Molecular characterization of extracellular medium-chain-length poly(3-hydroxyalkanoate) depolymerase genes from Pseudomonas alcaligenes strains.
A bacterial strain M4-7 capable of degrading various polyesters, such as poly(epsilon-caprolactone), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxyoctanoate), and poly(3-hydroxy-5-phenylvalerate), was isolated from a marine environment and identified as Pseudomonas alcaligenes. The relative molecular mass of a purified extracellular medium-chain-length poly(3-hydroxyalkanoate) (MCL-PHA) depolymerase (PhaZ(PalM4-7)) from P. alcaligenes M4-7 was 28.0 kDa, as determined by SDS-PAGE. The PhaZ(PalM4-7) was most active in 50 mM glycine-NaOH buffer (pH 9.0) at 35 degrees C. It was insensitive to dithiothreitol, sodium azide, and iodoacetamide, but susceptible to p-hydroxymercuribenzoic acid, N-bromosuccinimide, acetic anhydride, EDTA, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, Tween 80, and Triton X-100. In this study, the genes encoding MCL-PHA depolymerase were cloned, sequenced, and characterized from a soil bacterium, P. alcaligenes LB19 (Kim et al., 2002, Biomacromolecules 3, 291-296) as well as P. alcaligenes M4-7. The structural gene (phaZ(PalLB19)) of MCL-PHA depolymerase of P. alcaligenes LB19 consisted of an 837 bp open reading frame (ORF) encoding a protein of 278 amino acids with a deduced M((r)) of 30,188 Da. However, the MCL-PHA depolymerase gene (phaZ(PalM4-7)) of P. alcaligenes M4-7 was composed of an 834 bp ORF encoding a protein of 277 amino acids with a deduced Mr of 30,323 Da. Amino acid sequence analyses showed that, in the two different polypeptides, a substrate-binding domain and a catalytic domain are located in the N-terminus and in the C-terminus, respectively. The PhaZ(PalLB19) and the PhaZ(PalM4-7) commonly share the lipase box, GISSG, in their catalytic domains, and utilize 111Asn and 110Ser residues, respectively, as oxyanions that play an important role in transition-state stabilization of hydrolytic reactions.
Kim
Do Young
DY
Department of Microbiology, School of Bioscience and Biotechnology, Chungnam National University, Daejeon 305-764, Republic of Korea.
Kim
Hyun Chul
HC
Kim
Sun Young
SY
Rhee
Young Ha
YH
D000595:Amino Acid Sequence; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003470:Culture Media; D008969:Molecular Sequence Data; D011091:Polyesters / Q000378:metabolism*; D044202:Pseudomonas alcaligenes / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D003470:Culture Media; D011091:Polyesters; D002265:Carboxylic Ester Hydrolases; C077226:poly(3-hydroxyalkanoic acid) depolymerase