Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Nov;179(22):6965–6970. doi: 10.1128/jb.179.22.6965-6970.1997

Structure and function of poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1.

M Nojiri 1, T Saito 1
PMCID: PMC179635  PMID: 9371441

Abstract

Poly(3-hydroxybutyrate) (PHB) depolymerase from Alcaligenes faecalis T1 is composed of three domains: the catalytic (C) domain, the fibronectin type III-like (F) domain, and the substrate-binding (S) domain. We constructed domain deletion, inversion, chimera, and extra-F-domain mutants and examined their enzyme activity and PHB-binding ability. In addition, we performed substitution of 214Asp and 273His with glycine and aspartate, respectively, to examine their participation in a catalytic triad together with 139Ser. The mutant with both the F and S domains deleted and the trypsin-digested enzyme showed no PHB-hydrolyzing activity and less PHB-binding ability than that of the wild-type enzyme but retained D-(-)-3-hydroxybutyrate trimer-hydrolyzing activity at a level similar to that of the wild-type enzyme. The mutant with the F domain deleted and the mutant which had the order of the F and S domains inverted retained PHB-binding ability and trimer-hydrolyzing activity at levels similar to those of the wild-type enzyme but lost PHB-hydrolyzing activity. The chimera mutant, in which the F domain was substituted with a Thr-rich domain of PHB depolymerase A from Pseudomonas lemoignei, and the extra-F-domain mutant, with an additional F domain, retained trimer- and PHB-hydrolyzing activities and PHB-binding ability at levels similar to those of the wild-type enzyme. Two mutants (D214G and H273D) showed no enzymatic activity toward trimer and PHB, and they were not labeled with [3H]diisopropylfluorophosphate.

Full Text

The Full Text of this article is available as a PDF (779.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson A. J., Dawes E. A. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev. 1990 Dec;54(4):450–472. doi: 10.1128/mr.54.4.450-472.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Behrends A., Klingbeil B., Jendrossek D. Poly(3-hydroxybutyrate) depolymerases bind to their substrate by a C-terminal located substrate binding site. FEMS Microbiol Lett. 1996 Oct 1;143(2-3):191–194. doi: 10.1111/j.1574-6968.1996.tb08479.x. [DOI] [PubMed] [Google Scholar]
  3. Fukui T., Narikawa T., Miwa K., Shirakura Y., Saito T., Tomita K. Effect of limited tryptic modification of a bacterial poly(3-hydroxybutyrate) depolymerase on its catalytic activity. Biochim Biophys Acta. 1988 Jan 29;952(2):164–171. doi: 10.1016/0167-4838(88)90112-4. [DOI] [PubMed] [Google Scholar]
  4. Gilkes N. R., Warren R. A., Miller R. C., Jr, Kilburn D. G. Precise excision of the cellulose binding domains from two Cellulomonas fimi cellulases by a homologous protease and the effect on catalysis. J Biol Chem. 1988 Jul 25;263(21):10401–10407. [PubMed] [Google Scholar]
  5. Jaeger K. E., Ransac S., Dijkstra B. W., Colson C., van Heuvel M., Misset O. Bacterial lipases. FEMS Microbiol Rev. 1994 Sep;15(1):29–63. doi: 10.1111/j.1574-6976.1994.tb00121.x. [DOI] [PubMed] [Google Scholar]
  6. Jendrossek D., Frisse A., Behrends A., Andermann M., Kratzin H. D., Stanislawski T., Schlegel H. G. Biochemical and molecular characterization of the Pseudomonas lemoignei polyhydroxyalkanoate depolymerase system. J Bacteriol. 1995 Feb;177(3):596–607. doi: 10.1128/jb.177.3.596-607.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Jendrossek D., Müller B., Schlegel H. G. Cloning and characterization of the poly(hydroxyalkanoic acid)-depolymerase gene locus, phaZ1, of Pseudomonas lemoignei and its gene product. Eur J Biochem. 1993 Dec 1;218(2):701–710. doi: 10.1111/j.1432-1033.1993.tb18424.x. [DOI] [PubMed] [Google Scholar]
  8. Jendrossek D., Schirmer A., Schlegel H. G. Biodegradation of polyhydroxyalkanoic acids. Appl Microbiol Biotechnol. 1996 Dec;46(5-6):451–463. doi: 10.1007/s002530050844. [DOI] [PubMed] [Google Scholar]
  9. Kanematsu M., Imaeda T., Yamawaki Y., Hirose Y., Inoue A., Goto H., Doi H. Hepatocellular carcinoma with extrahepatic primary neoplasms. Gastrointest Radiol. 1992 Winter;17(1):53–57. doi: 10.1007/BF01888509. [DOI] [PubMed] [Google Scholar]
  10. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Saito T., Suzuki K., Yamamoto J., Fukui T., Miwa K., Tomita K., Nakanishi S., Odani S., Suzuki J., Ishikawa K. Cloning, nucleotide sequence, and expression in Escherichia coli of the gene for poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis. J Bacteriol. 1989 Jan;171(1):184–189. doi: 10.1128/jb.171.1.184-189.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Semimaru T., Goto M., Furukawa K., Hayashida S. Functional analysis of the threonine- and serine-rich Gp-I domain of glucoamylase I from Aspergillus awamori var. kawachi. Appl Environ Microbiol. 1995 Aug;61(8):2885–2890. doi: 10.1128/aem.61.8.2885-2890.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Shinohe T., Nojiri M., Saito T., Stanislawski T., Jendrossek D. Determination of the active sites serine of the poly (3-hydroxybutyrate) depolymerases of Pseudomonas lemoignei (PhaZ5) and of Alcaligenes faecalis. FEMS Microbiol Lett. 1996 Jul 15;141(1):103–109. doi: 10.1111/j.1574-6968.1996.tb08370.x. [DOI] [PubMed] [Google Scholar]
  15. Shirakura Y., Fukui T., Saito T., Okamoto Y., Narikawa T., Koide K., Tomita K., Takemasa T., Masamune S. Degradation of poly(3-hydroxybutyrate) by poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1. Biochim Biophys Acta. 1986 Jan 15;880(1):46–53. doi: 10.1016/0304-4165(86)90118-2. [DOI] [PubMed] [Google Scholar]
  16. Shirakura Y., Fukui T., Tanio T., Nakayama K., Matsuno R., Tomita K. An extracellular D(-)-3-hydroxybutyrate oligomer hydrolase from Alcaligenes faecalis. Biochim Biophys Acta. 1983 Oct 28;748(2):331–339. doi: 10.1016/0167-4838(83)90310-2. [DOI] [PubMed] [Google Scholar]
  17. Tanio T., Fukui T., Shirakura Y., Saito T., Tomita K., Kaiho T., Masamune S. An extracellular poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis. Eur J Biochem. 1982 May;124(1):71–77. doi: 10.1111/j.1432-1033.1982.tb05907.x. [DOI] [PubMed] [Google Scholar]
  18. Tomme P., Driver D. P., Amandoron E. A., Miller R. C., Jr, Antony R., Warren J., Kilburn D. G. Comparison of a fungal (family I) and bacterial (family II) cellulose-binding domain. J Bacteriol. 1995 Aug;177(15):4356–4363. doi: 10.1128/jb.177.15.4356-4363.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tomme P., Van Tilbeurgh H., Pettersson G., Van Damme J., Vandekerckhove J., Knowles J., Teeri T., Claeyssens M. Studies of the cellulolytic system of Trichoderma reesei QM 9414. Analysis of domain function in two cellobiohydrolases by limited proteolysis. Eur J Biochem. 1988 Jan 4;170(3):575–581. doi: 10.1111/j.1432-1033.1988.tb13736.x. [DOI] [PubMed] [Google Scholar]
  20. Watanabe T., Ito Y., Yamada T., Hashimoto M., Sekine S., Tanaka H. The roles of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation. J Bacteriol. 1994 Aug;176(15):4465–4472. doi: 10.1128/jb.176.15.4465-4472.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES