weiss RL, Roberts JD, et al. Role of glutamate dehydrogenase in ammonia assimilation in nitrogen-fixing Bacillus macerans[J].J Bacteriol,1987,169(10):4692-4695.
    [7]
    Magasanik B.Genetic control of nitrogen assimilation in bacteria[J].Annu Rev Genet,1982,16(1):135-168.
    [8]
    Du LX,Jia SJ,Lu FP,et al.Morphological changes of Rhizopus chinesis 12 in submerged culture and its relationship with antibiotic production[J].Process Biochem,2003,38(12):1643-1646.
    [9]
    Gu WL,Zhang XC,Jiao RS,et al.Resarch on nitrogen metabolism of validamycin biosynthesis from Streptomyces hygroscopicus[J].Chin Sci Bull(科学通报),1984,13:15.
    [10]
    Liao YQ,Wei ZH,Zhong JJ,et al.Effect of fermentation temperature on validamycin A production by Streptomyces hygroscopicus 5008[J].J Biotechnol,2009,142(3):271-274.
    [11]
    Iwasa T,Higashide E,Shibata M,et al.Studies on validamycins,new antibiotics.III[J].J Antibiot,1971,24(2):114-118.
    [12]
    Hodgson DA.Primary metabolism and its control in streptomycetes:a most unusual group of bacteria[J].Adv Microb Physiol,2000,42:47-238.
    [13]
    Lee S, Egelkrout E. Biosynthetic studies on the α-glucosidase inhibitor acarbose in Actinoplanes sp.:glutamate is the primary source of the nitrogen in acarbose[J].J Antibiot,1998,51(2):225-227.
    [14]
    Wu H, Bai LQ, Deng ZX, et al. Genomic and transcriptomic insights into the thermo-regulated biosynthesis of validamycin in Streptomyces hygroscopicus 5008[J].BMC Genomics,2012,13(1):337.
    [15]
    He LW,Liu ZM,Cui L,et al.High throughput screening method and application for L-glutamate specific aminotransferase[J].China Biotech(中国生物工程杂志),2017,37(8):59-65.
    [16]
    Roy A,Kucukural A,Zhang Y,et al.I-TASSER:a unified platform for automated protein structure and function prediction[J].Nat Protoc,2010,5(4):725.
    [17]
    Smith TJ,Peterson PE,Schmidt T,et al.Structures of bovine glutamate dehydrogenase complexes elucidate the mechanism of purine regulation[J].J Mol Biol,2001,307(2):707-720.
    [18]
    Britton KL, Baker PJ, Engel PC, et al. Evolution of substrate diversity in the superfamily of amino acid dehydrogenases:prospects for rational chiral synthesis[J].J Mol Biol,1993,234(4):938-945.
    [19]
    Miñambres B,Olivera ER,Jensen RA,et al.A new class of glutamate dehydrogenases(GDH)biochemical and genetic characterization of the first member,the AMP-requiring nad-specific GDH of Streptomyces clavuligerus[J].J Biol Chem,2000,275(50):39529-39542.
    [20]
    Britton KL, Baker PJ, Rice DW, et al. Structural relationship between the hexameric and tetrameric family of glutamate dehydrogenases[J].FEBS J,1992,209(3):851-859.
    [21]
    Lesk AM.NAD-binding domains of dehydrogenases[J].Curr Opin Struct Biol,1995,5(6):775-783.
    [22]
    Baker PJ,Farrants GW,Rice DW,et al.Recent progress on the structure and function of glutamate dehydrogenase[J].Biochem Soc Trans,1987,15(4):748-751.
    [23]
    Peterson PE,Smith TJ.The structure of bovine glutamate dehydrogenase provides insights into the mechanism of allostery[J].Structure,1999,7(7):769-782.
    [24]
    Baker PJ, Britton KL, Engel PC, et al. Subunit assembly and active site location in the structure of glutamate dehydrogenase[J].Proteins,1992,12(1):75-86.
  • Related Articles

    [1]XIE Jing, FAN Chunlin, XU Jie, ZHANG Jian, YE Wencai, ZHANG Xiaoqi. Alkaloids of Ervatamia pandacaqui[J]. Journal of China Pharmaceutical University, 2021, 52(3): 287-292. DOI: 10.11665/j.issn.1000-5048.20210304
    [2]LI Linzhen, WEI Xi, LIU Lu, LI Yongjun, LIANG Jingyu. Chemical constituents from the stems of Clerodendrum trichotomum Thunb.[J]. Journal of China Pharmaceutical University, 2019, 50(5): 544-548. DOI: 10.11665/j.issn.1000-5048.20190506
    [3]LIN Qinghua, XU Jian, FENG Feng. Chemical constituents from the stems of Picrasma quassioides Bennet[J]. Journal of China Pharmaceutical University, 2017, 48(6): 675-679. DOI: 10.11665/j.issn.1000-5048.20170607
    [4]HUANG Qilong, ZHANG Wanjin, LI Yan, CHEN Juan, ZHOU Baoping, ZOU Xiaohan, ZHANG Chunlei, CAO Zhengyu. Alkaloid constituents from Corydalis decumbens[J]. Journal of China Pharmaceutical University, 2017, 48(5): 563-567. DOI: 10.11665/j.issn.1000-5048.20170509
    [5]XU Yunhui, JIANG Xueyang, XU Jian, JIANG Renwang, ZHANG Jie, XIE Zijian, FENG Feng. Chemical constituents from Callicarpa kwangtungensis Chun[J]. Journal of China Pharmaceutical University, 2016, 47(3): 299-302. DOI: 10.11665/j.issn.1000-5048.20160309
    [6]MA Lin, ZHANG Rongfei, YU Shule, WU Zhengfeng, ZHAO Shouxun, Wang Lei, YE Wencai, ZHANG Jian, YIN Zhiqi. Chemical constituents of Fructus Gleditsiae Abnormalis[J]. Journal of China Pharmaceutical University, 2015, 46(2): 188-193. DOI: 10.11665/j.issn.1000-5048.20150209
    [7]LI Linzhen, WANG Menghua, SUN Jianbo, LIANG Jingyu. Chemical constituents from Aletris spicata[J]. Journal of China Pharmaceutical University, 2014, 45(2): 175-177. DOI: 10.11665/j.issn.1000-5048.20140208
    [8]CHANG Bo, XIAO Linjing, ZHANG Jian, ZHAO Shouxun, YE Wencai, YIN Zhiqi. Chemical constituents from Abies ernestii var.salouenensis[J]. Journal of China Pharmaceutical University, 2014, 45(1): 43-47. DOI: 10.11665/j.issn.1000-5048.20140107
    [9]LI Jiu-hui, CHEN Guang-ying, HAN Chang-ri, MO Zheng-rong, SONG Xiao-ping. Chemical constituents from the stems of Vatica mangachpoi Blanco[J]. Journal of China Pharmaceutical University, 2012, 43(1): 25-27.
    [10]Chemical constituents from Senecio nemorensis.[J]. Journal of China Pharmaceutical University, 2010, 41(1): 26-28.

Catalog

    Article views (649) PDF downloads (1145) Cited by()

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return