Glutamate dehydrogenase(GDH)plays an important role in the ammonium assimilation and nitrogen metabolism by catalyzing the reversible oxidative deamination of L-glutamate toα-ketoglutarate.In the present study,the po...Glutamate dehydrogenase(GDH)plays an important role in the ammonium assimilation and nitrogen metabolism by catalyzing the reversible oxidative deamination of L-glutamate toα-ketoglutarate.In the present study,the potential functions of GDH in response to heat stre ss were explored in the scleractinian coral Pocillopora damicornis(designated as PdGDH).The cDNA of PdGDH contained an open reading frame of 1611 bp encoding a polypeptide of 536 amino acids,which exhibited the highest sequence identity to GDH of Stylophora pistillata(96%identity),and the deduced PdGDH protein was predicted to contain one GdhA domain(from Val95 to Tyr525).The recombinant protein of PdGDH(rPdGDH)was expressed in Escherichia coli BL21(DE3)-Transetta,and its catalytic activity was measured under different temperatures,pH conditions and epigallocatechin-3-gallate(EGCG,a GDH inhibitor)concentrations.The purified rPdGDH only used reduced coenzyme nicotinamide adenine dinucleotide(NADH)as coenzyme,and its highe st activity was observed at 35℃and pH 7.5,re spectively.The rPdGDH activity was negatively correlated with the concentration of EGCG,and was inhibited by more than half(65%,P<0.05)at 10mol/L EGCG.No significant alteration of PdGDH mRNA expression was detected at 12 h after exposure to heat and ammonium(P>0.05).Furthermore,the activities of NADH-GDH in the scleractinian coral P.damicornis increased significantly at 12 h after the heat and ammonium stress,and the NADH-GDH activity in the heat stress group(32.66 U/mg,P<0.05)was significantly higher than that in the heat and ammonium stress group(11.26 U/mg).These results collectively suggested that PdGDH,as a homologue of glutamate dehydrogenase in the scleractinian coral P.damicornis,could respond to heat stress at the protein level,which would have ability to further promote ammonium assimilation to increase the heat acclimatization of the coral-Symbiodiniaceae symbiotic association.展开更多
基金Supported by the Major Science and Technology Program of Hainan Province (No. ZDKJ2019011)the ISF-NSFC Joint Scientifi c Research Program (No. 42161144006 or 3511/21)+1 种基金the National Natural Science Foundation of China (Nos. 31772460, 42076145)the Hainan Provincial Natural Science Foundation of China (Nos. 2019RC067, 420CXTD432)
文摘Glutamate dehydrogenase(GDH)plays an important role in the ammonium assimilation and nitrogen metabolism by catalyzing the reversible oxidative deamination of L-glutamate toα-ketoglutarate.In the present study,the potential functions of GDH in response to heat stre ss were explored in the scleractinian coral Pocillopora damicornis(designated as PdGDH).The cDNA of PdGDH contained an open reading frame of 1611 bp encoding a polypeptide of 536 amino acids,which exhibited the highest sequence identity to GDH of Stylophora pistillata(96%identity),and the deduced PdGDH protein was predicted to contain one GdhA domain(from Val95 to Tyr525).The recombinant protein of PdGDH(rPdGDH)was expressed in Escherichia coli BL21(DE3)-Transetta,and its catalytic activity was measured under different temperatures,pH conditions and epigallocatechin-3-gallate(EGCG,a GDH inhibitor)concentrations.The purified rPdGDH only used reduced coenzyme nicotinamide adenine dinucleotide(NADH)as coenzyme,and its highe st activity was observed at 35℃and pH 7.5,re spectively.The rPdGDH activity was negatively correlated with the concentration of EGCG,and was inhibited by more than half(65%,P<0.05)at 10mol/L EGCG.No significant alteration of PdGDH mRNA expression was detected at 12 h after exposure to heat and ammonium(P>0.05).Furthermore,the activities of NADH-GDH in the scleractinian coral P.damicornis increased significantly at 12 h after the heat and ammonium stress,and the NADH-GDH activity in the heat stress group(32.66 U/mg,P<0.05)was significantly higher than that in the heat and ammonium stress group(11.26 U/mg).These results collectively suggested that PdGDH,as a homologue of glutamate dehydrogenase in the scleractinian coral P.damicornis,could respond to heat stress at the protein level,which would have ability to further promote ammonium assimilation to increase the heat acclimatization of the coral-Symbiodiniaceae symbiotic association.
