Zhang Jin, Friedrich Peter, Pierik Antonio J, Martins Berta M, Buckel Wolfgang
Laboratorium für Mikrobiologie, Fachbereich Biologie and Synmikro, Philipps-Universität, Marburg, Germany Max-Plank-Institut für terrestrische Mikrobiologie, Marburg.
Institut für Zytobiologie, Philipps-Universität, Marburg, Germany.
Appl Environ Microbiol. 2015 Feb;81(3):1071-84. doi: 10.1128/AEM.03099-14. Epub 2014 Dec 1.
4-Hydroxybutyryl-coenzyme A (CoA) dehydratase (4HBD) from Clostridium aminobutyricum catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA and the irreversible isomerization of vinylacetyl-CoA to crotonyl-CoA. 4HBD is an oxygen-sensitive homotetrameric enzyme with one 4Fe-4S cluster and one flavin adenine dinucleotide (FAD) in each subunit. Upon the addition of crotonyl-CoA or the analogues butyryl-CoA, acetyl-CoA, and CoA, UV-visible light and electron paramagnetic resonance (EPR) spectroscopy revealed an internal one-electron transfer to FAD and the 4Fe-4S cluster prior to hydration. We describe an active recombinant 4HBD and variants produced in Escherichia coli. The variants of the cluster ligands (H292C [histidine at position 292 is replaced by cysteine], H292E, C99A, C103A, and C299A) had no measurable dehydratase activity and were composed of monomers, dimers, and tetramers. Variants of other potential catalytic residues were composed only of tetramers and exhibited either no measurable (E257Q, E455Q, and Y296W) hydratase activity or <1% (Y296F and T190V) dehydratase activity. The E455Q variant but not the Y296F or E257Q variant displayed the same spectral changes as the wild-type enzyme after the addition of crotonyl-CoA but at a much lower rate. The results suggest that upon the addition of a substrate, Y296 is deprotonated by E455 and reduces FAD to FADH·, aided by protonation from E257 via T190. In contrast to FADH·, the tyrosyl radical could not be detected by EPR spectroscopy. FADH· appears to initiate the radical dehydration via an allylic ketyl radical that was proposed 19 years ago. The mode of radical generation in 4HBD is without precedent in anaerobic radical chemistry. It differs largely from that in enzymes, which use coenzyme B12, S-adenosylmethionine, ATP-driven electron transfer, or flavin-based electron bifurcation for this purpose.
来自氨基丁酸梭菌的4-羟基丁酰辅酶A（CoA）脱水酶（4HBD）催化4-羟基丁酰-CoA可逆脱水生成巴豆酰-CoA，以及乙烯基乙酰-CoA不可逆异构化为巴豆酰-CoA。4HBD是一种对氧敏感的同四聚体酶，每个亚基含有一个4Fe-4S簇和一个黄素腺嘌呤二核苷酸（FAD）。添加巴豆酰-CoA或其类似物丁酰-CoA、乙酰-CoA和CoA后，紫外-可见光谱和电子顺磁共振（EPR）光谱显示，在水化之前，内部发生了单电子转移至FAD和4Fe-4S簇。我们描述了一种在大肠杆菌中产生的活性重组4HBD及其变体。簇配体的变体（H292C [292位的组氨酸被半胱氨酸取代]、H292E、C99A、C103A和C299A）没有可测量的脱水酶活性，由单体、二聚体和四聚体组成。其他潜在催化残基的变体仅由四聚体组成，要么没有可测量的（E257Q、E455Q和Y296W）水合酶活性，要么脱水酶活性<1%（Y296F和T190V）。添加巴豆酰-CoA后，E455Q变体而非Y296F或E257Q变体显示出与野生型酶相同的光谱变化，但速率要低得多。结果表明，添加底物后，Y296被E455去质子化并将FAD还原为FADH·，在E257经T190质子化的辅助下完成。与FADH·不同，EPR光谱无法检测到酪氨酸自由基。FADH·似乎通过19年前提出的烯丙基酮基自由基引发自由基脱水。4HBD中自由基产生的模式在厌氧自由基化学中尚无先例。它与那些为此目的使用辅酶B12、S-腺苷甲硫氨酸、ATP驱动的电子转移或黄素基电子分叉的酶有很大不同。
