細胞膜非血基質二鐵烷類羥化酶 (alkane hydroxylase, alkB)來自Pseudomonas oleovorans可羥基化中等直鏈烷類，蛋白質序列比對和定點突變均顯示alkB和許多真核或原核的膜蛋白desaturase具有多個組胺酸可與活化中心的鐵離子配位。此外，還有兩個酵素rubredoxin-2 和 rubredoxin reductase在細胞質中，負責將電子從NADH傳遞至膜上的烷類羥化酶。 我們成功的從大腸桿菌中表現並且藉由金屬離子親和層析法(metal-affinity chromatography)純化具有His-tag的重組alkane hydroxylase和rubredoxin-2。經由光譜分析得知，所純化的His-tagged rubredoxin-2僅有一個三價鐵離子在C端，可被spinach ferredoxin reductase還原成二價鐵，而此還原過程還造成蛋白質二級結構的改變。在純化具有His-tag的重組alkane hydroxylase時，發現需要比一般His-tag的重組蛋白還要更高濃度的咪唑(imidazole)才可將其從Ni2+ column沖提出來。藉由Ni2+ column純化野生型alkane hydroxylase，我們得知在蛋白質表面與鐵催化中心配位的多個組胺酸是造成His-tag的重組alkB與Ni2+ column會有較高親和力的原因。加入FeSO4之後，野生型與His-tag重組的alkane hydroxylase均可將1,7-octadiene環氧化成1,2-epoxy-7-octene，當spinach ferredoxin reductase、NADPH和自行純化僅有一個鐵離子的His-tag重組rubredoxin-2均存在的活性測試中。這顯示了alkB中與鐵離子配位的多個組胺酸活化中心暴露在蛋白質表面，而藉由金屬離子親和層析法可以簡易的純化野生型膜蛋白alkane hydroxylase。 The integral membrane non-heme diiron alkane hydroxylase (alkB) from Pseudomonas oleovorans is able to hydroxylate medium-chain-length alkane. Primary protein sequence alignment and site-directed mutagenesis demonstrated that alkB and numbers of eukaryotic or prokaryotic integral-membrane desaturase comprise evolutionarily histidine-rich motifs probably coordinating the active site iron center. The other two components in the cytoplasm of the alkane hydroxylase complex, rubredoxin-2 and rubredoxin reductase, are responsible for reducing equivalent electron transfer from NADH to ω-hydroxylase. We successfully expressed and purified the recombinant His-tagged integral-membrane alkane hydroxylase and cytosolic rubredoxin-2 from E. coli lysates by a simple metal-affinity chromatography. On the basis of spectroscopic data, the isolated His-tagged rubredoxin-2 containing only one iron atom located at the C-terminal domain can be reduced by NADPH-dependent spinach ferredoxin reductase and subsequently results in conformational change of secondary structure. During purification, the recombinant His6-tagged ω-hydroxylase required more competing imidazole to elute out from Ni2+ column than the usual His6-tagged protein. After wild type alkane hydroxylase purification by Ni2+ column, we revealed that additional Ni2+ affinity for the recombinant His-tagged alkB come form surface poly-histidine residue motifs of ω-hydroxylase for catalytic iron coordination. In activity assay, both recombinant His-tagged and wild type alkane hydroxylase, after addition of FeSO4, are able to convert 1,7-octadiene to 1,2-epoxy-7-octene in the present of recombinant His-tagged 1Fe-rubredoxin-2, NADPH and spinach ferredoxin reductase. It supports the active site iron coordinating His-rich motifs of alkB are exposed to the protein surface, and simple purification of alkane hydroxylase was achieved by metal-affinity chromatography.
