Locations of selected residues for pEthF incorporation in the three-dimensional structure of the mDHFR-WT.

<p>Valine at the 43rd position and phenylalanine at the 179th position are highlighted in red (left) and blue (right), respectively. The cofactor NADPH (magenta) and the inhibitor (orange) are shown in stick representation (PDB ID: 3D80).</p

Enzymatic activity of the mDHFR after site-specific biotinylation and immobilization.

<p>(A) Activity of the mDHFR-43biotin versus the mDHFR-43pEthF. Error bars represent standard errors (n = 3). (B) Activity of the immobilized mDHFR-43biotin. Streptavidin-coated wells were incubated with 50 µL of the mDHFR-43biotin and the mDHFR-43pEthF at 1 mg/mL, separately, for 30 min at RT. After washing, the enzymatic reaction was initiated at 25°C by adding 200 µL of assay buffer, and monitored by spectrometry. Error bars represent standard errors (n = 3).</p

THPTA-assisted CuAAC reaction and its effect on the enzymatic activity.

<p>(A) In-gel fluorescence of reaction products from TBTA- and THPTA-assisted dye labeling via CuAAC. The mDHFR-pEthF (30 µM) was reacted at 25°C for 15 min with sulforhodamine-azide (60 µM) in the presence of 1 mM CuSO₄, 1 mM TBTA, 2 mM ascorbate in a phosphate buffer (pH 8.0) containing 30% (v/v) DMSO or in the presence of 1 mM CuSO₄, 1 mM THPTA, 2 mM ascorbate in a DMSO-free phosphate buffer (pH 8.0). (B) Effect of THPTA incubation on activity of the mDHFR-43pEthF. After incubation of 30 µM protein with (open circle) or without (close circle) 1 mM THPTA in 10 µl of DMSO-free phosphate buffer (pH 8.0) for 15 min at 25°C, the mixture was diluted 36-fold with the assay buffer for activity assay. Error bars represent standard errors (n = 3). (C) Effect of THPTA incubation on activity of the mDHFR-179pEthF (n = 3).</p

Effect of various reductants on CuAAC reaction rates and activities of the mDHFR-43pEthF and the mDHFR-179pEthF.

<p>(A) Time course of CuAAC reactions initiated by ascorbate, TCEP, and DTT. Reactions were performed at 25°C by adding 2 mM reductant to a phosphate-buffered (pH 8.0) mixture containing 30 µM of the mDHFR-43pEthF (black) or the mDHFR-179pEthF (gray), 60 µM of azidocoumarin, 1 mM of CuSO₄, 1 mM THPTA. Fluorescence evolution was recorded at λ_ex = 400 nm and λ_em = 470 nm. (B) Relative loss of enzymatic activity after incubation with various CuAAC systems in the absence of azidocoumarin (<b>1</b>–<b>4</b>). Incubation times were 15 min for system <b>1</b> and <b>4</b>, 12 h for system <b>2</b>, and 2 h for system <b>3</b>. Activity losses were normalized to that in system <b>1</b>. Error bars represent standard errors (n = 3). Two-sided Student's t-tests were applied to the data (*P<0.05). (C) Effect of ascorbate-driven dye labeling of the mDHFR-43pEthF on the enzymatic activity. The labeling was performed at 25°C for 15 min in the presence of 30 µM of the mDHFR-43pEthF, 60 µM of azidocoumarin, 1 mM of CuSO₄, 1 mM THPTA, and 2 mM ascorbate. Error bars represent standard errors (n = 3).</p

Chemical structures.

<p>(A) <i>p</i>-ethynylphenylalanine, (B) Cu(I)-chelating ligands, and (C) azide-functionalized reagents.</p

Enzyme activities of FDHs.

<p>Enzyme activities of FDHs for A) formate oxidation and B) CO₂ reduction at different pH values.</p

Determination of kinetic parameters.

<p>Correlation between the measured and calculated initial rates of TsFDH-catalyzed A) formate oxidation and B) CO₂ reduction; CbFDH-catalyzed C) formate oxidation and D) CO₂ reduction.</p

Formate production through FDH-catalyzed CO₂ reduction.

<p>Formate production by (•) TsFDH and (▴) CbFDH in 100 mM sodium phosphate buffer, pH 7.0.</p

The kinetic parameters of FDHs^[a].

[a]<p>Kinetic parameters for the forward (formate oxidation) and reverse (CO₂ reduction) reactions were calculated by fitting the initial rates to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103111#pone.0103111.e001" target="_blank">equation (1</a>).</p>[b]<p>Formate oxidation (A: NAD⁺, B: sodium formate).</p>[c]<p>CO₂ reduction (A: NADH, B: sodium bicarbonate).</p

Structural comparison of TsFDH and CbFDH.

<p>Structural comparison of the A) N- and B) C-terminal loops of TsFDH (green, modeled using 2NAD) and CbFDH (cyan, pdb code: 2FSS). The elongated N- and C-terminal loops are shown in blue.</p