J Biol Chem, Vol. 274, Issue 53, 37800-37808, December 31, 1999

Local Sequence Dependence of Polyhydroxyalkanoic Acid Degradation in Hydrogenophaga pseudoflava^*

Sung Chul Yoon and Mun Hwan Choi

From the Biomaterials Science Laboratory, Division of Life Science, Gyeongsang National University, Chinju 660-701, Korea

The first order intracellular degradation of various polyhydroxyalkanoic acid (PHA) inclusions in Hydrogenophaga pseudoflava cells was investigated by analyzing the compositional and microstructural changes of the PHA using gas chromatography, ¹³C NMR spectroscopy, and differential scanning calorimetry. Two types of PHA, copolymers and blend-type polymers, were separately accumulated in cells for comparison. The constituent monomers were 3-hydroxybutyric acid (3HB), 4-hydroxybutyric acid (4HB), and 3-hydroxyvaleric acid (3HV). It was found that the 3HB-4HB copolymer was degraded only when the polymer contained a minimal level of 3HB units. With the cells containing a 3HB/4HB blend-type polymer, only poly(3HB) was degraded, whereas poly(4HB) was not degraded, indicating the totally inactive nature of the intracellular depolymerase against poly(4HB). On the basis of the magnitude of the first order degradation rate constants, the relative substrate specificity of the depolymerase toward the constituting monomer units was determined to decrease in the order 3HB > 3HV > 4HB. ¹³C NMR resonances of the tetrad, triad, and dyad sequences were analyzed for the samples isolated before and after degradation experiments. The results showed that the intracellular degradation depended on the local monomer sequence of the copolymers. The relative substrate specificity of the depolymerase determined from the NMR local sequence analysis agreed well with that obtained from the kinetics analysis. It is suggested that, without isolation and purification of the intracellular PHA depolymerase and "native" PHA substrates, the relative specificity of the enzyme as well as the microstructural heterogeneity of the PHA could be determined by measuring in situ the first order degradation rate constants of the PHA in cells.