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PMID:15489436

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Citation

Handrick, R, Reinhardt, S, Kimmig, P and Jendrossek, D (2004) The "intracellular" poly(3-hydroxybutyrate) (PHB) depolymerase of Rhodospirillum rubrum is a periplasm-located protein with specificity for native PHB and with structural similarity to extracellular PHB depolymerases. J. Bacteriol. 186:7243-53

Abstract

Rhodospirillum rubrum possesses a putative intracellular poly(3-hydroxybutyrate) (PHB) depolymerase system consisting of a soluble PHB depolymerase, a heat-stable activator, and a 3-hydroxybutyrate dimer hydrolase (J. M. Merrick and M. Doudoroff, J. Bacteriol. 88:60-71, 1964). In this study we reinvestigated the soluble R. rubrum PHB depolymerase (PhaZ1). It turned out that PhaZ1 is a novel type of PHB depolymerase with unique properties. Purified PhaZ1 was specific for amorphous short-chain-length polyhydroxyalkanoates (PHA) such as native PHB, artificial PHB, and oligomer esters of (R)-3-hydroxybutyrate with 3 or more 3-hydroxybutyrate units. Atactic PHB, (S)-3-hydroxybutyrate oligomers, medium-chain-length PHA, and lipase substrates (triolein, tributyrin) were not hydrolyzed. The PHB depolymerase structural gene (phaZ1) was cloned. Its deduced amino acid sequence (37,704 Da) had no significant similarity to those of intracellular PHB depolymerases of Wautersia eutropha or of other PHB-accumulating bacteria. PhaZ1 was found to have strong amino acid homology with type-II catalytic domains of extracellular PHB depolymerases, and Ser(42), Asp(138), and His(178) were identified as catalytic-triad amino acids, with Ser(42) as the putative active site. Surprisingly, the first 23 amino acids of the PHB depolymerase previously assumed to be intracellular revealed features of classical signal peptides, and Edman sequencing of purified PhaZ1 confirmed the functionality of the predicted cleavage site. Extracellular PHB depolymerase activity was absent, and analysis of cell fractions unequivocally showed that PhaZ1 is a periplasm-located enzyme. The previously assumed intracellular activator/depolymerase system is unlikely to have a physiological function in PHB mobilization in vivo. A second gene, encoding the putative true intracellular PHB depolymerase (PhaZ2), was identified in the genome sequence of R. rubrum.

Links

PubMed PMC523223 Online version:10.1128/JB.186.21.7243-7253.2004

Keywords

Amino Acid Sequence; Carboxylic Ester Hydrolases/antagonists & inhibitors; Carboxylic Ester Hydrolases/chemistry; Carboxylic Ester Hydrolases/genetics; Carboxylic Ester Hydrolases/metabolism; Hydroxybutyrates/metabolism; Molecular Sequence Data; Periplasm/enzymology; Polyesters/metabolism; Rhodospirillum rubrum/enzymology; Rhodospirillum rubrum/genetics; Rhodospirillum rubrum/growth & development; Solubility; Substrate Specificity

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

RHORU:Q6YNP7

GO:0050526: poly(3-hydroxybutyrate) depolymerase activity

ECO:0000314:

F

Table 3 shows substrate specificity of PHB depolymerase PhaZ1

complete
CACAO 3343

RHORU:Q6YNP7

enables

GO:0050526: poly(3-hydroxybutyrate) depolymerase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete


See also

References

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