Poly(3-hydroxyvalerate) depolymerase of Pseudomonas lemoignei

Pseudomonas lemoignei is equipped with at least five polyhydroxyalkanoate ( PHA) depolymerase structural genes (phaZ1 to phaZ5) which enable the bacter ium to utilize extracellular poly(3-hydroxybutyrate) (PHB), poly(3-hydroxyv alerate) (PHV), and related polyesters consisting of short-chain-length hxd roxyalkanoates (PHA,,,) as the sole sources of carbon and energy. Four gene s (phaZ1, phaZ2, phaZ3, and phaZ5) encode PHB depolymerases C, B, D, and A, respectively, It was speculated that the remaining gene, phaZ4, encodes th e PHV depolymerase (D, Jendrossek, A. Frisse, A. Behrends, M. Andermann, H. D. Kratzin, T. Stanislawski, and H. G. Schlegel, J. Bacteriol. 177:596-607 , 1995). However, in this study, we show that phaZ4 codes for another PHB d epolymeraes (i) by disagreement of 5 out of 41 amino acids that had been de termined by Edman degradation of the PHV depolymerase and of four endoprote inase GluC-generated internal peptides with the DNA-deduced sequence of pha Z4? (ii) by the lack of immunological reaction of purified recombinant PhaZ 4 with PHV depolymerase-specific antibodies, and (iii) by the low activity of the PhaZ4 depolymerase with PHV as a substrate. The true PHV depolymeras e-encoding structural gene, phaZ6, was identified by screening a genomic li brary of P. lemoignei in Escherichia coli for clearing zone formation on PH V agar, The DNA sequence of phaZ6 contained all 41 amino acids of the GluC- generated peptide fragments of the PW depolymerase. PhaZ6 was expressed and purified from recombinant E. coli and showed immunological identity to the wild-type PHV depolymerase and had high specific activities with PHB and P HV as substrates. To our knowledge, this is the first report on a PHA(SCL) depolymerase gene that is expressed during growth on PHV or odd-numbered ca rbon sources and that encodes a protein,vith high PHV depolymerase activity . Amino acid analysis revealed that PhaZ6 (relative molecular mass [M-r], 4 3,610 Da) resembles precursors of other extracellular PHA(SCL) depolymerase s (28 to 50% identical amino acids). The mature protein (M-r, 41,048) is co mposed of (i) a large catalytic domain including a catalytic triad of S-136 , D-211, and H-269 similar to serine hydrolases; (ii) a linker region highl y enriched in threonine residues and other amino acids with hydroxylated or small side chains (Thr-rich region); and (iii) a C-terminal domain similar in sequence to the substrate-binding domain of PHA(SCL) depolymerases. Dif ferences in the codon usage of phaZ6 for some codons from the average codon usage of P. lemoignei indicated that phaZ6 might be derived from other org anisms by gene transfer. Multialignment of separate domains of bacterial PH A(SCL) depolymerases suggested that not only complete depolymerase genes bu t also individual domains might have been exchanged between bacteria during evolution of PHA(SCL) depolymerases.