K. Sudesh1,*, Y. M. Normi1,2, Y. Kikawa2, Z. Gan3, K. Matsumoto2, T. Hiraishi2, and Y. Doi2,4
1 School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, MALAYSIA
2 Polymer Chemistry Laboratory, RIKEN Institute, Hirosawa 2-1, Wako-shi, 351-0198, Saitama, JAPAN
3 CAS Key Laboratory of Engineering Plastics, Joint Laboratory of Polymer Science and Materials, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, CHINA
4 Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, JAPAN.
Keywords: polyhydroxyalkanoate, granule, AFM, polymer chain, fibril
The most intriguing aspect of poly(3-hydroxybutyrate) (PHB) is its native structure in life bacterial cells. PHB is stored in cell cytoplasm as discrete water insoluble granules. Upon purification from cellular materials, PHB is a solid exhibiting almost 70% crystallinity. In vivo however, PHB is maintained in an amorphous mobile form, which is the only form accessible to the intracellular depolymerase enzymes. It is thus of paramount importance to prevent crystallization of PHB granules in vivo. How bacteria manage to achieve this has long been debated. The involvement of effective plasticizing agents has been proposed. In addition, other unknown mechanisms were thought to be involved to prevent PHB crystallization in vivo. At present, it is generally accepted that PHB granules in vivo remain amorphous simply because of slow nucleation kinetics that are operative for small particles. We have recently provided direct evidence for the existence of highly organized PHB chains in the form of fibrillar aggregates in the native granules. Here we provide further evidence by observing the initial stages of polymerization. We have successfully observed by using atomic force microscopy the formation of PHB chains in vitro. For this we have used purified PHB synthase. Before the addition of the substrate, R-3-hydroxybutyryl-CoA, the enzyme existed mainly as single subunits. Upon the addition of substrate, the enzyme formed complexes that were frequently associated with fibrillar structures. The synthesis and organization of the polymer chains into fibrillar structures occurred simultaneously. Of particular importance is the finding that both ends of a growing polymer chain were attached to the PHB synthase complex. We propose that this fibrillar organization is important to prevent crystallization and maintain the polymer chains in an amorphous/mobile state. Water molecules may be involved in the organization of the polymer chains. We believe that we have finally revealed the actual mechanism used by bacteria to prevent PHB crystallisation in vivo.
論文來源：International Symposium on Biological Polyesters ,Auguest 22-27, 2004
