Putignani Lorenza, Ambrosi Cecilia, Ascenzi Paolo, Visca Paolo
Unità di Microbiologia Molecolare, Istituto Nazionale per le Malattie Infettive I.R.C.C.S. Lazzaro Spallanzani, Via Portuense 292, 00149 Roma, Italy.
Biochem Biophys Res Commun. 2004 Jan 9;313(2):245-57. doi: 10.1016/j.bbrc.2003.11.116.
Omega-amino acid monooxygenases (EC 1.14.13.-), catalysing the formation of hydroxamate precursors of microbial siderophores (e.g., pyoverdine), have so far eluded structural and biochemical characterisation. Here, the expression of recombinant L-ornithine-Ndelta-oxygenase (PvdA) from Pseudomonas aeruginosa PAO1 is reported. A library of eight monoclonal antibodies (MAbs) directed against PvdA has been generated. Two MAb families recognising the N- and C-terminal regions of PvdA were identified. The MAbs made it possible to demonstrate that 45-48 kDa PvdA homologues are expressed in response to iron limitation by different species and strains of fluorescent pseudomonads. Despite the different degrees in sequence similarity between P. aeruginosa PvdA and putative homologues from Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, Burkholderia cepacia, and Ralstonia solanacearum, in silico domain scanning predicts an impressive conservation of putative cofactor and substrate binding domains. The MAb library was also used to monitor PvdA expression during the transition of P. aeruginosa from iron-sufficient to iron-deficient growth.
ω-氨基酸单加氧酶（EC 1.14.13.-）催化微生物铁载体（如绿脓菌素）异羟肟酸前体的形成，迄今为止尚未得到结构和生化特性的描述。本文报道了铜绿假单胞菌PAO1重组L-鸟氨酸-Nδ-加氧酶（PvdA）的表达情况。已构建了一个针对PvdA的8种单克隆抗体（MAb）文库。鉴定出了两个识别PvdA N端和C端区域的单克隆抗体家族。这些单克隆抗体能够证明，45 - 48 kDa的PvdA同源物在不同种类和菌株的荧光假单胞菌对铁限制的应答中表达。尽管铜绿假单胞菌PvdA与荧光假单胞菌、恶臭假单胞菌、丁香假单胞菌、洋葱伯克霍尔德菌和青枯雷尔氏菌的推定同源物在序列相似性上存在不同程度的差异，但通过计算机结构域扫描预测，推定的辅因子和底物结合结构域具有惊人的保守性。该单克隆抗体文库还用于监测铜绿假单胞菌从铁充足生长转变为铁缺乏生长过程中PvdA的表达。
