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structure with acetylene or an inhibitor bound [ 21 , 27 ], an acetylene molecule
docked computationally to the AH structure gave an excellent fit in the pocket of
the hydrophobic ring with its carbon atoms positioned directly above the oxygen
ligand and the carboxylic acid group of Asp13 [ 21 ].
Crucial for deriving a reaction mechanism for AH from the X-ray structure is the
nature of the oxygen ligand of the tungsten ion. The bond length of 2.04
observed
in the structure falls between the values expected for a hydroxo ligand (OH
1.9-2.1
Å
)[ 21 ]. Seiffert et al. [ 21 ]
decided for a water molecule, since the close proximity of the heavy scatterer
tungsten may distort the distance observed in the X-ray data by Fourier series
termination and a simulation of this effect resulted in a true ligand distance of 2.25
Å
) and a coordinated water molecule (2.0-2.3
Å
Å
.
4.6 Site-Directed Mutagenesis
The development of a protocol for the heterologous expression of AH in
Escherichia coli allowed for further studies towards the reaction mechanism by
site-directed mutagenesis [ 22 ]. Initially, the metal and cofactor content of the
expressed protein was quite low compared to that of the native AH from
P. acetylenicus (0.06 mol W and 1.22 mol Fe versus 0.4 mol W and 3.7 mol Fe
per mol enzyme) [ 22 ]. This problem was partially solved by addition of an
N-terminal chaperone-binding sequence of the soluble nitrate reductase NarG
from E. coli . According to the literature this 30 amino acid long sequence is one
of two binding sites for chaperones during insertion of the bis-MGD cofactor during
protein biosynthesis [ 30 ]. The resulting fusion variant of AH had a higher content of
W (0.14 mol per mol enzyme) and Fe (3.5 mol per mol enzyme) and therefore an
increased activity compared to the heterologously expressed AH. Overall, when
normalized to their W content, the activity of the heterologously expressed AH
was nearly identical to that of the native enzyme purified from P. acetylenicus
(Table 1 )[ 22 ].
Table 1 Specific and relative activity of acetylene hydratase and variants [ 22 ].
W
mol/mol AH
Fe
mol/mol AH
Specific activity
[
Relative
activity W a
mol*min 1 *mg 1 ]
Acetylene hydratase
ʼ
P. acetylenicus AH
0.37
0.04
3.69
0.04
14.2
0.9
38.4
E. coli AH
0.06
0.02
1.22
0.26
2.6
0.8
43.3
E. coli AH D13A
0.09 0.02
1.17 0.29
0.2 0.1
2.2
E. coli AH D13E
0.05 0.01
1.11 0.30
2.5 0.3
50.0
E. coli NarG-AH
0.14
0.06
3.17
0.49
9.7
1.9
69.3
E. coli NarG-AH K48A
0.15
0.01
3.56
0.31
7.2
0.3
48.0
E. coli NarG-AH I142A
0.18
0.02
3.20
0.22
2.2
0.2
12.2
a
Relative to tungsten concentration, in nmol.
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