Birke Jakob, Röther Wolf, Jendrossek Dieter
Institute of Microbiology, University of Stuttgart, Germany.
Institute of Microbiology, University of Stuttgart, Germany
Appl Environ Microbiol. 2017 Jun 30;83(14). doi: 10.1128/AEM.00721-17. Print 2017 Jul 15.
Only two types of rubber oxygenases, rubber oxygenase (RoxA) and latex clearing protein (Lcp), have been described so far. RoxA proteins (RoxAs) are -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 C 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 -type cytochromes, and cleave polyisoprene to a mixture of C, C, C, and higher oligo-isoprenoids as end products. In this article, we purified a new type of rubber oxygenase, RoxB (RoxB of sp. strain 35Y). RoxB is distantly related to RoxAs and resembles RoxAs with respect to molecular mass (70.3 kDa for mature protein) and cofactor content (2 -type hemes). However, RoxB differs from all currently known RoxAs in having a distinctive product spectrum of C, C, C, and higher oligo-isoprenoids that has been observed only for Lcps so far. Purified RoxB revealed the highest specific activity of 4.5 U/mg (at 23°C) of all currently known rubber oxygenases and exerts a synergistic effect on the efficiency of polyisoprene cleavage by RoxA RoxB homologs were identified in several other Gram-negative rubber-degrading species, pointing to a prominent function of RoxB for the biodegradation of rubber in Gram-negative bacteria. The enzymatic cleavage of rubber (polyisoprene) is of high environmental importance given that enormous amounts of rubber waste materials are permanently released (e.g., by abrasion of tires). Research from the last decade has discovered rubber oxygenase A, RoxA, and latex clearing protein (Lcp) as being responsible for the primary enzymatic attack on the hydrophobic and water-insoluble biopolymer poly(-1,4-isoprene) in Gram-negative and Gram-positive rubber-degrading bacteria, respectively. Here, we provide evidence that a third type of rubber oxygenase is present in Gram-negative rubber-degrading species. Due to its characteristics, we suggest the designation RoxB for the new type of rubber oxygenase. Bioinformatic analysis of genome sequences indicates the presence of homologs in other Gram-negative rubber degraders.
到目前为止，仅发现了两种橡胶氧化酶，即橡胶氧化酶（RoxA）和乳胶清除蛋白（Lcp）。RoxA蛋白（RoxAs）是由革兰氏阴性橡胶降解细菌产生的约70 kDa的β型细胞色素，它们将聚异戊二烯切割成12-氧代-4,8-二甲基十三碳-4,8-二烯-1-醛（ODTD），一种C13寡聚异戊二烯，作为主要终产物。Lcps在革兰氏阳性橡胶降解菌中普遍存在，与RoxAs没有氨基酸序列相似性。此外，Lcps的分子量要小得多（约40 kDa），是α型细胞色素，并且将聚异戊二烯切割成C11、C12、C13和更高的寡聚异戊二烯混合物作为终产物。在本文中，我们纯化了一种新型橡胶氧化酶，RoxB（来自sp.菌株35Y的RoxB）。RoxB与RoxAs的亲缘关系较远，在分子量（成熟蛋白为70.3 kDa）和辅因子含量（2个β型血红素）方面与RoxAs相似。然而，RoxB与所有目前已知的RoxAs不同，其具有独特的C11、C12、C13和更高寡聚异戊二烯产物谱，到目前为止仅在Lcps中观察到。纯化的RoxB在所有目前已知的橡胶氧化酶中显示出最高的比活性，为4.5 U/mg（在23°C下），并且对RoxA切割聚异戊二烯的效率具有协同作用。在其他几种革兰氏阴性橡胶降解物种中鉴定出了RoxB同源物，这表明RoxB在革兰氏阴性细菌中橡胶生物降解中具有重要作用。鉴于大量橡胶废料不断被释放（例如通过轮胎磨损），橡胶（聚异戊二烯）的酶促切割具有高度的环境重要性。过去十年的研究发现橡胶氧化酶A（RoxA）和乳胶清除蛋白（Lcp）分别是革兰氏阴性和革兰氏阳性橡胶降解细菌中对疏水性和水不溶性生物聚合物聚（-1,4-异戊二烯）进行初次酶促攻击的原因。在此，我们提供证据表明革兰氏阴性橡胶降解物种中存在第三种橡胶氧化酶。由于其特性，我们建议将这种新型橡胶氧化酶命名为RoxB。基因组序列的生物信息学分析表明在其他革兰氏阴性橡胶降解菌中存在RoxB同源物。
