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Influence of growth stage on activities of polyhydroxyalkanoate (PHA) polymerase and PHA depolymerase in Pseudomonas putida U.

Ren Q, de Roo G, Witholt B, Zinn M, Thöny-Meyer L - BMC Microbiol. (2010)

Bottom Line: In addition, it was found that after eliminating phasins (PhaF and PhaI) from the granules PhaC activity decreased further.It was found that in P. putida PhaC and PhaZ are concomitantly active, resulting in parallel synthesis and degradation of PHA.Moreover PhaC activity was found to be decreased, whereas PhaZ activity increased during growth.

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Affiliation: Laboratory for Biomaterials, Swiss Federal Laboratories for Materials Science and Technology (Empa), CH-9014 St, Gallen, Switzerland. qun.ren@empa.ch

ABSTRACT

Background: Medium chain length (mcl-) polyhydroxyalkanoates (PHA) are synthesized by many bacteria in the cytoplasm as storage compounds for energy and carbon. The key enzymes for PHA metabolism are PHA polymerase (PhaC) and depolymerase (PhaZ). Little is known of how mcl-PHA accumulation and degradation are controlled. It has been suggested that overall PHA metabolism is regulated by the β-oxidation pathway of which the flux is governed by intracellular ratios of [NADH]/[NAD] and [acetyl-CoA]/[CoA]. Another level of control could relate to modulation of the activities of PhaC and PhaZ. In order to investigate the latter, assays for in vitro activity measurements of PhaC and PhaZ in crude cell extracts are necessary.

Results: Two in vitro assays were developed which allow the measurement of PhaC and PhaZ activities in crude cell extracts of Pseudomonas putida U. Using the assays, it was demonstrated that the activity of PhaC decreased 5-fold upon exponential growth on nitrogen limited medium and octanoate. In contrast, the activity of PhaZ increased only 1.5-fold during growth. One reason for the changes in the enzymatic activity of PhaC and PhaZ could relate to a change in interaction with the phasin surface proteins on the PHA granule. SDS-PAGE analysis of isolated PHA granules demonstrated that during growth, the ratio of [phasins]/[PHA] decreased. In addition, it was found that after eliminating phasins (PhaF and PhaI) from the granules PhaC activity decreased further.

Conclusion: Using the assays developed in this study, we followed the enzymatic activities of PhaC and PhaZ during growth and correlated them to the amount of phasins on the PHA granules. It was found that in P. putida PhaC and PhaZ are concomitantly active, resulting in parallel synthesis and degradation of PHA. Moreover PhaC activity was found to be decreased, whereas PhaZ activity increased during growth. Availability of phasins on PHA granules affected the activity of PhaC.

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Consumption of R-3-hydroxyoctanoyl-CoA in crude cell extracts of P. putida U and P. putida U::phaC1-. Panel A: Influence of free CoA on R-3-hydroxyoctanoyl-CoA thioesterase activity in a crude cell extract of P. putida U::phaC1-. Assay conditions: 100 mM Tris-HCl, pH 8, 1 mg/ml BSA, 0.5 mM MgCl2, 0.5 mM R-3-hydroxyoctanoyl-CoA, 0.1 mg/ml crude cell extract of P. putida U::phaC1- with no CoA (filled diamond) or 1 mM CoA (open diamond). Data represent the average of two measurements. Panel B: R-3-hydroxyoctanoyl-CoA consumption in crude cell extracts of P. putida U::phaC1- and P. putida U in the presence of free CoA. Assay conditions: 100 mM Tris-HCl, pH 8, 1 mg/ml BSA, 0.5 mM MgCl2, 0.5 mM R-3-hydroxyoctanoyl-CoA, 1 mM CoA, 4 mg/ml crude cell extract of either P. putida U::phaC1- (open diamond) or P. putida U (open square). R-3-hydroxyoctanoyl-CoA depletion was measured by HPLC. Data represent the average of two measurements.
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Figure 1: Consumption of R-3-hydroxyoctanoyl-CoA in crude cell extracts of P. putida U and P. putida U::phaC1-. Panel A: Influence of free CoA on R-3-hydroxyoctanoyl-CoA thioesterase activity in a crude cell extract of P. putida U::phaC1-. Assay conditions: 100 mM Tris-HCl, pH 8, 1 mg/ml BSA, 0.5 mM MgCl2, 0.5 mM R-3-hydroxyoctanoyl-CoA, 0.1 mg/ml crude cell extract of P. putida U::phaC1- with no CoA (filled diamond) or 1 mM CoA (open diamond). Data represent the average of two measurements. Panel B: R-3-hydroxyoctanoyl-CoA consumption in crude cell extracts of P. putida U::phaC1- and P. putida U in the presence of free CoA. Assay conditions: 100 mM Tris-HCl, pH 8, 1 mg/ml BSA, 0.5 mM MgCl2, 0.5 mM R-3-hydroxyoctanoyl-CoA, 1 mM CoA, 4 mg/ml crude cell extract of either P. putida U::phaC1- (open diamond) or P. putida U (open square). R-3-hydroxyoctanoyl-CoA depletion was measured by HPLC. Data represent the average of two measurements.

Mentions: An improved assay was developed in which thioesterases activity is suppressed by addition of free CoA. This is illustrated in Figure 1A in which a crude extract of a polymerase knock-out mutant P. putida U::PhaC1- was used. This mutant was found to grow well on fatty acids but was unable to produce PHA. Due to the presence of interfering acyl-CoA thioesterases in the extract, R-3-hydroxyoctanoyl-CoA was rapidly depleted. However, addition of CoA reduced the consumption of acyl-CoA by 90%, probably due to product inhibition of the thioesterases [22]. Although PhaC itself is known to be slightly inhibited by free CoA, with a Ki of 0.715 mM [23], the assay permitted measuring PhaC activity in crude cell extracts. This was demonstrated by comparison of the rate of R-3-hydroxyoctanoyl-CoA consumption by crude extracts of P. putida U and P. putida U::PhaC1- (Figure 1B).


Influence of growth stage on activities of polyhydroxyalkanoate (PHA) polymerase and PHA depolymerase in Pseudomonas putida U.

Ren Q, de Roo G, Witholt B, Zinn M, Thöny-Meyer L - BMC Microbiol. (2010)

Consumption of R-3-hydroxyoctanoyl-CoA in crude cell extracts of P. putida U and P. putida U::phaC1-. Panel A: Influence of free CoA on R-3-hydroxyoctanoyl-CoA thioesterase activity in a crude cell extract of P. putida U::phaC1-. Assay conditions: 100 mM Tris-HCl, pH 8, 1 mg/ml BSA, 0.5 mM MgCl2, 0.5 mM R-3-hydroxyoctanoyl-CoA, 0.1 mg/ml crude cell extract of P. putida U::phaC1- with no CoA (filled diamond) or 1 mM CoA (open diamond). Data represent the average of two measurements. Panel B: R-3-hydroxyoctanoyl-CoA consumption in crude cell extracts of P. putida U::phaC1- and P. putida U in the presence of free CoA. Assay conditions: 100 mM Tris-HCl, pH 8, 1 mg/ml BSA, 0.5 mM MgCl2, 0.5 mM R-3-hydroxyoctanoyl-CoA, 1 mM CoA, 4 mg/ml crude cell extract of either P. putida U::phaC1- (open diamond) or P. putida U (open square). R-3-hydroxyoctanoyl-CoA depletion was measured by HPLC. Data represent the average of two measurements.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Consumption of R-3-hydroxyoctanoyl-CoA in crude cell extracts of P. putida U and P. putida U::phaC1-. Panel A: Influence of free CoA on R-3-hydroxyoctanoyl-CoA thioesterase activity in a crude cell extract of P. putida U::phaC1-. Assay conditions: 100 mM Tris-HCl, pH 8, 1 mg/ml BSA, 0.5 mM MgCl2, 0.5 mM R-3-hydroxyoctanoyl-CoA, 0.1 mg/ml crude cell extract of P. putida U::phaC1- with no CoA (filled diamond) or 1 mM CoA (open diamond). Data represent the average of two measurements. Panel B: R-3-hydroxyoctanoyl-CoA consumption in crude cell extracts of P. putida U::phaC1- and P. putida U in the presence of free CoA. Assay conditions: 100 mM Tris-HCl, pH 8, 1 mg/ml BSA, 0.5 mM MgCl2, 0.5 mM R-3-hydroxyoctanoyl-CoA, 1 mM CoA, 4 mg/ml crude cell extract of either P. putida U::phaC1- (open diamond) or P. putida U (open square). R-3-hydroxyoctanoyl-CoA depletion was measured by HPLC. Data represent the average of two measurements.
Mentions: An improved assay was developed in which thioesterases activity is suppressed by addition of free CoA. This is illustrated in Figure 1A in which a crude extract of a polymerase knock-out mutant P. putida U::PhaC1- was used. This mutant was found to grow well on fatty acids but was unable to produce PHA. Due to the presence of interfering acyl-CoA thioesterases in the extract, R-3-hydroxyoctanoyl-CoA was rapidly depleted. However, addition of CoA reduced the consumption of acyl-CoA by 90%, probably due to product inhibition of the thioesterases [22]. Although PhaC itself is known to be slightly inhibited by free CoA, with a Ki of 0.715 mM [23], the assay permitted measuring PhaC activity in crude cell extracts. This was demonstrated by comparison of the rate of R-3-hydroxyoctanoyl-CoA consumption by crude extracts of P. putida U and P. putida U::PhaC1- (Figure 1B).

Bottom Line: In addition, it was found that after eliminating phasins (PhaF and PhaI) from the granules PhaC activity decreased further.It was found that in P. putida PhaC and PhaZ are concomitantly active, resulting in parallel synthesis and degradation of PHA.Moreover PhaC activity was found to be decreased, whereas PhaZ activity increased during growth.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Biomaterials, Swiss Federal Laboratories for Materials Science and Technology (Empa), CH-9014 St, Gallen, Switzerland. qun.ren@empa.ch

ABSTRACT

Background: Medium chain length (mcl-) polyhydroxyalkanoates (PHA) are synthesized by many bacteria in the cytoplasm as storage compounds for energy and carbon. The key enzymes for PHA metabolism are PHA polymerase (PhaC) and depolymerase (PhaZ). Little is known of how mcl-PHA accumulation and degradation are controlled. It has been suggested that overall PHA metabolism is regulated by the β-oxidation pathway of which the flux is governed by intracellular ratios of [NADH]/[NAD] and [acetyl-CoA]/[CoA]. Another level of control could relate to modulation of the activities of PhaC and PhaZ. In order to investigate the latter, assays for in vitro activity measurements of PhaC and PhaZ in crude cell extracts are necessary.

Results: Two in vitro assays were developed which allow the measurement of PhaC and PhaZ activities in crude cell extracts of Pseudomonas putida U. Using the assays, it was demonstrated that the activity of PhaC decreased 5-fold upon exponential growth on nitrogen limited medium and octanoate. In contrast, the activity of PhaZ increased only 1.5-fold during growth. One reason for the changes in the enzymatic activity of PhaC and PhaZ could relate to a change in interaction with the phasin surface proteins on the PHA granule. SDS-PAGE analysis of isolated PHA granules demonstrated that during growth, the ratio of [phasins]/[PHA] decreased. In addition, it was found that after eliminating phasins (PhaF and PhaI) from the granules PhaC activity decreased further.

Conclusion: Using the assays developed in this study, we followed the enzymatic activities of PhaC and PhaZ during growth and correlated them to the amount of phasins on the PHA granules. It was found that in P. putida PhaC and PhaZ are concomitantly active, resulting in parallel synthesis and degradation of PHA. Moreover PhaC activity was found to be decreased, whereas PhaZ activity increased during growth. Availability of phasins on PHA granules affected the activity of PhaC.

Show MeSH
Related in: MedlinePlus