Abstract
Pseudomonas lemoignei has five different polyhydroxyalkanoate (PHA) depolymerase genes (phaZ1 to phaZ5), which encode the extracellularly localized poly(3-hydroxybutyrate) (PHB) depolymerases C, B, and D, poly(3-hydroxyvalerate) (PHV) depolymerase, and PHB depolymerase A, respectively. Four of the five genes (phaZ1 to phaZ4) have been cloned, and one of them (phaZ1) was studied in detail earlier (D. Jendrossek, B. Müller, and H. G. Schlegel, Eur. J. Biochem. 218:701-710, 1993). The fifth PHA depolymerase gene (phaZ5) was identified by colony hybridization of recombinant Escherichia coli clones with a phaZ5-specific oligonucleotide. The nucleotide sequence of a 3,704-bp EcoRI fragment was determined and found to contain two large open reading frames (ORFs) which coded for a polypeptide with significant similarities to glycerol-3-phosphate dehydrogenases of various sources (313 amino acids; M(r), 32,193) and for the precursor of PHB depolymerase A (PhaZ5; 433 amino acids; M(r), 44,906). The PHV depolymerase gene (phaZ4) was subcloned, and the nucleotide sequence of a 3,109-bp BamHI fragment was determined. Two large ORFs (ORF3 and ORF4) that represent putative coding regions were identified. The deduced amino acid sequence of ORF3 (134 amino acids; M(r), 14,686) revealed significant similarities to the branched-chain amino acid aminotransferase (IlfE) of enterobacteria. ORF4 (1,712 bp) was identified as the precursor of a PHV depolymerase (567 amino acids; M(r), 59,947). Analysis of primary structures of the five PHA depolymerases of P. lemoignei and of the PHB depolymerases of Alcaligenes faecalis and Pseudomonas pickettii revealed homologies of 25 to 83% to each other and a domain structure: at their N termini, they have typical signal peptides of exoenzymes. The adjacent catalytic domains are characterized by several conserved amino acids that constitute putative catalytic triads which consist of the consensus sequence of serine-dependent hydrolases including the pentapeptide G-X-S-X-G, a conserved histidine and aspartate, and a conserved region resembling the oxyanion hole of lipases. C terminal of the catalytic domain an approximately 40-amino-acid-long threonine-rich region (22 to 27 threonine residues) is present in PhaZ1, PhaZ2, PhaZ3, and PhaZ5. Instead of the threonine-rich region PhaZ4 and the PHB depolymerases of A. faecalis and P. pickettii contain an approximately 90-amino-acid-long sequence resembling the fibronectin type III module of eucaryotic extracellular matrix proteins. The function of the fibronectin type III module in PHA depolymerases remains obscure. Two types of C-terminal sequences apparently represent substrate-binding sites; the PHB type is present in the PHB depolymerases of A. faecalis and P. pickettii and in PhaZ2, PhaZ3, and PhaZ5 and the PHV type is present in the PHV-hydrolyzing depolymerases (PhaZ4 and PhaZ1). phaZ1 was transferred to A. eutrophus H16 and JMP222. All transconjugants of both strains were able to grow with extracellular PHB as a carbon source and produced translucent halos on PHB-containing solid media. PhaZ1, PhaZ2, PhaZ4, and PhaZ5 were purified from P. lemoignei and from recombinant E. coli; the processing sites of the precursors in E. coli were the same as in P. lemoignei, and similar substrate specificities were determined for the wild-type and the recombinant proteins. All PHA depolymerases hydrolyzed PHB at high specific activities. PhaZ1 and PhaZ4 additionally cleaved PHV, and PhaZ4 hydrolyzed poly(4-hydroxybutyrate). None of the depolymerases was able to hydrolyze polyactide or PHA consisting of monomers with more than five carbon atoms. While the wild-type depolymerase proteins were glycosylated and found to contain glucose and N-acetylglucosamine, none of the recombinant proteins was glycosylated. PHB hydrolysis was dependent on divalent cations such as Ca2+ and was inhibited by the presence of EDTA.
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- 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]
- Blaak H., Schnellmann J., Walter S., Henrissat B., Schrempf H. Characteristics of an exochitinase from Streptomyces olivaceoviridis, its corresponding gene, putative protein domains and relationship to other chitinases. Eur J Biochem. 1993 Jun 15;214(3):659–669. doi: 10.1111/j.1432-1033.1993.tb17966.x. [DOI] [PubMed] [Google Scholar]
- Bork P., Doolittle R. F. Proposed acquisition of an animal protein domain by bacteria. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):8990–8994. doi: 10.1073/pnas.89.19.8990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Briese B. H., Jendrossek D., Schlegel H. G. Degradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by aerobic sewage sludge. FEMS Microbiol Lett. 1994 Mar 15;117(1):107–111. doi: 10.1111/j.1574-6968.1994.tb06750.x. [DOI] [PubMed] [Google Scholar]
- Brucato C. L., Wong S. S. Extracellular poly(3-hydroxybutyrate) depolymerase from Penicillium funiculosum: general characteristics and active site studies. Arch Biochem Biophys. 1991 Nov 1;290(2):497–502. doi: 10.1016/0003-9861(91)90572-z. [DOI] [PubMed] [Google Scholar]
- CHOWDHURY A. A. POLY-BETA-HYDROXYBUTTERSAEURE ABBAUENDE BAKTERIEN UND EXOENZYM. Arch Mikrobiol. 1963 Dec 10;47:167–200. [PubMed] [Google Scholar]
- Delafield F. P., Doudoroff M., Palleroni N. J., Lusty C. J., Contopoulos R. Decomposition of poly-beta-hydroxybutyrate by pseudomonads. J Bacteriol. 1965 Nov;90(5):1455–1466. doi: 10.1128/jb.90.5.1455-1466.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Don R. H., Pemberton J. M. Properties of six pesticide degradation plasmids isolated from Alcaligenes paradoxus and Alcaligenes eutrophus. J Bacteriol. 1981 Feb;145(2):681–686. doi: 10.1128/jb.145.2.681-686.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Gilkes N. R., Henrissat B., Kilburn D. G., Miller R. C., Jr, Warren R. A. Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. Microbiol Rev. 1991 Jun;55(2):303–315. doi: 10.1128/mr.55.2.303-315.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Grunstein M., Wallis J. Colony hybridization. Methods Enzymol. 1979;68:379–389. doi: 10.1016/0076-6879(79)68027-8. [DOI] [PubMed] [Google Scholar]
- Hanna Z., Fregeau C., Préfontaine G., Brousseau R. Construction of a family of universal expression plasmid vectors. Gene. 1984 Oct;30(1-3):247–250. doi: 10.1016/0378-1119(84)90128-8. [DOI] [PubMed] [Google Scholar]
- Hansen C. K. Fibronectin type III-like sequences and a new domain type in prokaryotic depolymerases with insoluble substrates. FEBS Lett. 1992 Jun 29;305(2):91–96. doi: 10.1016/0014-5793(92)80871-d. [DOI] [PubMed] [Google Scholar]
- Haywood G. W., Anderson A. J., Ewing D. F., Dawes E. A. Accumulation of a Polyhydroxyalkanoate Containing Primarily 3-Hydroxydecanoate from Simple Carbohydrate Substrates by Pseudomonas sp. Strain NCIMB 40135. Appl Environ Microbiol. 1990 Nov;56(11):3354–3359. doi: 10.1128/aem.56.11.3354-3359.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Huisman G. W., Wonink E., Meima R., Kazemier B., Terpstra P., Witholt B. Metabolism of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas oleovorans. Identification and sequences of genes and function of the encoded proteins in the synthesis and degradation of PHA. J Biol Chem. 1991 Feb 5;266(4):2191–2198. [PubMed] [Google Scholar]
- 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]
- Jaeger K. E., Ransac S., Koch H. B., Ferrato F., Dijkstra B. W. Topological characterization and modeling of the 3D structure of lipase from Pseudomonas aeruginosa. FEBS Lett. 1993 Oct 11;332(1-2):143–149. doi: 10.1016/0014-5793(93)80501-k. [DOI] [PubMed] [Google Scholar]
- 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]
- Knauf V. C., Nester E. W. Wide host range cloning vectors: a cosmid clone bank of an Agrobacterium Ti plasmid. Plasmid. 1982 Jul;8(1):45–54. doi: 10.1016/0147-619x(82)90040-3. [DOI] [PubMed] [Google Scholar]
- 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]
- Lusty C. J., Doudoroff M. Poly-beta-hydroxybutyrate depolymerases of Pseudomonas lemoignei. Proc Natl Acad Sci U S A. 1966 Sep;56(3):960–965. doi: 10.1073/pnas.56.3.960. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Matavulj M., Molitoris H. P. Fungal degradation of polyhydroxyalkanoates and a semiquantitative assay for screening their degradation by terrestrial fungi. FEMS Microbiol Rev. 1992 Dec;9(2-4):323–331. doi: 10.1111/j.1574-6968.1992.tb05854.x. [DOI] [PubMed] [Google Scholar]
- Matuschek M., Burchhardt G., Sahm K., Bahl H. Pullulanase of Thermoanaerobacterium thermosulfurigenes EM1 (Clostridium thermosulfurogenes): molecular analysis of the gene, composite structure of the enzyme, and a common model for its attachment to the cell surface. J Bacteriol. 1994 Jun;176(11):3295–3302. doi: 10.1128/jb.176.11.3295-3302.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nakayama K., Saito T., Fukui T., Shirakura Y., Tomita K. Purification and properties of extracellular poly(3-hydroxybutyrate) depolymerases from Pseudomonas lemoignei. Biochim Biophys Acta. 1985 Jan 21;827(1):63–72. doi: 10.1016/0167-4838(85)90101-3. [DOI] [PubMed] [Google Scholar]
- Neu H. C., Heppel L. A. The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem. 1965 Sep;240(9):3685–3692. [PubMed] [Google Scholar]
- Persson B., Bengtsson-Olivecrona G., Enerbäck S., Olivecrona T., Jörnvall H. Structural features of lipoprotein lipase. Lipase family relationships, binding interactions, non-equivalence of lipase cofactors, vitellogenin similarities and functional subdivision of lipoprotein lipase. Eur J Biochem. 1989 Jan 15;179(1):39–45. doi: 10.1111/j.1432-1033.1989.tb14518.x. [DOI] [PubMed] [Google Scholar]
- Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- SCHLEGEL H. G., GOTTSCHALK G., VON BARTHA R. Formation and utilization of poly-beta-hydroxybutyric acid by Knallgas bacteria (Hydrogenomonas). Nature. 1961 Jul 29;191:463–465. doi: 10.1038/191463a0. [DOI] [PubMed] [Google Scholar]
- SCHLEGEL H. G., KALTWASSER H., GOTTSCHALK G. [A submersion method for culture of hydrogen-oxidizing bacteria: growth physiological studies]. Arch Mikrobiol. 1961;38:209–222. [PubMed] [Google Scholar]
- 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]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schirmer A., Jendrossek D. Molecular characterization of the extracellular poly(3-hydroxyoctanoic acid) [P(3HO)] depolymerase gene of Pseudomonas fluorescens GK13 and of its gene product. J Bacteriol. 1994 Nov;176(22):7065–7073. doi: 10.1128/jb.176.22.7065-7073.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schirmer A., Jendrossek D., Schlegel H. G. Degradation of poly(3-hydroxyoctanoic acid) [P(3HO)] by bacteria: purification and properties of a P(3HO) depolymerase from Pseudomonas fluorescens GK13. Appl Environ Microbiol. 1993 Apr;59(4):1220–1227. doi: 10.1128/aem.59.4.1220-1227.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schrag J. D., Li Y. G., Wu S., Cygler M. Ser-His-Glu triad forms the catalytic site of the lipase from Geotrichum candidum. Nature. 1991 Jun 27;351(6329):761–764. doi: 10.1038/351761a0. [DOI] [PubMed] [Google Scholar]
- Steinbüchel A., Hustede E., Liebergesell M., Pieper U., Timm A., Valentin H. Molecular basis for biosynthesis and accumulation of polyhydroxyalkanoic acids in bacteria. FEMS Microbiol Rev. 1992 Dec;9(2-4):217–230. doi: 10.1111/j.1574-6968.1992.tb05841.x. [DOI] [PubMed] [Google Scholar]
- Stinson M. W., Merrick J. M. Extracellular enzyme secretion by Pseudomonas lemoignei. J Bacteriol. 1974 Jul;119(1):152–161. doi: 10.1128/jb.119.1.152-161.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stroud R. M. A family of protein-cutting proteins. Sci Am. 1974 Jul;231(1):74–88. doi: 10.1038/scientificamerican0774-74. [DOI] [PubMed] [Google Scholar]
- 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]
- Timm A., Steinbüchel A. Cloning and molecular analysis of the poly(3-hydroxyalkanoic acid) gene locus of Pseudomonas aeruginosa PAO1. Eur J Biochem. 1992 Oct 1;209(1):15–30. doi: 10.1111/j.1432-1033.1992.tb17256.x. [DOI] [PubMed] [Google Scholar]
- Timm A., Steinbüchel A. Formation of polyesters consisting of medium-chain-length 3-hydroxyalkanoic acids from gluconate by Pseudomonas aeruginosa and other fluorescent pseudomonads. Appl Environ Microbiol. 1990 Nov;56(11):3360–3367. doi: 10.1128/aem.56.11.3360-3367.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
- Worsey M. J., Williams P. A. Metabolism of toluene and xylenes by Pseudomonas (putida (arvilla) mt-2: evidence for a new function of the TOL plasmid. J Bacteriol. 1975 Oct;124(1):7–13. doi: 10.1128/jb.124.1.7-13.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yamada K., Mukai K., Doi Y. Enzymatic degradation of poly(hydroxyalkanoates) by Pseudomonas pickettii. Int J Biol Macromol. 1993 Aug;15(4):215–220. doi: 10.1016/0141-8130(93)90040-s. [DOI] [PubMed] [Google Scholar]