Engineering enhanced thermostability into the Geobacillus pallidus nitrile hydratase

Abstract

Nitrile hydratases (NHases) are important biocatalysts for the enzymatic conversion of nitriles to industrially-important amides such as acrylamide and nicotinamide. Although thermostability in this enzyme class is generally low, there is not sufficient understanding of its basis for rational enzyme design. The gene expressing the Co-type NHase from the moderate thermophile, Geobacillus pallidus RAPc8 (NRRL B-59396), was subjected to random mutagenesis. Four mutants were selected that were 3 to 15-fold more thermostable than the wild-type NHase, resulting in a 3.4-7.6 ​kJ/mol increase in the activation energy of thermal inactivation at 63 ​°C. High resolution X-ray crystal structures (1.15-1.80 ​Å) were obtained of the wild-type and four mutant enzymes. Mutant 9E, with a resolution of 1.15 ​Å, is the highest resolution crystal structure obtained for a nitrile hydratase to date. Structural comparisons between the wild-type and mutant enzymes illustrated the importance of salt bridges and hydrogen bonds in enhancing NHase thermostability. These additional interactions variously improved thermostability by increased intra- and inter-subunit interactions, preventing cooperative unfolding of α-helices and stabilising loop regions. Some hydrogen bonds were mediated via a water molecule, specifically highlighting the significance of structured water molecules in protein thermostability. Although knowledge of the mutant structures makes it possible to rationalize their behaviour, it would have been challenging to predict in advance that these mutants would be stabilising.

Keywords: Crystal structure; Directed evolution; Electrostatic interactions; Nitrile hydratase; Protein engineering; Protein stability; Random mutagenesis; Thermophile; Thermostability.

Graphical abstract

Fig. 1

Effect of thermal inactivation temperature on wild-type NHase thermostability using cell free extract (CFE). The error bars indicate the standard deviation between duplicate measurements.

Fig. 2

The hydroxamic assay performed in microtitre plate format shows a positive result obtained from screening Lib 0.1 for NHases with improved thermostability. (A) The activity of the enzymes was determined directly at 37°C. (B) The enzymes were then subjected to thermal inactivation at 60°C for 10 ​min and the activity was measured at 37°C. Potential thermostabilised NHases are indicated with blue arrows.

Fig. 3

The effect of temperature on the extent of partial thermal inactivation on wild-type and mutant NHases. The enzyme samples were incubated in the absence of substrate for 10 ​min at the indicated temperatures. Residual activity was determined under standard assay conditions. The error bars indicate the standard deviation between triplicate experiments.

Fig. 4

Thermal inactivation of wild-type and mutant NHases. Cell extracts were incubated without substrate at 63 ​°C for different periods of time. The percentage residual activity was determined under standard assay conditions. The error bars indicate the standard deviation between duplicate measurements. The first-order rate constant (k_inact) for each enzyme is given by the gradients of the lines generated by linear regression of the data.

Fig. 5

Comparison of thermal inactivation between 9C and αS169R. Cell extracts were incubated without substrate at 65 ​°C for different periods of time. The percentage residual activity was determined under standard assay conditions. Error bars indicate the standard deviation between duplicate measurements. The first-order rate constant (k_inact) for each enzyme is given by the gradients of the lines generated by linear regression of the data.

Fig. 6

Protein sequence of the G. pallidus NHase α and β subunits with associated secondary protein features. The distribution of amino acid changes that occur in thermostabilised NHases is highlighted in cyan. The parts of the protein that cannot be visualized in the structures are highlighted in red. Helices are written in red, β sheets in blue and 3₁₀ helices in maroon. Solvent accessible residues are in upper case and solvent inaccessible residues are in lower case. The figure was produced using JOY (Mizuguchi et al., 1998). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Electron density map showing the water-mediated hydrogen bond between αS47 and αE33 in mutant 7D. The electron density map was contoured at 1σ (1σ is one standard deviation) to ensure that the data were significant. The amino acid residues are shown as sticks where the atoms are: C ​= ​white, O ​= ​red, N ​= ​blue, H₂O ​= ​red spheres. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

Electron density map showing the salt bridge between αK195 and αE192 as a result of the M→V (α188) mutation in mutant 8C. The electron density map was contoured at 1σ (1σ is one standard deviation) to ensure that the data were significant. The amino acid residues are shown in ball and stick format where the atoms are: C ​= ​white, O ​= ​red, N ​= ​blue, S ​= ​yellow. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 9

Electron density map showing the double salt bridge between αR28 and βE96 and the hydrogen bond between βE96 and βE92 in mutant 8C. The electron density map was contoured at 1σ (1σ is one standard deviation) to ensure that the data were significant. The amino acid residues are shown as sticks in the mutant and lines in the wild-type where the atoms are: C ​= ​white, O ​= ​red, N ​= ​blue, H₂O ​= ​red spheres. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 10

Electron density map showing the salt bridge between αR169 and βD218 in mutant 9C. The electron density map was contoured at 1σ (1σ is one standard deviation) to ensure that the data were significant. The amino acid residues are shown as sticks in the mutant and lines in the wild-type where the atoms are: C ​= ​white, O ​= ​red, N ​= ​blue, S ​= ​yellow, H₂O ​= ​red spheres, Co ​= ​cyan sphere. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 11

Electron density map showing the hydrogen bond between βK43 and βK50 in mutant 9C. The electron density map was contoured at 1σ (1σ is one standard deviation) to ensure that the data were significant. The amino acid residues are shown as sticks where the atoms are: C ​= ​white, O ​= ​red, N ​= ​blue, H₂O ​= ​red spheres. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 12

Superimposition of the wild-type (brown) and mutant 9E (green) electron density maps showing the water-mediated hydrogen bond between αS103 and αS23. The water molecule is only present in the density attributed to the mutant. The electron density maps were contoured at 1σ (1σ is one standard deviation) to ensure that the data were significant. The amino acid residues are shown as sticks in the mutant and lines in the wild-type, where the atoms are: C ​= ​white, O ​= ​red, N ​= ​blue, H₂O ​= ​red sphere. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 13

Electron density map showing hydrogen bond formation of βY127 with αR49 and αE57 in the wild-type NHase. The replacement of βY127 with βN127 in mutant 9E results in the resultant space being filled with water. The electron density map was contoured at 1σ (1σ is one standard deviation) to ensure that the data is significant. All the amino acid residues are shown as ball and stick. In the wild-type the atoms are: C ​= ​white, O ​= ​red, N ​= ​blue, H₂O ​= ​red spheres while in the mutant the both the amino acid residues and the water molecules are coloured cyan. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)