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

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.

FIG. 1.

Influence of temperature on activity and stability of purified soluble PHB depolymerase. Purified PhaZ1 was assayed at different temperatures (circles). For determination of temperature stability, purified PhaZ1 (0.6 μg) was incubated in assay buffer at 50°C for the times indicated before residual activity was determined (columns).

FIG. 2.

Amino acid sequence of soluble PHB depolymerase of R. rubrum PhaZ1 and alignment with extracellular PHB depolymerases harboring catalytic type II domains. PhaZAspTP4, Acidovorax sp. (accession no. AB015309.1); PhaZDac, Delftia acidovorans (accession no. AB003186); PhaZCsp, Comamonas sp. (accession no. AAA87070); PhaZCte, Comamonas testosteroni (accession no. AB000508); PhaZLspHS, Lepthotrix sp. strain HS (Caldimonas manganoxidans) (accession no. AB038647); PhaZSex, Streptomyces exfoliatus (accession no. U58990); PhaZRr, Rhodospirillum rubrum (accession no. AY061637). Amino acids that have been determined by Edman degradation of purified PhaZ1 or isolated peptides of PhaZ1 of R. rubrum are underlined. Alignment was performed with Clustalw 1.7 (gonnet series [49]). Increasing darkness of shading indicates increasing degrees of identity among PHB depolymerases at the respective position. Boldfaced amino acids indicate the putative catalytic triad and the oxyanion pocket amino acids. Asterisks, hydrophobic residues. Values in the consensus line represent phylogenetic tree scores for conserved residues; a lower value is better (i.e., there is less evolutionary cost).

FIG. 3.

Domain structure of PHA_SCL depolymerases. A schematic model of PHA_SCL depolymerases with different combinations of the catalytic domain, linker domain, and substrate-binding domain is shown. Abbreviations: Thr, threonine-rich region; Fn3, fibronectin type 3-like domain; Cad, cadherin-like domain; SBD, substrate-binding domain.