Cloning,Expression and Characterization of a Novel Acyltransferase GPAT

doi:10.13560/j.cnki.biotech.bull.1985.2020-1508

Abstract

Abstract:

Lithocarpins,a new polyketide skeleton compound isolated from marine fungus Phomopsis littocarpus,has the potential to be developed as lead compound of novel antitumor drugs. Based on genome sequencing and bioinformatics analysis,the biosynthetic gene cluster of lithocarpins was predicted. The unknown functional gene g7779 was amplified and successfully expressed in Escherichia coli. The recombinant protein was purified by Ni-NTA affinity chromatography. The function of the purified protein was analyzed by mass spectrometry,bioinformatics analysis combined with enzyme activity detection. The results showed that g7779 encoded a novel bi-functional enzyme（glutamic-pyruvic transaminase-acyltransferase,GPAT）with strong acyltransferase activity and certain glutamic pyruvic transaminase activity,its purity was 98.6%. Structural analysis showed that the protein was composed of 326 amino acids with a molecular weight of 36 kD. The secondary structure of GPAT consisted of 55.52% α-helix,29.45% random coil,9.82% extend strand and 5.21% β-turn. The activity of acyltransferase demonstrated that the optimal reaction temperature of GPAT for acetyl CoA was 35℃,and the highest enzyme activity was at pH 7.0. GPAT showed good thermal stability at 40℃,and the residual enzyme activity was > 80% after 2 h treatment at this temperature;but the thermal stability was poor at 50℃,and the residual enzyme activity was < 10% after 30 min. The values of K_m and V_max of GPAT towards acetyl CoA were 761.57 μmol/L and 29 370 μmol/（mg·min）,respectively. The results may provide molecular biological basis for the elucidation of lithocarpins biosynthesis pathway.

Key words: marine fungus, Phomopsis lithocarpus, heterologous expression, acyltransferase, enzymatic properties, bioinformatics

LIU Shan, YE Wei, ZHU Mu-zi, LI Sai-ni, DENG Zhang-shuang, ZHANG Wei-min. Cloning,Expression and Characterization of a Novel Acyltransferase GPAT[J]. Biotechnology Bulletin, 2021, 37(11): 257-266.

Figures/Tables 9

Fig.1 PCR verification of gene g7779 A:Cloning of gene g7779. B:PCR verification of bacterial solution. M:DNA marker 2K Plus. 1:Gene g7779. P:Positive control. 2-8:Positive monoclonal of pET28a-g7779

Fig.2 Purification and Western-Blot analysis of GPAT A:Optimization of the expression condition of GPAT. M:Blue Plus II protein marker. 1, 3, 5, 7, 9 and 11:Supernatant of condition ①, ②, ③, ④, ⑤ and ⑥. 2, 4, 6, 8, 10 and 12:Sediment of condition ①, ②, ③, ④, ⑤ and ⑥. B:Purification of GPAT. M:Blue Plus Ⅱ protein marker. 1:Uninduced total protein. 2:Total protein of supernatant after induction. 3-5:Binding buffer. 6:68 mmol/L imidazole eluent. 7:116 mmol/L imidazole eluent. C:Western-Blot analysis of GPAT. M:EasySee Western marker. 1:Uninduced total protein. 2:Total protein of supernatant after induction. 3:Binding buffer. 4:68 mmol/L imidazole eluent. 5:116 mmol/L imidazole eluent

Fig.3 MALDI-TOF result of representative peptides in GPAT

Fig.4 Matching of GPAT with mass spectrometry result The red part is the matching part

Fig.5 Characterization of enzymatic properties of GPAT A:Optimal reaction temperature of GPAT. B:Optimal reaction pH of GPAT. C:Thermostability of GPAT. D:Enzyme kinetics of GPAT

Fig.6 Hydrophilicity/hydrophobicity analysis of GPAT

Fig.7 Secondary structure analysis of GPAT The order of vertical lines from long to short are:α - helix（blue）,extend strand（red）,β - turn（green）and random coil（purple）

Fig.8 Prediction of phosphorylation sites of GPAT

Fig.9 Functional inference of GPAT in the biosynthetic pathway of lithocarpins

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