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Frontiers of Chemical Science and Engineering

ISSN 2095-0179

ISSN 2095-0187(Online)

CN 11-5981/TQ

Postal Subscription Code 80-969

2018 Impact Factor: 2.809

Front. Chem. Sci. Eng.    2016, Vol. 10 Issue (2) : 238-244    https://doi.org/10.1007/s11705-016-1566-2
RESEARCH ARTICLE
Functional characterization of a thermostable methionine adenosyltransferase from Thermus thermophilus HB27
Yanhui Liu,Biqiang Chen,Zheng Wang,Luo Liu(),Tianwei Tan()
Beijing Key Laboratory of Bioprocess, Beijing University of Chemical Technology, Beijing 100029, China
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Abstract

MATTt (a thermostable methionine adenosyltransferase from Thermus thermophilus HB27) was overexpressed in Escherchia coli and purified using Ni-NTA affinity column. The enzymatic activity of MATTt was investigated in a temperature range from 30 °C to 90 °C, showing that MATTt exhibited a high enzymatic activity and good thermostability at 80 °C. Circular dichroism spectra reveals that MATTt contains high portion of β-sheet structures contributing to the thermostability of MATTt. The kinetic parameter, Km is 4.19 mmol/L and 1.2 mmol/L for ATP and methionine, respectively. MATTt exhibits the highest enzymatic activity at pH 8. Cobalt (Co2+) and zinc ion (Zn2+) enhances remarkably the activity of MATTt compared to the magnesium ion (Mg2+). All these results indicated that the thermostable MATTt has great potential for industry applications.

Keywords ion-preference      methionine adenosyltransferase      secondary structure      thermostability      Thermus thermophilus     
Corresponding Author(s): Luo Liu,Tianwei Tan   
Online First Date: 01 April 2016    Issue Date: 19 May 2016
 Cite this article:   
Yanhui Liu,Biqiang Chen,Zheng Wang, et al. Functional characterization of a thermostable methionine adenosyltransferase from Thermus thermophilus HB27[J]. Front. Chem. Sci. Eng., 2016, 10(2): 238-244.
 URL:  
https://academic.hep.com.cn/fcse/EN/10.1007/s11705-016-1566-2
https://academic.hep.com.cn/fcse/EN/Y2016/V10/I2/238
Fig.1  The phylogenetic tree was constructed based on the multiple sequence alignments created using the Clustal X software [22], and drawn using program TreeView 1.6.6 [23]. The scale bottom left indicates the bootstrap scores according to the branch length. The sequence alignments show the similarity between the sequences (Fig. S1 in Supplementary information). The organisms for other MATs sequences and their respective GenBank accession numbers were as follows: Acanthamoeba castellanii, Q95032; Amoeba proteus, O09486; Arabidopsis thaliana, NP_192094; Ascobolus immersus, P50304; Aquifex aeolicus, NP_213786; Bacillus subtilis, NP_390933; Borrelia burgdorferi, NP_212510; Brassica juncea, P49611; Caenorhabditis elegans, NP_872086; Drosophila melanogaster, CAA54567; Escherichia coli, NP_289514; Haemophilus influenzae, NP_439330; Homo sapiens, NP_005902; Hordeum vulgare, P50299; Leishmania infantum, O43938; Lycopersicum esculentum, XP_004252944; Mycobacterium tuberculosis, NP_215908; Mycoplasma genitalium, NP_072707; Mycoplasma pneumoniae, NP_109748; Neurospora crassa, XP_965430; Rattus norvegicus, NP_599178; Staphylococcus aureus, P50307; Sulfolobus solfataricus, YP_005643019; Synechocystis sp., YP_007450090; Thermus thermophiles, YP_144908; Treponema pallidum, NP_219231; Methanocaldococcus jannaschii, Q58605
Fig.2  SDS gel analysis of the proteins. (A) M: molecular weight marker; 1: whole cell sample before induction; 2: whole cell sample 6 h after induction; 3: supernatant (10 µg proteins) after cell disruption; 4: diluted supernatant (1 µg proteins) after heat treatment for better determination of molecular weight; 5: supernatant (4.6 µg proteins) after heat treatment. (B) line 6: protein (5 µg proteins) after purification using His-tags.
Fig.3  Lineweaver-Burk plot. (A) The double bottom curve of ATP; (B) The double bottom curve of L-Met. The KM and Vmax were calculated according to the slopes.
Organism Vmax /(µmol·min–1·mg–1) Km (ATP) /(mmol/L) Km (methionine) /(mmol/L) Temperature /°C Ref.
T. thermophilus 0.84 4.19 1.2 70 This study
M. jannaschiia 3.0 0.25 0.14 70 [25]
E. coli 1.2 0.11 0.08 25 [26]
H. sapiens 0.2 0.03 0.003 37 [27]
B. subtilis 0.36 0.92 0.26 37 [14]
Tab.1  Comparison of MATTt with MATs from different organisms
Fig.4  The stability of MATTt at 70 °C (solid line), 80 °C (dash line), and 90 °C (dot line). The sample was incubated in a water bath and the residual activity was determined at scheduled time. The initial activity was expressed as 100%
Fig.5  Circular dichroism spectra of MATTt at RT, 70, 80 and 90 °C
Program Secondary structure Temperature /°C
25 70 80 90
K2D2 α-helix/% 15.97 12.52 3.44 3.69
β-sheet/% 30.83 35.2 47.08 44.51
SELCON3 α-helix/% 81.1 81.1 46.6 43.9
β-sheet/% 0.8 0.6 12.0 28.7
β-turns/% 7.1 6.7 24.2 21.7
Random coils/% 16.1 15.7 33.4 33.1
Tab.2  Contents of the secondary structures of MATTt at different temperatures
Fig.6  Effect of temperature on MATTt activity. The activity determined at 80 °C was expressed as 100%.
Fig.7  pH-preference of MATTt. The activity of MATTt determined at pH 8.0 was expressed as 100%
Fig.8  Ion-preference of MATTt. The activity of MATTt in the presence of Zn2+ was expressed as 100%
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