Physical and chemical characterization of the recombinant protein

Molecular mass determination

According to SDS-PAGE (Fig. 3-2), the AbfD monomer revealed a mass of about 54 kDa. The quaternary structure of holoenzyme was determined by gel filtration chromatography, which separated the proteins based on their mass. As standards, aldolase, catalase, ferritin and thyroglobin were loaded individually onto a Superdex 60 column with a flowrate of 0.5 ml/min.

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A calibration curve was made with their elution volume parameters and used for the determination molecular mass of recombinant AbfD. The apparent molecular mass of the recombinant AbfD amounts to ca. 230 kDa (Fig. 3-3). Therefore, a homotetrameric structure appears to be most likely, which confirmed the molecular properties of native AbfD from C.

aminobutyricum [45].

y = -50.419x + 1786.5

0 100 200 300 400 500 600 700 800

20 22 24 26 28 30 32 34

Molecular mass (kDa)

Elution volume (ml)

Molecular mass calibration curve

calibration AbfD

Figure 3-3. Calibration curve of molecular mass determination.

Specific activity and cofactor determination

AbfD was assayed spectrophotometrically with 4-hydroxybutyrate CoA-transferase (AbfT) and the ‘enzyme pool’ from A. fermentans, which is an enzyme mixture containing crotonase, 3-hydroxybutyryl-CoA dehydrogenase, thiolase and phosphate acetyltransferase [108]. The produced NADH was measured at 340 nm on the spectrophotometer. The obtained specific

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activity of recombinant AbfD (0.5 mg/ml) was calculated to be about 2.2 U/mg, this corresponds to the value of native AbfD from C. aminobutyricum (Tab. 3-2) [52].

In order to compare the properties of the native enzyme with those of the recombinant enzyme, the cofactor contents were quantified. Non-heme iron from recombinant AbfD was determined with Ferene as 11.8 mol iron per mol homotetrameric enzyme. As evidence from previous results, E. coli cannot offer optimal conditions for the iron sulfur cluster assembly compared to C.

aminobutyricum, and therefore an iron reconstitution technique was used to improve the iron stoichiometry of the enzyme. After incubation of recombinant AbfD with iron(III) chloride and sodium sulfide for 2 h, the iron content increased to 14.8 mol/mol enzyme and the specific activity to 4.5 U/mg. The FAD content was determined spectrophotometrically. The result indicated that the homotetrameric enzyme consists of 4.4 ± 0.2 mol FAD per mol enzyme as expected.

Table 3-2. Comparison of recombinant AbfD properties with native enzyme Spec. activity

(U/mg)

Iron content (mol/mol protein)

Flavin content (mol/mol protein)

Structure

AbfD from C.aminobutyricum

(Irfan Çinkaya)

2 – 16.7 12.0 – 13.4 4 tetramer

AbfD from E.coli 2.2 ± 0.3 11.8 ± 0.1 4.4 ± 0.2 tetramer AbfD after iron

reconstitution

4.5 ± 0.3 14.8 n.d tetramer

n.d: not determined

MALDI-TOF mass spectrometry

It was used to analyze the organic molecules during the dehydration reaction. 4-Hydroxybutyryl-CoA as substrate, which was synthesized using 4-Hydroxybutyryl-CoA-transferase, 4-hydroxybutyrate and acetyl-CoA, incubated with active AbfD in D2O for 30 min. After purification of the reaction mixture

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using a SepPak^TM hydrophobic column, the products were identified by MALDI-TOF. In comparison with control (Fig 3-4, 1), the peak at 854 Da (4-hydroxybutyryl-CoA) dropped down and a peak at 839 Da revealed the production of CoA. The theoretical mass of crotonyl-CoA amounts 836 Da, which was shifted to 839 Da (Fig 3-4, 2), as protons were deuterium-exchanged during dehydration procedure. The peaks at 857 Da and 858 Da could be considered as 4-hydroxybutyryl-CoA carrying three or four deuterium atoms. Due to its instability, only slight peaks could be observed.

Figure 3-4. MALDI TOF mass spectrometry using 4-hydroxybutyryl-CoA as substrate.

1, Synthesized 4-hydroxybutyryl-CoA (854 Da) in D2O as control; 2, 4-Hydroxybutyryl-CoA was incubated anaerobically with purified AbfD in D₂O.

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To characterize the reverse reaction of AbfD, crotonyl-CoA was incubated with AbfD in D2O for 30 min. The mixture was purified with the same column as described below. Different to the control (Fig 3-5, 1), with active AbfD treated sample revealed that the peak of crotonyl-CoA (836 Da) was shifted to 839 Da (Fig. 3-5, 2), which was due to the proton-exchange by deuterium atoms. Unexpectedly, the mass spectrometry indicated an unclear peak corresponding to 4-hydroxybutyryl-CoA (854 Da). It is likely to attribute to the instability of 4-4-hydroxybutyryl-CoA, it could cleave to free CoA and 4-hydroxybutyrate or reconstruct to lactone.

Figure 3-5. MALDI TOF mass spectrometry using crotonyl-CoA as substrate.

1, Synthesized crotonyl-CoA (836 Da) in D2O as control. 2, Crotonyl-CoA was incubated anaerobically with purified AbfD in D₂O.

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Inactivation by air

As an oxygen sensitive enzyme in anaerobic microorganisms, it was shown previously that AbfD lost the dehydratase activity very quickly at room temperature during exposure to air, within 5 hours dehydratase activity disappeared completely. The native AbfDs from C. aminobutyricum became completely inactive within 70 min of being exposed to aerobic conditions. It has been also observed that exposure to air bleached the dark brown color. This could be an indication that the protein lost the essential iron. However, the yellow color was intensified, indicating that the flavin in the protein was still present.

Butyryl-CoA dehydrogenase activity

A fold similar to that seen in the AbfD crystal structure was found in FAD-containing medium chain acyl-CoA dehydrogenase (MCAD) from pig liver, which catalyzes the reversible oxidation of an acyl-CoA derivative to form the α,β double bond in the corresponding enoyl-CoA, although the amino sequence of these enzymes showed just 16 % identity. Because the non-activated β-hydrogen has to be removed by butyryl-CoA deβ-hydrogenase in a way analogous to the reaction catalyzed by AbfD, the dehydrogenase activity was tested using the purified active dehydratase or the inactivated enzyme by air. However, there was no dehydrogenase activity detectable. EPR spectroscopy has been also applied to detect the FAD radical of the oxidized AbfD as a hypothetic dehydrogenase. Compared to the sample without adding butyryl-CoA, no difference of flavin signal in EPR spectra was found, as shown in Fig 3-6,

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Figure 3-6. EPR spectra of inactivated AbfD by air at 77 K using butyryl-CoA as substrate.

Test using 4-hydroxypentanoate as inhibitor

4-Hydroxypentanoate, acts probably as an interesting inhibitor, was synthesized by incubating (R, S)-γ-valerolactone and sodium hydroxide at 60 ºC. After adding acetyl-CoA and AbfT in the synthesized 4-hydroxypentanoate, the products were detected by MALDI-TOF mass spectroscopy. The presence of 4-hydroxyvaleryl-CoA was inferred from the peak of 869 Da (Fig.

3-7). The resulting CoA ester was mixed with AbfD in potassium phosphate, and the products measured directly with a photometer at 290 nm. Obviously, no activity was observed. As shown in Fig 3-8, in order to detect the competition effect between hydroxybutyrate and 4-hydroxypentanoate, the coupled assay of AbfD was initiated by adding 4-hydroxybutyrate as substrate. After mixing with 4-hydroxypentanoate there was no variation of the reaction rate, which proved that the CoA ester derivate of 4-hydroxypentanoate acted not as an inhibitor of AbfD.

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Figure 3-7. MALDI-TOF mass spectrum showing the peak at 869 Da corresponding to 4-hydroxyvaleryl-CoA.

Figure 3-8. Inhibition test of AbfD using 4-hydroxypentanoyl-CoA.

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