Vangnai Alisa S, Arp Daniel J
Department of Biochemistry and Biophysics, Laboratory for Nitrogen Fixation Research1, and Department of Botany and Plant Pathology2, Oregon State University, Cordley 2082, Corvallis, 97331-2902 OR, USA.
Microbiology (Reading). 2001 Mar;147(Pt 3):745-756. doi: 10.1099/00221287-147-3-745.
Butane-grown "Pseudomonas butanovora" expressed two soluble alcohol dehydrogenases (ADHs), an NAD(+)-dependent secondary ADH and an NAD(+)-independent primary ADH. Two additional NAD(+)-dependent secondary ADHs could be detected when cells were grown on 2-butanol and lactate. The inducible NAD(+)-independent 1-butanol dehydrogenase (BDH) of butane-grown cells was primarily responsible for 1-butanol oxidation in the butane metabolism pathway. BDH was purified to near homogeneity and identified as a quinohaemoprotein, containing, per mol enzyme, 1.0 mol pyrroloquinoline quinone (PQQ) and 0.25 mol haem c as prosthetic groups. BDH was synthesized as a monomer of approximately 66 kDa. It has a broad substrate range, including primary alcohols, secondary alcohols, aldehydes, C(4) diols and aromatic alcohols. It exhibited the lowest K:(m) (7+/-1 microM) and highest k(cat)/K:(m) (72x10(4) M(-1) s(-1)) value towards 1-butanol. BDH exhibited ferricyanide-dependent ADH activity. Calcium ions (up to 10 mM) increased BDH activity substantially. Two BDH internal amino acid sequences showed 73 and 62% identity and 83 and 66% similarity, respectively, when compared with an amino acid sequence of ethanol dehydrogenase from Comamonas testosteroni. The presence of the inducible BDH and secondary ADH may indicate that the terminal and subterminal oxidation pathways are involved in butane degradation of butane-grown "P. butanovora".
以丁烷为生长底物的“丁烷假单胞菌”表达了两种可溶性醇脱氢酶（ADH），一种依赖NAD⁺的次级ADH和一种不依赖NAD⁺的初级ADH。当细胞在2-丁醇和乳酸上生长时，还能检测到另外两种依赖NAD⁺的次级ADH。以丁烷为生长底物的细胞中可诱导的不依赖NAD⁺的1-丁醇脱氢酶（BDH）在丁烷代谢途径中主要负责1-丁醇的氧化。BDH被纯化至接近均一，并被鉴定为一种醌血红蛋白，每摩尔酶含有1.0摩尔吡咯喹啉醌（PQQ）和0.25摩尔血红素c作为辅基。BDH以约66 kDa的单体形式合成。它具有广泛的底物范围，包括伯醇、仲醇、醛、C₄二醇和芳香醇。它对1-丁醇表现出最低的Kₘ（7±1 μM）和最高的kcat/Kₘ（72×10⁴ M⁻¹ s⁻¹）值。BDH表现出铁氰化物依赖性ADH活性。钙离子（高达10 mM）可显著提高BDH活性。与睾丸丛毛单胞菌乙醇脱氢酶的氨基酸序列相比，两个BDH内部氨基酸序列的同一性分别为73%和62%，相似性分别为83%和66%。可诱导的BDH和次级ADH的存在可能表明末端和亚末端氧化途径参与了以丁烷为生长底物的“丁烷假单胞菌”的丁烷降解过程。
