Cloning and characterization of histamine dehydrogenase from Nocardioide simplex
Cloning and characterization of histamine dehydrogenase from Nocardioide simplex
复制标题
DOI：
10.1016/j.abb.2004.11.024
复制
发表时间：
2005-04-01
影响因子：
3.9
通讯作者：
Klinman, JP
中科院分区：
文献类型：
--
作者：
Limburg, J;Mure, M;Klinman, JP
组胺脱氢酶（NSHADH）可从以组胺为唯一氮源的单纯诺卡依菌培养物中分离得到。此前有报道认为NSHADH可能含有醌辅助因子色氨酸（TTQ）。在这里。从单纯螺旋体基因组DNA中克隆出编码NSHADH的17111基因，并对分离得到的酶进行了全光谱表征。蛋白质序列比对显示NSHADH与三甲胺脱氢酶（TMADH: EC 1.5.99.7）有关，后者含有细菌铁氧化还原蛋白型[4Fe-4S]簇和6- s -半胱氨酸FMN辅助因子。NSHADH与任何含有胺脱氢酶的TTQ没有序列相似性。NSHADH每个亚基含有3.6 +/- 0.3 mol铁和3.7 +/- 0.2 mol酸不稳定S。NSHADH和TMADH的紫外/可见光谱比较显示出显著的相似性。组胺减少的NSHADH的EPR谱也支持黄素和[4Fe-4S]辅助因子的存在。重要的是，我们发现NSHADH具有狭窄的底物特异性，仅氧化组胺(k - M = 31 +/- 11 μ M, k(cat)/ k - M = 2.1 （+/- 0.4） × 10(5) M-1 s(-1)), agmatine (k - M = 37 +/- 6 μ M, k(cat)/ k - M = 6.0 （+/- 0.6） × 10（4) M-1 s(-1)）和腐胺(k - M = 1280 +/- 240 μ M, k（cat)/ k - M = 1500 +/- 200 M-1 s(-1)）。本文介绍了NSHADH氧化脱胺对组胺的动力学表征，包括k(cat)/ k -m（组胺）的pH依赖性和底物氘同位素效应(D)(k（cat)/ k -m（组胺））= 7.0 +/- 1.8的测量。k（cat）也依赖于pH值，并具有还原底物氘同位素(D)(k(cat)) = 1.3 0.2。(c) 2004 Elsevier Inc.版权所有。
Histamine dehydrogenase (NSHADH) can be isolated from cultures of Nocardioides simplex grown with histamine as the sole nitrogen source. A previous report suggested that NSHADH might contain the quinone cofactor tryptophan tryptophyl quinone (TTQ). Here. the 17,111 gene encoding NSHADH is cloned from the genomic DNA of N. simplex, and the isolated enzyme is subjected to a full spectroscopic characterization. Protein sequence alignment shows NSHADH to be related to trimethylamine dehydrogenase (TMADH: EC 1.5.99.7), where the latter contains a bacterial ferredoxin-type [4Fe-4S] cluster and 6-S-cysteinyl FMN cofactor. NSHADH has no sequence similarity to any TTQ containing amine dehydrogenases. NSHADH contains 3.6 +/- 0.3 mol Fe and 3.7 +/- 0.2 mol acid labile S per subunit. A comparison of the UV/vis spectra of NSHADH and TMADH shows significant similarity. The EPR spectrum of histaminc reduced NSHADH also supports the presence of the flavin and [4Fe-4S] cofactors. Importantly, we show that NSHADH has a narrow Substrate specificity, oxidizing only histamine (K-m = 31 +/- 11 mu M, k(cat)/K-m = 2.1 (+/- 0.4) x 10(5) M-1 s(-1)), agmatine (K-m = 37 +/- 6 mu M, k(cat)/K-m = 6.0 (+/- 0.6) x 10(4) M-1 s(-1)), and putrescine (K-m = 1280 +/- 240 mu M, k(cat)/K-m = 1500 +/- 200 M-1 s(-1)). A kinetic characterization of the oxidative deamination of histamine by NSHADH is presented that includes the pH dependence of k(cat)/K-m (histamine) and the measurement of a substrate deuterium isotope effect (D)(k(cat)/K-m (histamine)) = 7.0 +/- 1.8 at pH 8.5. k(cat) is also pH dependent and has a reduced substrate deuterium isotope of (D)(k(cat)) = 1.3 0.2. (c) 2004 Elsevier Inc. All rights reserved.