An extracellular protein with strong absorption at 406 nm was purified from cell-free culture fluid of latexgrown Xanthomonas sp. strain 35Y. This protein was identical to the gene product of a recently characterized gene cloned from Xanthomonas sp., as revealed by determination of m/z values and sequencing of selected isolated peptides obtained after trypsin fingerprint analysis. The purified protein degraded both natural rubber latex and chemosynthetic poly(cis-1,4-isoprene) in vitro by oxidative cleavage of the double bonds of poly(cis-1,4-isoprene). 12-Oxo-4,8-dimethyltrideca-4,8-diene-1-al (m/z 236) was identified and unequivocally characterized as the major cleavage product, and there was a homologous series of minor metabolites that differed from the major degradation product only in the number of repetitive isoprene units between terminal functions, CHO-CH 2 O and OCH 2 -COCH 3 . An in vitro enzyme assay for oxidative rubber degradation was developed based on high-performance liquid chromatography analysis and spectroscopic detection of product carbonyl functions after derivatization with dinitrophenylhydrazone. Enzymatic cleavage of rubber by the purified protein was strictly dependent on the presence of oxygen; it did not require addition of any soluble cofactors or metal ions and was optimal around pH 7.0 at 40°C. Carbon monoxide and cyanide inhibited the reaction; addition of catalase had no effect, and peroxidase activity could not be detected. The purified protein was specific for natural rubber latex and chemosynthetic poly ( Natural rubber (NR) is a biopolymer that is synthesized by many plants and some fungi. This polymer has been commercially exploited for more than 100 years by cultivating and tapping the rubber tree (Hevea brasiliensis). NR is a polymer of many isoprene units [poly(cis-1,4-isoprene)] that, after cross-linking of the linear polymer chains by sulfur bridges (vulcanization), has superior physical properties. Despite the development of chemosynthetic rubbers, NR is still a necessary raw material for products such as tires, latex gloves, condoms, and seals.NR does not accumulate in the environment. Many reports on the biodegradability of rubbers were published during the last century (for recent studies see references 2, 9, 11, 12, and 18 and references therein). Even chemically cross-linked (vulcanized) rubbers have been shown to be biodegradable (3,7,17). Two biological strategies for microbial NR degradation have been described so far. (i) A large number of bacteria, most of which belong to the actinomycetes, are able to grow and to produce clearing zones on agar media containing NR latex in the form of a milky opaque emulsion as a carbon source (9). So far, Xanthomonas sp. strain 35Y is the only known gram-negative NR-degrading bacterium belonging to this group (18). (ii) The members of the other group of NR-utilizing bacteria do not produce clearing zones on NR latex agar; rather, they are able to solubilize solid pieces of NR and to use the resulting emulsion as a carbon sou...

Natural rubber [poly-(cis-1,4-isoprene)] is cleaved to by rubber oxygenase A (RoxA) isolated from Xanthomonas sp. RoxA has two c-type haem centres that show two distinct a-bands at 549 and 553 nm in the dithionite-reduced state. A wellresolved midpoint potential (E 0 9) of -65 mV was determined for one haem by spectrophotometric titrations in the absence of dioxygen with dithionite and ferricyanide as reductant and oxidant, respectively. The midpoint potential of the second haem was not resolvable (E 0 9 about "130 to -160 mV). One of the two haems was reduced by NADH (549 nm a-band), similar to bacterial dihaem peroxidases. Evidence for an electron transfer between the two haems was provided by slow reduction of the second haem (553 nm a-band) upon incubation of the partially reduced enzyme at room temperature. Addition of imidazole or related compounds to RoxA led to UV/vis spectral features similar to those observed for partially reduced RoxA. Notably, reduction of RoxA with dithionite or NADH, or binding of compounds such as imidazole, resulted in a reversible inactivation of the enzyme, unlike dihaem peroxidases. In line with this result, RoxA did not show any peroxidase activity. EPR spectra of RoxA as isolated showed two low-spin Fe(III) haem centres, with apparent g-values of 3.39, 3.09, 2.23, 1.92 and 1.50. A weak signal in the g56 region resulting from a high-spin Fe(III) haem was also observed with a preparation-dependent intensity that disappeared in the presence of imidazole. Attempts to provide spectroscopic evidence for binding of the natural substrate (polyisoprene latex) to RoxA failed. However, experimental data are presented that RoxA is able to subtract redox equivalents from its substrate or from model compounds. In conclusion, RoxA is a novel type of dihaem dioxygenase with features clearly different from classical cytochrome c peroxidases.

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The recently discovered extracellular poly[(R)-3-hydroxybutyrate] (PHB) depolymerase PhaZ7 of Paucimonas lemoignei represents the first member of a new subgroup (EC 3.1.1.75) of serine hydrolases with no significant amino acid similarities to conventional PHB depolymerases, lipases or other hydrolases except for a potential lipase box-like motif (Ala-His-Ser 136 -Met-Gly) and potential candidates for catalytic triad and oxyanion pocket amino acids. In order to identify amino acids essential for activity 11 mutants of phaZ7 were generated by site-directed mutagenesis and expressed in recombinant protease-deficient Bacillus subtilis WB800. The wild-type depolymerase and 10 of the 11 mutant proteins (except for Ser 136 Cys) were expressed and efficiently secreted by B. subtilis as shown by Western blots of cell-free culture fluid proteins. No PHB depolymerase activity was detected in strains harbouring one of the following substitutions : His 47 Ala, Ser 136 Ala, Asp 242 Ala, Asp 242 Asn, His 306 Ala, indicating the importance of these amino acids for activity. Replacement of Ser 136 by Thr resulted in a decrease of activity to about 20% of the wild-type level and suggested that the hydroxy group of the serine side chain is important for activity but can be partially replaced by the hydroxy function of threonine. Alterations of Asp 256 to Ala or Asn or of the putative serine hydrolase pentapeptide motif (Ala-His-Ser 136 -Met-Gly) to a lipase box consensus sequence (Gly 134 -His-Ser 136 -Met-Gly) or to the PHB depolymerase box consensus sequence (Gly 134 -Leu 135 -Ser 136 -Met-Gly) had no significant effect on PHB depolymerase activity, indicating that these amino acids or sequence motifs were not essential for activity. In conclusion, the PHB depolymerase PhaZ7 is a serine hydrolase with a catalytic triad and oxyanion pocket consisting of His 47 , Ser 136 , Asp 242 and His 306 .

Polyhydroxyalkanoates (PHA) are biodegradable polyesters that have attracted commercial and academic interest as environmentally friendly materials. A number of enzymes are able to degrade polyhydroxyalkanoates to water-soluble products. PhaZ7 poly(3-hydroxybutyrate) (PHB) depolymerase (EC 3.1.1.75), a 342-amino-acid hydrolase from the PHA-degrading bacterium Paucimonas lemoignei, has been found to possess substrate specificity for amorphous PHA. PhaZ7 was crystallized by the microdialysis method. Thin rod-like crystals were grown in low ionic strength solution and found to belong to the monoclinic space group C2, with unit-cell parameters a = 225.8, b = 46.5, c = 171.3, beta = 128.9 degrees. A complete data set was collected to 2.75 A resolution at 100 K using synchrotron radiation.