DataSheet1_Improving the Stability and Activity of Arginine Decarboxylase at Alkaline pH for the Production of Agmatine.docx

Agmatine, involved in various modulatory actions in cellular mechanisms, is produced from arginine (Arg) by decarboxylation reaction using arginine decarboxylase (ADC, EC 4.1.1.19). The major obstacle of using wild-type Escherichia coli ADC (ADCes) in agmatine production is its sharp activity loss and instability at alkaline pH. Here, to overcome this problem, a new disulfide bond was rationally introduced in the decameric interface region of the enzyme. Among the mutants generated, W16C/D43C increased both thermostability and activity. The half-life (T1/2) of W16C/D43C at pH 8.0 and 60°C was 560 min, which was 280-fold longer than that of the wild-type, and the specific activity at pH 8.0 also increased 2.1-fold. Site-saturation mutagenesis was subsequently performed at the active site residues of ADCes using the disulfide-bond mutant (W16C/D43C) as a template. The best variant W16C/D43C/I258A displayed a 4.4-fold increase in the catalytic efficiency when compared with the wild-type. The final mutant (W16C/D43C/I258A) was successfully applied to in vitro synthesis of agmatine with an improved yield and productivity (>89.0% yield based on 100 mM of Arg within 5  h).</p

Fluorescence microscope image of s-GFP and deletion variants.

<p>s-GFP, s-N14 and s-C225 were employed in live cell imaging by expressing in <i>E. coli</i>. The images were captured using fluorescence microscope equipped with digital image analyzer.</p

Comparison of biophysical properties of s-GFP, n-GFP and variants.

[a]<p>The relative fluorescence (in arbitrary units) is defined as the whole cell fluorescence compared with fluorescence of cells expressing s-GFP. All the fluorescence values were normalized by the O.D_{600 nm} of expressed cells.</p>[b]<p>Specific activity (in arbitrary units/µM of purified protein) is the fluorescence of purified protein compared with the fluorescence of purified protein of s-GFP.</p>[c]<p>The excitation and emission maxima in units of nanometers.</p>[d]<p>The refolding rate constant for the fast phase calculated by fitting refolding curve in sigma plot.</p>[e]<p>Concentration of urea (M) at which 50% of the initial fluorescence is recovered during refolding of urea denatured protein.</p

Equilibrium refolding plot for s-GFP, n-GFP, s-N14 and s-C225.

<p>Protein samples were denatured in 8 M urea and diluted to different concentration of urea in refolding buffer. Recovered fluorescence normalized by dividing by fluorescence of corresponding non-denatured samples diluted in similar fashion.</p

Deletional Protein Engineering Based on Stable Fold

<div><p>Diversification of protein sequence-structure space is a major concern in protein engineering. Deletion mutagenesis can generate a protein sequence-structure space different from substitution mutagenesis mediated space, but it has not been widely used in protein engineering compared to substitution mutagenesis, because it causes a relatively huge range of structural perturbations of target proteins which often inactivates the proteins. In this study, we demonstrate that, using green fluorescent protein (GFP) as a model system, the drawback of the deletional protein engineering can be overcome by employing the protein structure with high stability. The systematic dissection of N-terminal, C-terminal and internal sequences of GFPs with two different stabilities showed that GFP with high stability (s-GFP), was more tolerant to the elimination of amino acids compared to a GFP with normal stability (n-GFP). The deletion studies of s-GFP enabled us to achieve three interesting variants viz. s-DL4, s-N14, and s-C225, which could not been obtained from n-GFP. The deletion of 191–196 loop sequences led to the variant s-DL4 that was expressed predominantly as insoluble form but mostly active. The s-N14 and s-C225 are the variants without the amino acid residues involving secondary structures around N- and C-terminals of GFP fold respectively, exhibiting comparable biophysical properties of the n-GFP. Structural analysis of the variants through computational modeling study gave a few structural insights that can explain the spectral properties of the variants. Our study suggests that the protein sequence-structure space of deletion mutants can be more efficiently explored by employing the protein structure with higher stability.</p> </div

Structural interactions of modeled structure of loop deleted variants.

<p>a) In s-DL1, the loop deletion eliminated hydrogen bonds involved by the residues K79 and H81. b) The residues F83, K85, A87 and M88 were deleted in the loop sequence of s-DL2.</p

Fluorescence excitation and emission spectra.

<p>a) Excitation and b) Emission spectra of s-GFP, s-N14, s-C225 and n-GFP, measured using fluorescence spectrometer. All amplitudes were arbitrarily normalized to a maximum value of 1.0 to show the difference in spectral wavelength not the spectral intensity.</p

The primary structure and secondary structure map of GFP

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051510#pone.0051510-Pedelacq1" target="_blank">[<b>27</b>]</a><b>.</b> Single-letter amino acid codes are used for representing the primary structure. Green color boxed residues ‘GYG’ represents the chromophore of GFP. Strands are represented in blue arrowhead and helices are indicated in red. The connecting black lines represent the turns and loops which conjoins the adjacent strands. The sequence of s-N14 is devoid of residues in box I. For s-C225, the residues in box VI are deleted. Residues in boxes II, III, IV and V are deleted in s-DL1, s-DL2, s-DL3 and s-DL4 respectively.</p

Comparison of relative whole cell fluorescence of s-GFP, n-GFP and deletion variants.

<p>The relative fluorescence (in arbitrary units) is defined as the whole cell fluorescence compared with fluorescence of cells expressing s-GFP^[a] and n-GFP^[b] respectively. All the fluorescence values were normalized by the O.D_{600 nm} of expressed cells. ^[c]Deletion of preceding residues in primary structure of n-GFP resulted in loss of fluorescence and experiments were not carried out. ^[+]Reported elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051510#pone.0051510-Li1" target="_blank">[6]</a> that these loop deletions makes the protein non-functional.</p

Representative plot of refolding kinetics for s-GFP, s-N14, s-C225 and n-GFP.

<p>Refolding kinetics was measured after denaturation in urea (8 M) followed by renaturation by dilution. Normalized fluorescence in arbitrary units (au) was plotted against time. Insert table showing the fast phase and slow phase rates of refolding process.</p