Difference between revisions of "Part:BBa K2286012"

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<partinfo>BBa_K2286012 short</partinfo>
 
<partinfo>BBa_K2286012 short</partinfo>
  
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This is a halohydrin dehalogenase from  Agrobacterium radiobacterAD1(HheC). HheC is an important enzyme in the halide dehalogenation reaction, which catalyzes o-halide to epoxides and hydrogen halides through intramolecular nucleophilic substitution mechanism. So it can catalyze many substrates, such as 1,3-dichloro propanol (2,3-DCP), 2,3-dichloropropane-1-ol (2,3-DCP), 3-chloropropane-1,2-diol (CPD) and so on.
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This is a halohydrin dehalogenase from  <em>Agrobacterium radiobacterAD1</em>(HheC). HheC is an important enzyme in the halide dehalogenation reaction, which catalyzes o-halide to epoxides and hydrogen halides through intramolecular nucleophilic substitution mechanism. So it can catalyze many substrates, such as 1,3-dichloro propanol (2,3-DCP), 2,3-dichloropropane-1-ol (2,3-DCP), 3-chloropropane-1,2-diol (CPD) and so on.
  
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2286012 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2286012 SequenceAndFeatures</partinfo>
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==Characterization==
 
==Characterization==
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[[File:T--UESTC-China--HheC-2,3-DCP-pH8.5.png|500px|thumb|center|'''Fig 3'''.The relationship between substrate concentration and reaction rate.Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃]]
 
[[File:T--UESTC-China--HheC-2,3-DCP-pH8.5.png|500px|thumb|center|'''Fig 3'''.The relationship between substrate concentration and reaction rate.Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃]]
  
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The results of pH 8 and pH 8.5 show that pH 8.5 is more suitable for HheC catalytic reaction of 2,3-DCP.
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===Michaelis constant of HheC toward CPD===
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Km=7.04277±2.07491mM
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Kcat=0.676±0.065 S-1
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[[File:T--UESTC-China--HheC-CPD-pH8.png|500px|thumb|center|'''Fig 4'''.The relationship between substrate concentration and reaction rate.Data were measured in 50mM Tris-H2SO4 at pH 8 and 37℃]]
  
  

Latest revision as of 17:50, 1 November 2017


HheC: halohydrin dehalogenase

This is a halohydrin dehalogenase from Agrobacterium radiobacterAD1(HheC). HheC is an important enzyme in the halide dehalogenation reaction, which catalyzes o-halide to epoxides and hydrogen halides through intramolecular nucleophilic substitution mechanism. So it can catalyze many substrates, such as 1,3-dichloro propanol (2,3-DCP), 2,3-dichloropropane-1-ol (2,3-DCP), 3-chloropropane-1,2-diol (CPD) and so on.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 718
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

Molecular weight

This halohydrin dehalogenase gene codes for a protein of 762 amino acids with a molecular mass of 29kDa[2].

Structure of HheC: homologous tetramer

It is reported that halohydrin dehalogenase HheC is a symmetric tetramer that can be thought of as a dimer. The four active centers of HheC tetramer are centrosymmetric. The active sites include substrate binding sites and halide binding sites, surrounded by four Loop regions, loop1 (F12-Q87), Loop2 (A133 (373), Loop3 (P175-F188) and Loop4 (F243-P253), while the Ser132-Tyr145-Arg149 catalytic triad is contained in these four Loops. There is a synergistic effect between the amino acid residues on the four Loop regions, which are involved in the change of the active site conformation during the enzymatic catalysis. At the same time, it is reported that the C-terminus of the HheC plays an important role in its catalytic activity and thermal stability [1].

Fig 1.3D model of HheC.

Michaelis constant of HheC toward 2,3-DCP

Michaelis constant of HheC at pH 8

Km=0.86642±0.10932mM Kcat=0.569±0.020S-1

Fig 2.The relationship between substrate concentration and reaction rate .Data were measured in 50mM Tris-H2SO4 at pH 8 and 37℃

Michaelis constant of HheC at pH 8.5

Km=3.03149±0.55904 mM Kcat=1.241±0.095 S-1

Fig 3.The relationship between substrate concentration and reaction rate.Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃

The results of pH 8 and pH 8.5 show that pH 8.5 is more suitable for HheC catalytic reaction of 2,3-DCP.

Michaelis constant of HheC toward CPD

Km=7.04277±2.07491mM Kcat=0.676±0.065 S-1

Fig 4.The relationship between substrate concentration and reaction rate.Data were measured in 50mM Tris-H2SO4 at pH 8 and 37℃