Structural and Biochemical Studies of a Moderately Thermophilic Exonuclease I from <i>Methylocaldum szegediense</i>

<div><p>A novel exonuclease, designated as MszExo I, was cloned from <i>Methylocaldum szegediense</i>, a moderately thermophilic methanotroph. It specifically digests single-stranded DNA in the 3ʹ to 5ʹ direction. The protein is composed of 479 amino acids, and it shares 47% sequence identity with <i>E. coli</i> Exo I. The crystal structure of MszExo I was determined to a resolution of 2.2 Å and it aligns well with that of <i>E. coli</i> Exo I. Comparative studies revealed that MszExo I and <i>E. coli</i> Exo I have similar metal ion binding affinity and similar activity at mesophilic temperatures (25–47°C). However, the optimum working temperature of MszExo I is 10°C higher, and the melting temperature is more than 4°C higher as evaluated by both thermal inactivation assays and DSC measurements. More importantly, two thermal transitions during unfolding of MszExo I were monitored by DSC while only one transition was found in <i>E. coli</i> Exo I. Further analyses showed that magnesium ions not only confer structural stability, but also affect the unfolding of MszExo I. MszExo I is the first reported enzyme in the DNA repair systems of moderately thermophilic bacteria, which are predicted to have more efficient DNA repair systems than mesophilic ones.</p></div

Structural alignment between <i>E. coli</i> Exo I and MszExo I.

<p>(A) Overall structure of MszExo I. Like <i>E. coli</i> Exo I, MszExo I can be divided into three domains, the N-terminal exonuclease domain (1–197) colored in yellow, the SH3-like domain (198–355) colored in green and the C-terminal helical region (356–479) colored in blue. The magnesium ion at the active site is represented as a green sphere. Two missing regions (173–177, 350–359) are represented as red dashed lines. (B) Structure alignment of <i>E. coli</i> Exo I and MszExo I. <i>E. coli</i> Exo I (PDB: 1FXX) is colored in grey, and MszExo I (PDB: 4RG8) colored in green. Images were generated using the Pymol program.</p

Tm value of MszExo I in different buffers.

<p>*, B1 is the buffer which is composed of 50 mM Glycine-NaOH, 200 mM NaCl, pH 9.5.</p><p>**, B2 is the buffer which is composed of 50 mM Glycine-NaOH, 1 mM EDTA, pH 9.5.</p><p>Tm value of MszExo I in different buffers.</p

Substrate specificity of MszExo I.

<p>(A) MszExo I (5 nM) was incubated with 3’-overhang and 5’-overhang DNAs (50 nM) for indicated time at 37°C. The positions labeled by a 6-FAM are indicated by an asterisk for the DNA substrates. The 28-nt, 22-nt, 15-nt, 10-nt, and 5-nt poly dAs labeled by a 6-FAM at 5’-end were used as markers. Other details are described in Materials and Methods. (B) Time course experiments of the exonuclease activity of MszExo I were performed using 6-FAM labeled ssDNAs (5FAM-dA28, 5FAM-dT28 and 5FAM-dC28) as substrates. MszExo I (5 nM) was incubated with the DNA substrates (50 nM) at 37°C for indicated time. The 28-nt, 22-nt, 15-nt, 10-nt, and 5-nt poly dAs labeled by a 6-FAM at 5’-end were used as markers. Other details are described in Materials and Methods.</p

Thermal stability of <i>E. coli</i> Exo I and MszExo I evaluated by DSC.

<p>Comparison of DSC curve of <i>E. coli</i> Exo I (filled circle) and MszExo I (open circle). The protein concentration was 70 μM. The <i>E. coli</i> Exo I data (filled circle) were fitted to two-state transition model (black solid line). The T_m value for <i>E. coli</i> Exo I was 49°C. The MszExo I data (open circle) were fitted to non-two-state transition model (grey solid line). Two peaks indicated the two individual transitions. T_m values of these two transitions were 53.1°C and 57.4°C, respectively.</p

Thermal profiling of <i>E. coli</i> Exo I and MszExo I.

<p>(A) Determining optimum working temperature for two Exo Is. The reaction conditions were 5 nM enzyme, 1 μM ssDNA substrate (half FRET-dT67 with half dT67), 50 mM Glycine-NaOH buffer, pH 9.5, 200 mM NaCl, 0.1 mg/ml BSA and 5mM MgCl₂ at given temperature varying from 25 to 67°C. Other details are described in Materials and Methods. MszExo I (open circle) showed broader working temperature than <i>E. coli</i> Exo I (filled circle). Errors were calculated from three parallel experiments. (B) Thermal stability of two Exo Is evaluated by thermal inactivation assay. In this assay, 15 nM enzyme in standard reaction buffer was incubated at given temperature varying from 25 to 62°C for 10 min, then cooled on ice, and finally activity was determined at 37°C. The data were normalized by taking the activity of enzymes incubated at 37°C as 100%. Errors came from three parallel experiments. Thermal stability of MszExo I was by 5°C higher than that of <i>E. coli</i> Exo I.</p

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

DSC measurements of MszExo I under different conditions.

<p>(A) DSC measurements of MszExo I as a function of magnesium ions concentration. DSC measurements of MszExo I in three different buffers were performed. The base buffer was 50 mM Glycine-NaOH, 200 mM NaCl, pH 9.5. Open circle represents the base buffer in the presence of 1mM EDTA, and black line represents the best fit to two-state transition model. Open square represents the base buffer in addition of 5 mM MgCl₂, and grey line represents the best fit to non-two-state transition model. Plus sign represents the base buffer in addition of 20 mM MgCl₂, and red line represents the best fit to non-two-state transition model. All Tm values are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117470#pone.0117470.t002" target="_blank">Table 2</a>. (B) DSC measurements of MszExo I as a function of salt concentration. DSC measurements of MszExo I in three different buffers were performed. The base buffer was 50 mM Glycine-NaOH, 1 mM EDTA, pH 9.5. Open circle represents the base buffer, open square represents the base buffer in addition of 200 mM NaCl, and plus sign represents the base buffer in addition of 600 mM NaCl. DSC data were fitted to two-state transition model. Tm values are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117470#pone.0117470.t002" target="_blank">Table 2</a>. (C) The unfolded fraction of MszExo I monitored by the transition enthalpy. DSC Data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117470#pone.0117470.g007" target="_blank">Fig. 7B</a> were transformed into F_U-T plots by integration of the Cp versus T curve.</p