Dieter Jendrossek | Universität Stuttgart (original) (raw)

Papers by Dieter Jendrossek

Fems Microbiology Letters, 1996

Applied and Environmental Microbiology, Apr 1, 1999

The relationship between extracellular poly(3-hydroxybutyrate) (PHB) depolymerase synthesis and t... more The relationship between extracellular poly(3-hydroxybutyrate) (PHB) depolymerase synthesis and the unusual properties of a succinate uptake system was investigated in Pseudomonas lemoignei. Growth on and uptake of succinate were highly pH dependent, with optima at pH 5.6. Above pH 7, growth on and uptake of succinate were strongly reduced with concomitant derepression of PHB depolymerase synthesis. The specific succinate uptake rates were saturable by high concentrations of succinate, and maximal transport rates of 110 nmol/mg of cell protein per min were determined between pH 5.6 and 6.8. The apparent K S0.5 values increased with increasing pH from 0.2 mM succinate at pH 5.6 to more than 10 mM succinate at pH 7.6. The uptake of [ 14 C]succinate was strongly inhibited by several monocarboxylates. Dicarboxylates also inhibited the uptake of succinate but only at pH values near the dissociation constant of the second carboxylate function (pK a 2). We conclude that the succinate carrier is specific for the monocarboxylate forms of various carboxylic acids and is not able to utilize the dicarboxylic forms. The inability to take up succinate 2؊ accounts for the carbon starvation of P. lemoignei observed during growth on succinate at pH values above 7. As a consequence the bacteria produce high levels of extracellular PHB depolymerase activity in an effort to escape carbon starvation by utilization of PHB hydrolysis products.

Macromolecules, 1997

The samples examined were thin films of approximately 200 µm thickness produced by melt casting. ... more The samples examined were thin films of approximately 200 µm thickness produced by melt casting. Since only the amorphous regions of the semicrystalline films contribute to the 1 H NMR image intensity, this technique provides unique information regarding the degradation process in the amorphous regions of the films when combined with total weight loss measurements. It was found that although the total weight loss rate of both the PHB and PHB/V films was constant, as previously reported, the initial amorphous material consumption rate was exponential. During the initial stages of the degradation process, up to 40-60 h, preferential consumption of amorphous material by depolymerase B was found to take place. At later stages the preference for amorphous material diminished, and both crystalline and amorphous phases were degraded indiscriminately. This initial consumption of amorphous material supports evidence that this stage is necessary to provide access to lamellar crystalline regions. The initial amorphous polymer consumption was verified by optical microscopy of the PHB film surface, which revealed the well-known circular erosion pattern associated with this type of enzymatic activity. Values of 0.020 and 0.049 h-1 for the rate constant of amorphous PHB and PHB/V consumption by depolymerase B were calculated from the 1 H imaging data during the early stages of degradation. The factors responsible for the observed behavior of the depolymerase B enzyme and the implications for the mechanism of enzymatic degradation in PHAs are discussed.

Macromolecular Symposia, 1998

... [35] Y. Kanewasa, N. Tanahashi, Y. Doi & T. Saito, Polym Degrad Stab, 45, 179 (1994). [36... more ... [35] Y. Kanewasa, N. Tanahashi, Y. Doi & T. Saito, Polym Degrad Stab, 45, 179 (1994). [36] J. Mergaert, A. Webb, C. Anderson, A. Wouters & J. Swings, J. Environm. Polym. ... Microbiol. 13, 807 (1994). [57] P. Tomme, D. P. Driver, E. A. Amarandoron, R. C. Jr. ...

Journal of Environmental Polymer Degradation, 1994

Four polyhydroxyalkanoate (PHA) depolymerases were purified from the culture fluid ofPseudomonas ... more Four polyhydroxyalkanoate (PHA) depolymerases were purified from the culture fluid ofPseudomonas lemoignei: poly(3-hydroxybutyrate) (PHB), depolymerase A (Mr, 55,000), and PHB depolymerase B (Mr, 67,000) were specific for PHB and copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) as substrates. The third depolymerase additionally hydrolyzed poly(3-hydroxyvalerate) (PHV) at high rates (PHV depolymerase;Mr, 54,000). The N-terminal amino acid sequences of the three purified

Journal of Bacteriology, 2007

Poly(3-hydroxybutyrate) (PHB) granules isolated in native form (nPHB granules) from Ralstonia eut... more Poly(3-hydroxybutyrate) (PHB) granules isolated in native form (nPHB granules) from Ralstonia eutropha catalyzed formation of PHB from 14 C-labeled acetyl coenzyme A (CoA) in the presence of NADPH and concomitantly released CoA, revealing that PHB biosynthetic proteins (acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, and PHB synthase) are present and active in isolated nPHB granules in vitro. nPHB granules also catalyzed thiolytic cleavage of PHB in the presence of added CoA, resulting in synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) from PHB. Synthesis of 3HB-CoA was also shown by incubation of artificial (protein-free) PHB with CoA and PhaZa1, confirming that PhaZa1 is a PHB depolymerase catalyzing the thiolysis reaction. Acetyl-CoA was the major product detectable after incubation of nPHB granules in the presence of NAD + , indicating that downstream mobilizing enzyme activities were also present and active in isolated nPHB granules. We propose that intracellular concentration...

Journal of Bacteriology, 2005

The Mms16 protein has been previously found to be associated with isolated magnetosomes from two ... more The Mms16 protein has been previously found to be associated with isolated magnetosomes from two Magnetospirillum strains. A function of this protein as a magnetosome-specific GTPase involved in the formation of intracellular magnetosome membrane vesicles was suggested (Y. Okamura, H. Takeyama, and T. Matsunaga, J. Biol. Chem. 276: 48183-48188, 2001). Here we present a study of the Mms16 protein from Magnetospirillum gryphiswaldense to clarify its function. Insertion-duplication mutagenesis of the mms16 gene did not affect the formation of magnetosome particles but resulted in the loss of the ability of M. gryphiswaldense cell extracts to activate poly(3-hydroxybutyrate) (PHB) depolymerization in vitro, which was coincident with loss of the most abundant 16-kDa polypeptide from preparations of PHB granule-bound proteins. The mms16 mutation could be functionally complemented by enhanced yellow fluorescent protein (EYFP) fused to ApdA, which is a PHB granule-bound protein (phasin) in ...

Journal of Applied Microbiology, 2010

Aims: Natural rubber (poly-[cis-1,4-isoprene]) can be cleaved into 12-oxo-4,8-dimethyltrideca-4,8... more Aims: Natural rubber (poly-[cis-1,4-isoprene]) can be cleaved into 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al by rubber oxygenase A (RoxA) isolated from Xanthomonas sp. RoxA is a novel type of dihaem dioxygenase with unknown cleavage mechanism of the rubber carbon backbone. Analysis of mutant RoxA after mutagenesis could be a way to investigate the function of selected amino acids of RoxA during catalysis. Unfortunately, expression of functional RoxA in recombinant Escherichia coli or in recombinant c-Proteobacteria such as Pseudomonas putida was not possible in our hands. Therefore, expression of recombinant RoxA in the homologous host, Xanthomonas, was performed. Methods and Results: A transformation system via electroporation was established, and a conjugation system was optimized for Xanthomonas sp. Inactivation of the chromosomal roxA gene by insertional mutagenesis resulted in inability of Xanthomonas sp. to produce active RoxA and to utilize rubber as a sole source of carbon and energy. When an intact copy of roxA was cloned under control of a rhamnose-inducible promoter in a broad host range vector and was transferred to Xanthomonas sp., high expression levels of functional RoxA in the presence of rhamnose were obtained. Conclusions and Significance and Impact of the Study: Purification of recombinantly expressed RoxA was simplified because of drastically shortened fermentation times and because separation of RoxA from remaining rubber latex particles was not necessary with rhamnose-induced cultures. About 6 mg purified RoxA were obtained from 1 l of cell-free culture fluid. Purified recombinant RoxA was highly active and revealed comparable spectral properties as RoxA purified from the wild type. The results of our study are the methodical basis for molecular biological manipulation in Xanthomonas sp. and will simplify investigation into the biochemical mechanisms by which rubber can be biodegraded in the environment by this novel extracellular dihaem dioxygenase RoxA.

FEMS Microbiology Letters, 1996

Mutational analysis of the poly(3-hydroxybutyrate) (PHB) depolymerase A of Pseudomonas lemoignei ... more Mutational analysis of the poly(3-hydroxybutyrate) (PHB) depolymerase A of Pseudomonas lemoignei and of the poly(3hydroxybutyrate) depolymerase of Alcaligenes faecalis revealed that S138 (P. lemoignet) and S13' (A. fuecalis) are essential for activity. Both serines are part of a strictly conserved pentapeptide sequence which is present in all poly(3-hydroxybutyrate) depolymerases analyzed so far (G-L-S-S(A)-G) and which resembles the lipase box of lipases and other serine hydrolases (G-X-S-X-G). Mutation of another conserved serine, namely S lQ5 (P. lemoignei) and Slg6 (A. faecalis), resulted in mutant proteins with almost full activity and proved that Slg5 and S'% are not essential for activity. The results indicate the structural and functional relationship of poly(3-hydroxybutyrate) depolymerases to the family of serine hydrolases.

FEMS Microbiology Letters, 1994

The degradation of sheets of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (BIOPOL ®) by aerobic s... more The degradation of sheets of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (BIOPOL ®) by aerobic sewage sludge was analyzed. Degradation of the polymer was highly dependent on the pH of the culture medium and was maximal between pH 7 and pH 8.5. Below pH 6 and above pH 9 the degradation rate was very low. Agitation of the culture fluid had relatively little influence on the rates of degradation. 1.2/105 aerobic polymer-degrading bacteria per ml sewage sludge were identified by halo formation on solid poly(3-hydroxybutyrate) (PHB)-containing media. The number of PHB-degrading bacteria in other ecosystems amounted to 3.8 × 103 per ml sludge of a freshwater lake, 9.2 x 105 per g garden-soil, 1.3 x 106 per g field-soil and 4.3 x 106 per g compost.

Applied Microbiology and Biotechnology, 2007

Pseudomonas putida GP01 cells that had accumulated medium-chain-length polyhydroxyalkanoates (PHA... more Pseudomonas putida GP01 cells that had accumulated medium-chain-length polyhydroxyalkanoates (PHA(MCL)) secreted 3-hydroxyoctanoate and 3-hydroxyhexanoate when incubated in alkaline buffers. The release of acids strongly decreased the pH resulting in less efficient secretion of 3HA(MCL) at neutral pH. To increase the yield of secreted MCL-hydroxyalkanoates, experiments at constant pH in a pH stat apparatus were performed. High acid releasing rates were recorded for the wild type GP01 at pH 9.2 (0.60 mmol acid h(-1) g(-1) cellular dry weight [cdw]). At more alkaline constant pH values (pH 9.3-11), the initial acid secretion rates were even higher but rapidly decreased by time. When acid secretion of PHA depolymerase mutant GPo500 was tested (pH 9.2), considerably lower rates compared to wild type were recorded (0.18 mmol acid h(-1) g(-1) cdw). Determination of dissolved oxygen during acid release indicated different respiratoric activity in wild type (low) and mutant (high). Acid release of mutant, but not of the wild type, could be enhanced by aeration. Determination of PHA content of cells after alkaline incubation showed that the wild type had lost most of its accumulated PHA, whereas the PHA content of the depolymerase mutant was not significantly reduced. Considerable amounts of 3HA(MCL) were secreted by the wild type, but only little 3HA(MCL) were found for the depolymerase mutant. In summary, 3HA(MCL) can be more efficiently produced at constant high pH than by incubation without pH control. High PHA depolymerase activity enabled the wild type to compensate for the high external pH by secretion of PHA hydrolysis products, whereas production of protons at aerobic conditions presumably was responsible for the major portion of the observed acid releasing rates in the depolymerase mutant.

Applied and Environmental Microbiology, 2012

RoxA is an extracellular c -type diheme cytochrome secreted by Xanthomonas sp. strain 35Y during ... more RoxA is an extracellular c -type diheme cytochrome secreted by Xanthomonas sp. strain 35Y during growth on rubber. RoxA cleaves poly( cis -1,4-isoprene) to 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). Analysis of the RoxA structure revealed that Phe317 is located in close proximity (≈5 Å) to the N-terminal heme that presumably represents the active site. To find evidence of whether Phe317 is important for catalysis, we changed it to tyrosine, tryptophan, leucine, histidine, or alanine. All five RoxA muteins were expressed after integration of the respective gene into the chromosome of a Xanthomonas sp. ΔroxA strain. Residual clearing zone formation on opaque latex agar was found for Xanthomonas sp. strains expressing the Phe317Leu, Phe317Ala, or Phe317His variant (wild type > Leu > Ala > His). Strains in which Phe317 was changed to tyrosine or tryptophan were inactive. Phe317Ala and Phe312Leu RoxA muteins were purified, and polyisoprene cleavage activities were reduced...

Applied and Environmental Microbiology, 2011

Hopanoids and sterols are members of a large group of cyclic triterpenoic compounds that have imp... more Hopanoids and sterols are members of a large group of cyclic triterpenoic compounds that have important functions in many prokaryotic and eukaryotic organisms. They are biochemically synthesized from linear precursors (squalene, 2,3-oxidosqualene) in only one enzymatic step that is catalyzed by squalene-hopene cyclase (SHC) or oxidosqualene cyclase (OSC). SHCs and OSCs are related in amino acid sequences and probably are derived from a common ancestor. The SHC reaction requires the formation of five ring structures, 13 covalent bonds, and nine stereo centers and therefore is one of the most complex one-step enzymatic reactions. We summarize the knowledge of the properties of triterpene cyclases and details of the reaction mechanism of Alicyclobacillus acidocaldarius SHC. Properties of other SHCs are included.

Applied and Environmental Microbiology, 2006

Two methods for accurate poly(3-hydroxybutyrate) (PHB) depolymerase activity determination and qu... more Two methods for accurate poly(3-hydroxybutyrate) (PHB) depolymerase activity determination and quantitative and qualitative hydrolysis product determination are described. The first method is based on online determination of NaOH consumption rates necessary to neutralize 3-hydroxybutyric acid (3HB) and/or 3HB oligomers produced during the hydrolysis reaction and requires a pH-stat apparatus equipped with a software-controlled microliter pump for rapid and accurate titration. The method is universally suitable for hydrolysis of any type of polyhydroxyalkanoate or other molecules with hydrolyzable ester bonds, allows the determination of hydrolysis rates of as low as 1 nmol/min, and has a dynamic capacity of at least 6 orders of magnitude. By applying this method, specific hydrolysis rates of native PHB granules isolated from Ralstonia eutropha H16 were determined for the first time. The second method was developed for hydrolysis product identification and is based on the derivatizati...

Applied and Environmental Microbiology, 2000

Streptomyces coelicolor 1A and Pseudomonas citronellolis were able to degrade synthetic high-mole... more Streptomyces coelicolor 1A and Pseudomonas citronellolis were able to degrade synthetic high-molecular-weight poly( cis -1,4-isoprene) and vulcanized natural rubber. Growth on the polymers was poor but significantly greater than that of the nondegrading strain Streptomyces lividans 1326 (control). Measurement of the molecular weight distribution of the polymer before and after degradation showed a time-dependent increase in low-molecular-weight polymer molecules for S. coelicolor 1A and P. citronellolis , whereas the molecular weight distribution for the control ( S. lividans 1326) remained almost constant. Three degradation products were isolated from the culture fluid of S. coelicolor 1A grown on vulcanized rubber and were identified as ( 6Z )-2,6-dimethyl-10-oxo-undec-6-enoic acid, ( 5Z )-6-methyl-undec-5-ene-2,9-dione, and ( 5Z , 9Z )-6,10-dimethyl-pentadec-5,9-diene-2,13-dione. An oxidative pathway from poly( cis -1,4-isoprene) to methyl-branched diketones is proposed. It inclu...

Applied and Environmental Microbiology, 2007

The recently finished genome sequence of Ralstonia eutropha H16 harbors nine genes that are thoug... more The recently finished genome sequence of Ralstonia eutropha H16 harbors nine genes that are thought to encode functions for intracellular depolymerization (mobilization) of storage poly(3-hydroxybutyrate) (PHB). Based on amino acid similarities, the gene products belong to four classes (PhaZa1 to PhaZa5, PhaZb, PhaZc, and PhaZd1/PhaZd2). However, convincing direct evidence for the in vivo roles of the gene products is poor. In this study, we selected four candidate genes ( phaZa1 , phaZb , phaZc , and phaZd1 ) representing the four classes and investigated the physiological function of the gene products (i) with recombinant Escherichia coli strains and (ii) with R. eutropha null mutants. Evidence for weak but significant PHB depolymerase activity was obtained only for PhaZa1. The physiological roles of the other potential PHB depolymerases remain uncertain.

Applied and Environmental Microbiology, 2006

Geranyl-coenzyme A (CoA)-carboxylase (GCase; AtuC/AtuF) and methylcrotonyl-CoA-carboxylase (MCase... more Geranyl-coenzyme A (CoA)-carboxylase (GCase; AtuC/AtuF) and methylcrotonyl-CoA-carboxylase (MCase; LiuB/LiuD) are characteristic enzymes of the catabolic pathway of acyclic terpenes (citronellol and geraniol) and of saturated methyl-branched compounds, such as leucine or isovalerate, respectively. Proteins encoded by two gene clusters ( atuABCDEFGH and liuRABCDE ) of Pseudomonas aeruginosa PAO1 were essential for acyclic terpene utilization (Atu) and for leucine and isovalerate utilization (Liu), respectively, as revealed by phenotype analysis of 10 insertion mutants, two-dimensional gel electrophoresis, determination of GCase and MCase activities, and Western blot analysis of wild-type and mutant strains. Analysis of the genome sequences of other pseudomonads ( P. putida KT2440 and P. fluorescens Pf-5) revealed candidate genes for Liu proteins for both species and candidate genes for Atu proteins in P. fluorescens . This result concurred with the finding that P. fluorescens , but n...

The TRPM8 ion channel is expressed in sensory neurons and is responsible for sensing environmenta... more The TRPM8 ion channel is expressed in sensory neurons and is responsible for sensing environmental cues, such as cold temperatures and chemical compounds, including menthol and icilin. The channel functional activity is regulated by various physical and chemical factors and is likely to be preconditioned by its molecular composition. Our studies indicate that the TRPM8 channel forms a structural-functional complex with the polyester poly-(R)-3-hydroxybutyrate (PHB). We identified by mass spectrometry a number of PHB-modified peptides in the N terminus of the TRPM8 protein and in its extracellular S3-S4 linker. Removal of PHB by enzymatic hydrolysis and site-directed mutagenesis of both the serine residues that serve as covalent anchors for PHB and adjacent hydrophobic residues that interact with the methyl groups of the polymer resulted in significant inhibition of TRPM8 channel activity. We conclude that the TRPM8 channel undergoes posttranslational modification by PHB and that this modification is required for its normal function.

Applied and environmental microbiology, Jan 15, 2017

Only two types of rubber oxygenases, rubber oxygenase (RoxA) and latex clearing protein (Lcp), ha... more Only two types of rubber oxygenases, rubber oxygenase (RoxA) and latex clearing protein (Lcp), have been described so far. RoxA proteins (RoxAs) are c-type cytochromes of ≈70 kDa produced by Gram-negative rubber-degrading bacteria, and they cleave polyisoprene into 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD), a C15 oligo-isoprenoid, as the major end product. Lcps are common among Gram-positive rubber degraders and do not share amino acid sequence similarities with RoxAs. Furthermore, Lcps have much smaller molecular masses (≈40 kDa), are b-type cytochromes, and cleave polyisoprene to a mixture of C20, C25, C30, and higher oligo-isoprenoids as end products. In this article, we purified a new type of rubber oxygenase, RoxB Xsp (RoxB of Xanthomonas sp. strain 35Y). RoxB Xsp is distantly related to RoxAs and resembles RoxAs with respect to molecular mass (70.3 kDa for mature protein) and cofactor content (2 c-type hemes). However, RoxB Xsp differs from all currently known RoxAs in ...

Fems Microbiology Letters, 1996

Applied and Environmental Microbiology, Apr 1, 1999

The relationship between extracellular poly(3-hydroxybutyrate) (PHB) depolymerase synthesis and t... more The relationship between extracellular poly(3-hydroxybutyrate) (PHB) depolymerase synthesis and the unusual properties of a succinate uptake system was investigated in Pseudomonas lemoignei. Growth on and uptake of succinate were highly pH dependent, with optima at pH 5.6. Above pH 7, growth on and uptake of succinate were strongly reduced with concomitant derepression of PHB depolymerase synthesis. The specific succinate uptake rates were saturable by high concentrations of succinate, and maximal transport rates of 110 nmol/mg of cell protein per min were determined between pH 5.6 and 6.8. The apparent K S0.5 values increased with increasing pH from 0.2 mM succinate at pH 5.6 to more than 10 mM succinate at pH 7.6. The uptake of [ 14 C]succinate was strongly inhibited by several monocarboxylates. Dicarboxylates also inhibited the uptake of succinate but only at pH values near the dissociation constant of the second carboxylate function (pK a 2). We conclude that the succinate carrier is specific for the monocarboxylate forms of various carboxylic acids and is not able to utilize the dicarboxylic forms. The inability to take up succinate 2؊ accounts for the carbon starvation of P. lemoignei observed during growth on succinate at pH values above 7. As a consequence the bacteria produce high levels of extracellular PHB depolymerase activity in an effort to escape carbon starvation by utilization of PHB hydrolysis products.

Macromolecules, 1997

The samples examined were thin films of approximately 200 µm thickness produced by melt casting. ... more The samples examined were thin films of approximately 200 µm thickness produced by melt casting. Since only the amorphous regions of the semicrystalline films contribute to the 1 H NMR image intensity, this technique provides unique information regarding the degradation process in the amorphous regions of the films when combined with total weight loss measurements. It was found that although the total weight loss rate of both the PHB and PHB/V films was constant, as previously reported, the initial amorphous material consumption rate was exponential. During the initial stages of the degradation process, up to 40-60 h, preferential consumption of amorphous material by depolymerase B was found to take place. At later stages the preference for amorphous material diminished, and both crystalline and amorphous phases were degraded indiscriminately. This initial consumption of amorphous material supports evidence that this stage is necessary to provide access to lamellar crystalline regions. The initial amorphous polymer consumption was verified by optical microscopy of the PHB film surface, which revealed the well-known circular erosion pattern associated with this type of enzymatic activity. Values of 0.020 and 0.049 h-1 for the rate constant of amorphous PHB and PHB/V consumption by depolymerase B were calculated from the 1 H imaging data during the early stages of degradation. The factors responsible for the observed behavior of the depolymerase B enzyme and the implications for the mechanism of enzymatic degradation in PHAs are discussed.

Macromolecular Symposia, 1998

... [35] Y. Kanewasa, N. Tanahashi, Y. Doi & T. Saito, Polym Degrad Stab, 45, 179 (1994). [36... more ... [35] Y. Kanewasa, N. Tanahashi, Y. Doi & T. Saito, Polym Degrad Stab, 45, 179 (1994). [36] J. Mergaert, A. Webb, C. Anderson, A. Wouters & J. Swings, J. Environm. Polym. ... Microbiol. 13, 807 (1994). [57] P. Tomme, D. P. Driver, E. A. Amarandoron, R. C. Jr. ...

Journal of Environmental Polymer Degradation, 1994

Four polyhydroxyalkanoate (PHA) depolymerases were purified from the culture fluid ofPseudomonas ... more Four polyhydroxyalkanoate (PHA) depolymerases were purified from the culture fluid ofPseudomonas lemoignei: poly(3-hydroxybutyrate) (PHB), depolymerase A (Mr, 55,000), and PHB depolymerase B (Mr, 67,000) were specific for PHB and copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) as substrates. The third depolymerase additionally hydrolyzed poly(3-hydroxyvalerate) (PHV) at high rates (PHV depolymerase;Mr, 54,000). The N-terminal amino acid sequences of the three purified

Journal of Bacteriology, 2007

Poly(3-hydroxybutyrate) (PHB) granules isolated in native form (nPHB granules) from Ralstonia eut... more Poly(3-hydroxybutyrate) (PHB) granules isolated in native form (nPHB granules) from Ralstonia eutropha catalyzed formation of PHB from 14 C-labeled acetyl coenzyme A (CoA) in the presence of NADPH and concomitantly released CoA, revealing that PHB biosynthetic proteins (acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, and PHB synthase) are present and active in isolated nPHB granules in vitro. nPHB granules also catalyzed thiolytic cleavage of PHB in the presence of added CoA, resulting in synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) from PHB. Synthesis of 3HB-CoA was also shown by incubation of artificial (protein-free) PHB with CoA and PhaZa1, confirming that PhaZa1 is a PHB depolymerase catalyzing the thiolysis reaction. Acetyl-CoA was the major product detectable after incubation of nPHB granules in the presence of NAD + , indicating that downstream mobilizing enzyme activities were also present and active in isolated nPHB granules. We propose that intracellular concentration...

Journal of Bacteriology, 2005

The Mms16 protein has been previously found to be associated with isolated magnetosomes from two ... more The Mms16 protein has been previously found to be associated with isolated magnetosomes from two Magnetospirillum strains. A function of this protein as a magnetosome-specific GTPase involved in the formation of intracellular magnetosome membrane vesicles was suggested (Y. Okamura, H. Takeyama, and T. Matsunaga, J. Biol. Chem. 276: 48183-48188, 2001). Here we present a study of the Mms16 protein from Magnetospirillum gryphiswaldense to clarify its function. Insertion-duplication mutagenesis of the mms16 gene did not affect the formation of magnetosome particles but resulted in the loss of the ability of M. gryphiswaldense cell extracts to activate poly(3-hydroxybutyrate) (PHB) depolymerization in vitro, which was coincident with loss of the most abundant 16-kDa polypeptide from preparations of PHB granule-bound proteins. The mms16 mutation could be functionally complemented by enhanced yellow fluorescent protein (EYFP) fused to ApdA, which is a PHB granule-bound protein (phasin) in ...

Journal of Applied Microbiology, 2010

Aims: Natural rubber (poly-[cis-1,4-isoprene]) can be cleaved into 12-oxo-4,8-dimethyltrideca-4,8... more Aims: Natural rubber (poly-[cis-1,4-isoprene]) can be cleaved into 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al by rubber oxygenase A (RoxA) isolated from Xanthomonas sp. RoxA is a novel type of dihaem dioxygenase with unknown cleavage mechanism of the rubber carbon backbone. Analysis of mutant RoxA after mutagenesis could be a way to investigate the function of selected amino acids of RoxA during catalysis. Unfortunately, expression of functional RoxA in recombinant Escherichia coli or in recombinant c-Proteobacteria such as Pseudomonas putida was not possible in our hands. Therefore, expression of recombinant RoxA in the homologous host, Xanthomonas, was performed. Methods and Results: A transformation system via electroporation was established, and a conjugation system was optimized for Xanthomonas sp. Inactivation of the chromosomal roxA gene by insertional mutagenesis resulted in inability of Xanthomonas sp. to produce active RoxA and to utilize rubber as a sole source of carbon and energy. When an intact copy of roxA was cloned under control of a rhamnose-inducible promoter in a broad host range vector and was transferred to Xanthomonas sp., high expression levels of functional RoxA in the presence of rhamnose were obtained. Conclusions and Significance and Impact of the Study: Purification of recombinantly expressed RoxA was simplified because of drastically shortened fermentation times and because separation of RoxA from remaining rubber latex particles was not necessary with rhamnose-induced cultures. About 6 mg purified RoxA were obtained from 1 l of cell-free culture fluid. Purified recombinant RoxA was highly active and revealed comparable spectral properties as RoxA purified from the wild type. The results of our study are the methodical basis for molecular biological manipulation in Xanthomonas sp. and will simplify investigation into the biochemical mechanisms by which rubber can be biodegraded in the environment by this novel extracellular dihaem dioxygenase RoxA.

FEMS Microbiology Letters, 1996

Mutational analysis of the poly(3-hydroxybutyrate) (PHB) depolymerase A of Pseudomonas lemoignei ... more Mutational analysis of the poly(3-hydroxybutyrate) (PHB) depolymerase A of Pseudomonas lemoignei and of the poly(3hydroxybutyrate) depolymerase of Alcaligenes faecalis revealed that S138 (P. lemoignet) and S13' (A. fuecalis) are essential for activity. Both serines are part of a strictly conserved pentapeptide sequence which is present in all poly(3-hydroxybutyrate) depolymerases analyzed so far (G-L-S-S(A)-G) and which resembles the lipase box of lipases and other serine hydrolases (G-X-S-X-G). Mutation of another conserved serine, namely S lQ5 (P. lemoignei) and Slg6 (A. faecalis), resulted in mutant proteins with almost full activity and proved that Slg5 and S'% are not essential for activity. The results indicate the structural and functional relationship of poly(3-hydroxybutyrate) depolymerases to the family of serine hydrolases.

FEMS Microbiology Letters, 1994

The degradation of sheets of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (BIOPOL ®) by aerobic s... more The degradation of sheets of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (BIOPOL ®) by aerobic sewage sludge was analyzed. Degradation of the polymer was highly dependent on the pH of the culture medium and was maximal between pH 7 and pH 8.5. Below pH 6 and above pH 9 the degradation rate was very low. Agitation of the culture fluid had relatively little influence on the rates of degradation. 1.2/105 aerobic polymer-degrading bacteria per ml sewage sludge were identified by halo formation on solid poly(3-hydroxybutyrate) (PHB)-containing media. The number of PHB-degrading bacteria in other ecosystems amounted to 3.8 × 103 per ml sludge of a freshwater lake, 9.2 x 105 per g garden-soil, 1.3 x 106 per g field-soil and 4.3 x 106 per g compost.

Applied Microbiology and Biotechnology, 2007

Pseudomonas putida GP01 cells that had accumulated medium-chain-length polyhydroxyalkanoates (PHA... more Pseudomonas putida GP01 cells that had accumulated medium-chain-length polyhydroxyalkanoates (PHA(MCL)) secreted 3-hydroxyoctanoate and 3-hydroxyhexanoate when incubated in alkaline buffers. The release of acids strongly decreased the pH resulting in less efficient secretion of 3HA(MCL) at neutral pH. To increase the yield of secreted MCL-hydroxyalkanoates, experiments at constant pH in a pH stat apparatus were performed. High acid releasing rates were recorded for the wild type GP01 at pH 9.2 (0.60 mmol acid h(-1) g(-1) cellular dry weight [cdw]). At more alkaline constant pH values (pH 9.3-11), the initial acid secretion rates were even higher but rapidly decreased by time. When acid secretion of PHA depolymerase mutant GPo500 was tested (pH 9.2), considerably lower rates compared to wild type were recorded (0.18 mmol acid h(-1) g(-1) cdw). Determination of dissolved oxygen during acid release indicated different respiratoric activity in wild type (low) and mutant (high). Acid release of mutant, but not of the wild type, could be enhanced by aeration. Determination of PHA content of cells after alkaline incubation showed that the wild type had lost most of its accumulated PHA, whereas the PHA content of the depolymerase mutant was not significantly reduced. Considerable amounts of 3HA(MCL) were secreted by the wild type, but only little 3HA(MCL) were found for the depolymerase mutant. In summary, 3HA(MCL) can be more efficiently produced at constant high pH than by incubation without pH control. High PHA depolymerase activity enabled the wild type to compensate for the high external pH by secretion of PHA hydrolysis products, whereas production of protons at aerobic conditions presumably was responsible for the major portion of the observed acid releasing rates in the depolymerase mutant.

Applied and Environmental Microbiology, 2012

RoxA is an extracellular c -type diheme cytochrome secreted by Xanthomonas sp. strain 35Y during ... more RoxA is an extracellular c -type diheme cytochrome secreted by Xanthomonas sp. strain 35Y during growth on rubber. RoxA cleaves poly( cis -1,4-isoprene) to 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). Analysis of the RoxA structure revealed that Phe317 is located in close proximity (≈5 Å) to the N-terminal heme that presumably represents the active site. To find evidence of whether Phe317 is important for catalysis, we changed it to tyrosine, tryptophan, leucine, histidine, or alanine. All five RoxA muteins were expressed after integration of the respective gene into the chromosome of a Xanthomonas sp. ΔroxA strain. Residual clearing zone formation on opaque latex agar was found for Xanthomonas sp. strains expressing the Phe317Leu, Phe317Ala, or Phe317His variant (wild type > Leu > Ala > His). Strains in which Phe317 was changed to tyrosine or tryptophan were inactive. Phe317Ala and Phe312Leu RoxA muteins were purified, and polyisoprene cleavage activities were reduced...

Applied and Environmental Microbiology, 2011

Hopanoids and sterols are members of a large group of cyclic triterpenoic compounds that have imp... more Hopanoids and sterols are members of a large group of cyclic triterpenoic compounds that have important functions in many prokaryotic and eukaryotic organisms. They are biochemically synthesized from linear precursors (squalene, 2,3-oxidosqualene) in only one enzymatic step that is catalyzed by squalene-hopene cyclase (SHC) or oxidosqualene cyclase (OSC). SHCs and OSCs are related in amino acid sequences and probably are derived from a common ancestor. The SHC reaction requires the formation of five ring structures, 13 covalent bonds, and nine stereo centers and therefore is one of the most complex one-step enzymatic reactions. We summarize the knowledge of the properties of triterpene cyclases and details of the reaction mechanism of Alicyclobacillus acidocaldarius SHC. Properties of other SHCs are included.

Applied and Environmental Microbiology, 2006

Two methods for accurate poly(3-hydroxybutyrate) (PHB) depolymerase activity determination and qu... more Two methods for accurate poly(3-hydroxybutyrate) (PHB) depolymerase activity determination and quantitative and qualitative hydrolysis product determination are described. The first method is based on online determination of NaOH consumption rates necessary to neutralize 3-hydroxybutyric acid (3HB) and/or 3HB oligomers produced during the hydrolysis reaction and requires a pH-stat apparatus equipped with a software-controlled microliter pump for rapid and accurate titration. The method is universally suitable for hydrolysis of any type of polyhydroxyalkanoate or other molecules with hydrolyzable ester bonds, allows the determination of hydrolysis rates of as low as 1 nmol/min, and has a dynamic capacity of at least 6 orders of magnitude. By applying this method, specific hydrolysis rates of native PHB granules isolated from Ralstonia eutropha H16 were determined for the first time. The second method was developed for hydrolysis product identification and is based on the derivatizati...

Applied and Environmental Microbiology, 2000

Streptomyces coelicolor 1A and Pseudomonas citronellolis were able to degrade synthetic high-mole... more Streptomyces coelicolor 1A and Pseudomonas citronellolis were able to degrade synthetic high-molecular-weight poly( cis -1,4-isoprene) and vulcanized natural rubber. Growth on the polymers was poor but significantly greater than that of the nondegrading strain Streptomyces lividans 1326 (control). Measurement of the molecular weight distribution of the polymer before and after degradation showed a time-dependent increase in low-molecular-weight polymer molecules for S. coelicolor 1A and P. citronellolis , whereas the molecular weight distribution for the control ( S. lividans 1326) remained almost constant. Three degradation products were isolated from the culture fluid of S. coelicolor 1A grown on vulcanized rubber and were identified as ( 6Z )-2,6-dimethyl-10-oxo-undec-6-enoic acid, ( 5Z )-6-methyl-undec-5-ene-2,9-dione, and ( 5Z , 9Z )-6,10-dimethyl-pentadec-5,9-diene-2,13-dione. An oxidative pathway from poly( cis -1,4-isoprene) to methyl-branched diketones is proposed. It inclu...

Applied and Environmental Microbiology, 2007

The recently finished genome sequence of Ralstonia eutropha H16 harbors nine genes that are thoug... more The recently finished genome sequence of Ralstonia eutropha H16 harbors nine genes that are thought to encode functions for intracellular depolymerization (mobilization) of storage poly(3-hydroxybutyrate) (PHB). Based on amino acid similarities, the gene products belong to four classes (PhaZa1 to PhaZa5, PhaZb, PhaZc, and PhaZd1/PhaZd2). However, convincing direct evidence for the in vivo roles of the gene products is poor. In this study, we selected four candidate genes ( phaZa1 , phaZb , phaZc , and phaZd1 ) representing the four classes and investigated the physiological function of the gene products (i) with recombinant Escherichia coli strains and (ii) with R. eutropha null mutants. Evidence for weak but significant PHB depolymerase activity was obtained only for PhaZa1. The physiological roles of the other potential PHB depolymerases remain uncertain.

Applied and Environmental Microbiology, 2006

Geranyl-coenzyme A (CoA)-carboxylase (GCase; AtuC/AtuF) and methylcrotonyl-CoA-carboxylase (MCase... more Geranyl-coenzyme A (CoA)-carboxylase (GCase; AtuC/AtuF) and methylcrotonyl-CoA-carboxylase (MCase; LiuB/LiuD) are characteristic enzymes of the catabolic pathway of acyclic terpenes (citronellol and geraniol) and of saturated methyl-branched compounds, such as leucine or isovalerate, respectively. Proteins encoded by two gene clusters ( atuABCDEFGH and liuRABCDE ) of Pseudomonas aeruginosa PAO1 were essential for acyclic terpene utilization (Atu) and for leucine and isovalerate utilization (Liu), respectively, as revealed by phenotype analysis of 10 insertion mutants, two-dimensional gel electrophoresis, determination of GCase and MCase activities, and Western blot analysis of wild-type and mutant strains. Analysis of the genome sequences of other pseudomonads ( P. putida KT2440 and P. fluorescens Pf-5) revealed candidate genes for Liu proteins for both species and candidate genes for Atu proteins in P. fluorescens . This result concurred with the finding that P. fluorescens , but n...

The TRPM8 ion channel is expressed in sensory neurons and is responsible for sensing environmenta... more The TRPM8 ion channel is expressed in sensory neurons and is responsible for sensing environmental cues, such as cold temperatures and chemical compounds, including menthol and icilin. The channel functional activity is regulated by various physical and chemical factors and is likely to be preconditioned by its molecular composition. Our studies indicate that the TRPM8 channel forms a structural-functional complex with the polyester poly-(R)-3-hydroxybutyrate (PHB). We identified by mass spectrometry a number of PHB-modified peptides in the N terminus of the TRPM8 protein and in its extracellular S3-S4 linker. Removal of PHB by enzymatic hydrolysis and site-directed mutagenesis of both the serine residues that serve as covalent anchors for PHB and adjacent hydrophobic residues that interact with the methyl groups of the polymer resulted in significant inhibition of TRPM8 channel activity. We conclude that the TRPM8 channel undergoes posttranslational modification by PHB and that this modification is required for its normal function.

Applied and environmental microbiology, Jan 15, 2017

Only two types of rubber oxygenases, rubber oxygenase (RoxA) and latex clearing protein (Lcp), ha... more Only two types of rubber oxygenases, rubber oxygenase (RoxA) and latex clearing protein (Lcp), have been described so far. RoxA proteins (RoxAs) are c-type cytochromes of ≈70 kDa produced by Gram-negative rubber-degrading bacteria, and they cleave polyisoprene into 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD), a C15 oligo-isoprenoid, as the major end product. Lcps are common among Gram-positive rubber degraders and do not share amino acid sequence similarities with RoxAs. Furthermore, Lcps have much smaller molecular masses (≈40 kDa), are b-type cytochromes, and cleave polyisoprene to a mixture of C20, C25, C30, and higher oligo-isoprenoids as end products. In this article, we purified a new type of rubber oxygenase, RoxB Xsp (RoxB of Xanthomonas sp. strain 35Y). RoxB Xsp is distantly related to RoxAs and resembles RoxAs with respect to molecular mass (70.3 kDa for mature protein) and cofactor content (2 c-type hemes). However, RoxB Xsp differs from all currently known RoxAs in ...