Rubredoxins Involved in Alkane Oxidation

Rubredoxins (Rds) are essential electron transfer components of bacterial membrane-bound alkane hydroxylase systems. Several Rd genes associated with alkane hydroxylase or Rd reductase genes were cloned from gram-positive and gram-negative organisms able to grow on n-alkanes (Alk-Rds). Complementation tests in an Escherichia coli recombinant containing all Pseudomonas putida GPo1 genes necessary for growth on alkanes except Rd 2 (AlkG) and sequence comparisons showed that the Alk-Rds can be divided in AlkG1- and AlkG2-type Rds. All alkane-degrading strains contain AlkG2-type Rds, which are able to replace the GPo1 Rd 2 in n-octane hydroxylation. Most strains also contain AlkG1-type Rds, which do not complement the deletion mutant but are highly conserved among gram-positive and gram-negative bacteria. Common to most Rds are the two iron-binding CXXCG motifs. All Alk-Rds possess four negatively charged residues that are not conserved in other Rds. The AlkG1-type Rds can be distinguished from the AlkG2-type Rds by the insertion of an arginine downstream of the second CXXCG motif. In addition, the glycines in the two CXXCG motifs are usually replaced by other amino acids. Mutagenesis of residues conserved in either the AlkG1- or the AlkG2-type Rds, but not between both types, shows that AlkG1 is unable to transfer electrons to the alkane hydroxylase mainly due to the insertion of the arginine, whereas the exchange of the glycines in the two CXXCG motifs only has a limited effect.

红氧还蛋白（Rubredoxins, Rds）是细菌膜结合烷烃羟化酶系统中不可或缺的电子传递组分。从可利用正烷烃生长的革兰氏阳性与革兰氏阴性微生物中，已克隆得到多种与烷烃羟化酶或红氧还蛋白还原酶基因相关的红氧还蛋白基因（Alk-Rds）。通过在缺失Rd 2（AlkG）、且携带有恶臭假单胞菌GPo1（Pseudomonas putida GPo1）全部烷烃生长必需基因的重组大肠杆菌（Escherichia coli）中开展互补实验，并结合序列比对分析，可将Alk-Rds划分为AlkG1型与AlkG2型红氧还蛋白。所有可降解烷烃的菌株均携带AlkG2型Rds，该类蛋白可替代GPo1的Rd 2完成正辛烷羟化反应。多数菌株同时含有AlkG1型Rds，这类蛋白无法互补该缺失突变体，但在革兰氏阳性与阴性菌中高度保守。多数红氧还蛋白均具有两个铁结合CXXCG基序；所有Alk-Rds均带有4个在其他红氧还蛋白中未保守存在的带负电荷残基。AlkG1型与AlkG2型红氧还蛋白的差异体现为：AlkG1型在第二个CXXCG基序下游存在精氨酸插入位点，且其两个CXXCG基序中的甘氨酸通常会被其他氨基酸取代。对仅在AlkG1型或AlkG2型红氧还蛋白中保守、而两类间存在差异的残基进行诱变实验后证实，AlkG1无法向烷烃羟化酶传递电子的核心原因正是该精氨酸的插入，而两个CXXCG基序中甘氨酸的替换仅会产生有限影响。