Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125.
Proc Natl Acad Sci U S A. 2018 Sep 11;115(37):E8614-E8623. doi: 10.1073/pnas.1807984115. Epub 2018 Aug 13.
Arsenate respiration by bacteria was discovered over two decades ago and is catalyzed by diverse organisms using the well-conserved Arr enzyme complex. Until now, the mechanisms underpinning this metabolism have been relatively opaque. Here, we report the structure of an Arr complex (solved by X-ray crystallography to 1.6-Å resolution), which was enabled by an improved Arr expression method in the genetically tractable arsenate respirer sp. ANA-3. We also obtained structures bound with the substrate arsenate (1.8 Å), the product arsenite (1.8 Å), and the natural inhibitor phosphate (1.7 Å). The structures reveal a conserved active-site motif that distinguishes Arr [(R/K)GRY] from the closely related arsenite respiratory oxidase (Arx) complex (XGRGWG). Arr activity assays using methyl viologen as the electron donor and arsenate as the electron acceptor display two-site ping-pong kinetics. A Mo(V) species was detected with EPR spectroscopy, which is typical for proteins with a pyranopterin guanine dinucleotide cofactor. Arr is an extraordinarily fast enzyme that approaches the diffusion limit ( = 44.6 ± 1.6 μM, = 9,810 ± 220 seconds), and phosphate is a competitive inhibitor of arsenate reduction ( = 325 ± 12 μM). These observations, combined with knowledge of typical sedimentary arsenate and phosphate concentrations and known rates of arsenate desorption from minerals in the presence of phosphate, suggest that () arsenate desorption limits microbiologically induced arsenate reductive mobilization and () phosphate enhances arsenic mobility by stimulating arsenate desorption rather than by inhibiting it at the enzymatic level.
砷酸盐呼吸作用在二十多年前被发现，由多种生物体利用高度保守的 Arr 酶复合物进行催化。到目前为止，这种代谢的机制还相对不透明。在这里，我们报告了 Arr 复合物的结构（通过 X 射线晶体学解析至 1.6 Å 分辨率），这得益于在遗传上可操作的砷酸盐呼吸菌 sp. ANA-3 中改进的 Arr 表达方法。我们还获得了与底物砷酸盐（1.8 Å）、产物亚砷酸盐（1.8 Å）和天然抑制剂磷酸盐（1.7 Å）结合的结构。这些结构揭示了一个保守的活性位点模体，将 Arr [(R/K)GRY]与密切相关的亚砷酸盐呼吸氧化酶（Arx）复合物（XGRGWG）区分开来。使用甲基紫精作为电子供体和砷酸盐作为电子受体的 Arr 活性测定显示出双位点乒乓动力学。用电子顺磁共振光谱法检测到 Mo(V)物种，这是具有吡喃并嘌呤鸟嘌呤二核苷酸辅因子的蛋白质的典型特征。Arr 是一种非常快速的酶，接近扩散限制（ = 44.6 ± 1.6 μM， = 9,810 ± 220 秒），磷酸盐是砷酸盐还原的竞争性抑制剂（ = 325 ± 12 μM）。这些观察结果，结合典型沉积物中砷酸盐和磷酸盐的浓度以及已知在磷酸盐存在下从矿物中解吸砷酸盐的速率，表明 () 砷酸盐解吸限制微生物诱导的砷酸盐还原迁移， () 磷酸盐通过刺激砷酸盐解吸而不是在酶水平上抑制它来增强砷的迁移性。
