以結構生物觀點分析亞鐵離子與α-酮戊二酸依賴性酵素催化環丙烷化和羥基化的機制

Abstract

亞鐵離子/α-酮戊二酸依賴型酵素(Fe(II)/α-ketoglutarate-dependent enzymes; Fe/αKG enzymes)利用二價鐵作為輔因子，α-酮戊二酸作為輔受質，氧分子作為氧化劑來催化多種氧化反應。本型酵素家族的成員在結構上有一個共同的特徵：由雙層β-螺旋結構域(double-stranded β-helix fold)支撐其活性中心，並以2-His-1-carboxylate triad結合二價鐵作為其反應中心，利用α-酮戊二酸之氧化脱羧作用後產生的高反應性四價鐵超氧中間物(Fe(IV)-oxo intermediate)來推動各種化學反應。在本型酵素家族成員所能催化的反應中，對於環丙烷化反應(Cyclopropanation)在機制上的理解仍有待釐清之處。環丙烷是由高張力的三碳環構成，因其結構較不穩定而具有高反應性，也因此不易由化學方法合成。同屬亞鐵離子/α-酮戊二酸依賴型酵素的HrmJ與BelL分別在天然抗生素hormaomycins與belactosins的生合成中催化環丙烷化，以形成(1′R, 2′R)與(1′S, 2′S)構型的3-(2-nitrocyclopropyl)alanine (Ncpa)。雖然近年來的研究提出了BelL與HrmJ催化環丙烷化的反應機制，然而目前HrmJ與BelL都還欠缺受質與輔受質結合的結構資訊，因此尚無法了解詳細的催化機制。先前的研究透過對HrmJ、BelL及其同源蛋白的活性中心胺基酸位點進行點突變，發現了一些會影響反應之立體選擇性的位點，但這些酵素如何控制立體選擇性仍有許多未解之謎。因此，本研究的目標是希望能夠解出ScBelL與HrmJAw兩種酵素在反應不同階段的晶體結構。ScBelL為BelL的同源蛋白，具有催化6-nitronorleucine環丙烷化的功能，並選擇性的生成(1’R, 2’S)- Ncpa；HrmJAw則是HrmJ的同源蛋白，負責催化羥基化反應。我們成功的以液相層析法純化這兩種酵素，並嘗試獲得ScBelL和HrmJAw蛋白在apo形式以及包含不同組合的輔因子、輔受質和受質的晶體。然而，將ScBelL或HrmJAw加入Fe(II)和α-酮戊二酸、羥基草酸乙二胺或琥珀酸均導致嚴重沉澱。根據蛋白質熱位移分析，ScBelL與HrmJAw在同時結合Fe(II)和αKG 時，蛋白質的穩定度會降低。藉由調整緩衝液的成分，我們解決了ScBelL與HrmJAw在加入二價鐵與α-酮戊二酸等配體時嚴重沉澱的問題。目前，我們已經對apo ScBelL、apo HrmJAw以及與Fe(II)結合的HrmJAw進行多種蛋白質結晶條件篩選，並且持續地找尋合適的養晶條件。在HrmJAw的純化過程中，我們發現此蛋白會發生14個胺基酸斷裂，而此現象可能影響蛋白樣品的均質性，因此我們重新設計表達質體來表現HrmJAwΔ14，希望能藉由較穩定的蛋白來得到晶體，在對其進行蛋白質結晶條件篩選時，我們觀察到HrmJAwΔ14穩定性甚佳、不易發生沉澱現象，表示HrmJAwΔ14可能更適用於晶體培養。未來將嘗試找出適合HrmJAwΔ14的最佳緩衝液條件以提升蛋白質結晶條件篩選的成功率。

Fe(II)/α-ketoglutarate (αKG)-dependent enzymes use ferrous ion as a cofactor, αKG as a co-substrate, and dioxygen as an oxidant to catalyze various oxidative reactions. All members of this enzyme superfamily share a conserved structural feature: an iron-coordinating 2-His-1-carboxylate triad within a double-stranded β-helix (DSBH) fold, which harbors the active site. These enzymes utilize the high reactivity of the Fe(IV)-oxo intermediate, produced by oxidative decarboxylation of αKG, to drive different chemical transformations. Cyclopropanation is a notable reaction catalyzed by these enzymes, as the highly strained three-membered carbon ring is inherently unstable, highly reactive, and difficult to synthesize chemically. Two Fe(II)/αKG-dependent enzymes, HrmJ and BelL, are known to catalyze cyclopropanation in the biosynthesis of natural antibiotics hormaomycins and belactosins, which contain 3-(2-nitrocyclopropyl)alanine (Ncpa) with (1′R, 2′R) and (1′S, 2′S) configurations. Recent studies propose possible mechanism of BelL- and HrmJ-catalyzed cyclopropanation. However, no structural information is available for HrmJ and BelL in complexes with cofactor(s) and/or substrate to elucidate the detailed catalytic mechanism. Although point mutations of active site residues in HrmJ, BelL, and BelL homologues have led to the identification of certain residues affecting stereoconfiguration of their products, how these residues control the stereoselectivity remains largely unknown. Therefore, the aim of my thesis research is to reveal the structures of ScBelL and HrmJAw in different states of their catalytic cycles. ScBelL is a homologous protein of BelL, which catalyzes 6-nitronorleucine cyclopropanation and selectively produces (1’R, 2’S)-Ncpa, while HrmJAw is a homologous protein to HrmJ, which catalyzes the hydroxylation of 6-nitronorleucine. We successfully purified these two enzymes using liquid chromatography and then attempted to obtain ScBelL and HrmJAw protein crystals in their apo forms and in different combinations of cofactor, cosubstrate, substrate, product, or their analogs. However, incubating ScBelL or HrmJAw with Fe(II) plus αKG, noxalylglycine (NOG), or succinate resulted in severe precipitation. According to the thermal shift assay (TSA), ScBelL and HrmJAw are less stable when complexed with Fe(II) and αKG. Therefore, we adjusted the buffer composition to address this issue. Currently, we have performed protein crystallization on apo ScBelL, apo HrmJAw, and HrmJAw in complex with Fe(II), and we are continuing to search for suitable crystallization conditions. During the purification of HrmJAw, it was found that the recombinant protein tends to be cleaved at the fourteenth amino acid residue. To ensure sample homogeneity, we removed this protease-sensitive N-terminal fragment by designing a construct expressing HrmJAwΔ14, hoping to obtain crystals from this potentially more stable protein. During the initial screening of protein crystallization conditions, we observed that HrmJAwΔ14 tends to remain soluble even in the presence of precipitants, indicating that it may have higher solubility and stability, suitable for growing crystal crystals. In the future, we will identify suitable buffer conditions for HrmJAwΔ14 to facilitate protein crystallization.