Polyhydroxybutyrate synthase: Evidence for covalent catalysis

Journal of the American Chemical Society

Volumn 118, Issue 26, 1996, Pages 6319-6320

  Wodzinska, J ^a   Snell, K D ^a   Rhomberg, A ^a   Sinskey, A J ^a   Biemann, K ^a   Stubbe, J ^a  

^a MASSACHUSETTS INSTITUTE OF TECHNOLOGY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

POLYESTER;

ALCALIGENES; ARTICLE; ENZYME ACTIVITY; IN VITRO STUDY; POLYMERIZATION; REACTION ANALYSIS;

EID: 0030059466     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja961108a     Document Type: Article

References (18)

1. Ballard, D. G. H.; Holmes, P. A. Recent Adv. Mech. Synth. Aspects Polym., 1987, 215, 293-314.
2. Holmes, P. A. Phys. Technol. 1985, 16, 32-36.
3. Poirier, Y.; Newrath, C.; Somerville, C. Bio/Technology 1995, 13, 142-150.
4. Poirier, Y.; Douglas, E. D.; Klomparens, K.; Somerville, C. Science 1992, 256, 520-523.
5. Gemgross, T. U.; Snell, K. D.; Peoples, O. P.; Sinskey, A. J.; Csuhai, E.; Masamune, S.; Stubbe, J. Biochemistry 1994, 33, 9311-9320.
6. Liebergesell, M.; Sonomoto, K.; Madkour, M.; Mayer, F.; Steinbuchel, A. Eur. J. Biochem. 1994, 226, 71-80.
7. Gerngross, T. U.; Martin, D. P. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 6279-6283.
8. The reaction mixture contained in a final volume of 200 μL: PHB synthase (2.0 mg/mL, 31 μM), 1, 2, 3, or 4 (1 mM), Tris-HCl (45 mM including 4.5% glycerol and 0.045% Hecameg (6-O-(N-heptylcarbamoyl)-methyl α-D-glucopyranoside), pH 8.5). It was incubated for 15 min at 20 °C and then loaded onto two Superose 12 HR 10/30 (Pharmacia) columns connected in sequence equilibrated in 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl and 0.05% Hecameg at 4 °C. The synthase was eluted with the same buffer at a flow rate of 0.2 mL/min at 4 °C. The dimer eluted at 22.4 mL and the monomer at 24.0 mL. The molecular weight was determined on the basis of a calibration curve prepared using the following molecular weight standards: thyroglobulin (669 kDa), apoferrtin (443 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (64 kDa), and carbonic anhydrase (29 kDa).
9. -1) was measured after incubation of this mixture for 5 min at room temperature.
10. 3H]-4 (1 mM, 23.3 mCi/mmol) in 50 mM Tris-HCl (pH 8.5) containing 0.5% glycerol and 0.05% Hecameg for 15 min at 20 °C in a final volume of 275 μL. The protein was then precipiated by the addition of 300 μL acetonitrile, pelleted by centrifugation, and the supernatant removed. The protein pellet was washed three times with 1 mL of acetonitrile/water (ratio 5/1) being careful to resuspend the protein in each wash. The protein pellet was then resuspended in 50 mM ammonium bicarbonate (pH 7.8) and digested with trypsin (0.2 mg) for 3.5 h at 37 °C after which time a second portion of trypsin (0.2 mg) was added, and the mixture was incubated for an additional 17 h.
11. 10 was loaded on a Vydac C4 reversed phase column (220/4.6 mmD) equilibrated in 0.1% trifluoroacetic acid (TFA) and 0.1% Hecameg at 40 °C. The peptides were eluted with a linear gradient from O to 90% acetonitrile in 0.1% TFA and 0.1% Hecameg at a flow rate of 1 mL/min over a period of 90 min. Consecutive 1 mL fractions were collected automatically and analyzed by scintillation counting.
12. MALDI TOF mass spectra were obtained on a PerSeptive Biosystems Voyager Elite spectrometer operated in the linear mode. To 1 μL of the 1 mL fraction, 10 μL of matrix (sinapinic acid) solution was added, and 1 μL of this mixture applied on the sample probe.
13. Automated Edman sequencing was carried out by the MTT Biopolymers Laboratory using an Applied Biosystems Model 477A Protein Sequencer with on-line Model 120PTH Amino Acid Analyzer.
14. Ueda, S.; Yabutani, T.; Maehara, A.; Yamane, T. J. Bacteriol. 1996, 178, 774-779.
15. 8 was incubated with HBCoA (1 mM) in 150 mM phosphate (pH 7.0) in a final volume of 1.2 mL. The mixture also contained 11.8 mM Tris, 35.6 mM NaCl, 1.2% glycerol, and 0.012% Hecameg which were introduced with the enzyme. After incubation for 30 min at 20 °C, proteinase K (50 μg, 0.33 mg/mL) in 150 mM phosphate was added to the reaction mixture, and it was incubated at 37 °C for 17 h. The reaction mixture was then lyophilized, and the polymer was extracted with trifluoroethanol (TFb) (3 × 1 mL). Extracts were combined, and the solvent was evaporated to about 100 μL under a stream of air. This sample was loaded on a Shodex K807L GPC column equilibrated in TFE (1 mL/min) at 35 °C for at least 12 h and eluted with TFE at 1 mL/min. Fractions (0.5 mL) were collected and, after evaporation of the solvent, analyzed for radioactivity using scintillation counting. Radioactive polymer eluted between 8.0 and 11.5 mL TFE and contained 52% of recovered radioactivity. The remaining radioactivity eluted as small molecules between 12 and 13 mL of TFE.
16. 15 and submitted to an identical workup procedure. Radioactive polymer eluted between 8.0 and 11.5 mL TFE.
17. Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70-77.
18. Peoples, O.; Sinskey, A. J. J. Biol. Chem. 1989, 264, 15298-15303.