Cloning and expression of ntnD, encoding a novel NAD(P)+-independent 4-nitrobenzyl alcohol dehydrogenase from Pseudomonas sp strain TW3
Cloning and expression of ntnD, encoding a novel NAD(P)+-independent 4-nitrobenzyl alcohol dehydrogenase from Pseudomonas sp strain TW3
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DOI：
10.1128/jb.182.11.3136-3141.2000
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发表时间：
2000-06-01
影响因子：
3.2
通讯作者：
Williams, PA
中科院分区：
文献类型：
--
作者：
James, KD;Hughes, MA;Williams, PA
假单胞菌属菌株TW 3能够通过与TOL质粒的上游途径类似的途径将4-硝基甲苯有氧代谢为4-硝基苯甲酸酯，并将甲苯有氧代谢为苯甲酸酯。我们报告的苯甲醇脱氢酶基因（ntnD）的克隆和表征，该基因编码的酶的4-硝基苯甲醇和苯甲醇的催化剂4-硝基苯甲醛和苯甲醛，分别。该基因位于先前报道的ntn基因簇的下游。NtnD与类似的TOL质粒XyIB（苯甲醇脱氢酶）蛋白在其生物化学方面没有相似性，其是NAD（P）（+）非依赖性的并且需要通过染料连接的电子转移进行测定，或者在其推导的氨基酸序列方面没有相似性。然而，它的氨基酸序列与其他NAD（P）（+）-非依赖性醇氧化酶具有显著的相似性，并且含有III型黄素腺嘌呤二核苷酸依赖性醇氧化酶的特征性特征模式。逆转录-PCR表明，ntnD的转录生长过程中对4-硝基甲苯，虽然显然不是作为相同的转录本的一部分，作为其他ntn基因。从克隆和过表达的基因表达的酶的底物特异性是从4-硝基甲苯上生长的菌株TW 3表达的活性相似，提供证据表明，ntnD是以前未鉴定的基因在4-硝基甲苯催化剂的途径。对ntn基因周围14.8 kb区域的研究表明，一个或多个重组事件参与了它们当前组织的形成。
Pseudomonas sp. strain TW3 is able to metabolize 4-nitrotoluene to 4-nitrobenzoate and toluene to benzoate aerobically via a route analogous to the upper pathway of the TOL plasmids. We report the cloning and characterization of a benzyl alcohol dehydrogenase gene (ntnD) which encodes the enzyme for the catabolism of 4-nitrobenzyl alcohol and benzyl alcohol to 4-nitrobenzaldehyde and benzaldehyde, respectively. The gene is located downstream of the previously reported ntn gene cluster. NtnD bears no similarity to the analogous TOL plasmid XyIB (benzyl alcohol dehydrogenase) protein either in its biochemistry, being NAD(P)(+) independent and requiring assay via dye-linked electron transfer, or in its deduced amino acid sequence. It does, however, have significant similarity in its amino acid sequence to other NAD(P)(+)-independent alcohol dehydrogenases and contains signature patterns characteristic of type III flavin adenine dinucleotide-dependent alcohol oxidases. Reverse transcription-PCR demonstrated that ntnD is transcribed during growth on 4-nitrotoluene, although apparently not as part of the same transcript as the other ntn genes. The substrate specificity of the enzyme expressed from the cloned and overexpressed gene was similar to the activity expressed from strain TW3 grown on 4-nitrotoluene, providing evidence that ntnD is the previously unidentified gene in the pathway of 4-nitrotoluene catabolism. Examination of the 14.8-kb region around the ntn genes suggests that one or more recombination events have been involved in the formation of their current organization.