Department of Biochemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Biochemistry. 2012 Oct 9;51(40):7908-16. doi: 10.1021/bi300912n. Epub 2012 Sep 24.
Cyanobacterial aldehyde decarbonylases (ADs) catalyze the conversion of C(n) fatty aldehydes to formate (HCO(2)(-)) and the corresponding C(n-1) alk(a/e)nes. Previous studies of the Nostoc punctiforme (Np) AD produced in Escherichia coli (Ec) showed that this apparently hydrolytic reaction is actually a cryptically redox oxygenation process, in which one O-atom is incorporated from O(2) into formate and a protein-based reducing system (NADPH, ferredoxin, and ferredoxin reductase; N/F/FR) provides all four electrons needed for the complete reduction of O(2). Two subsequent publications by Marsh and co-workers [ Das, et al. ( 2011 ) Angew. Chem. Int. Ed. 50 , 7148 - 7152 ; Eser, et al. ( 2011 ) Biochemistry 50 , 10743 - 10750 ] reported that their Ec-expressed Np and Prochlorococcus marinus (Pm) AD preparations transform aldehydes to the same products more rapidly by an O(2)-independent, truly hydrolytic process, which they suggested proceeded by transient substrate reduction with obligatory participation by the reducing system (they used a chemical system, NADH and phenazine methosulfate; N/PMS). To resolve this discrepancy, we re-examined our preparations of both AD orthologues by a combination of (i) activity assays in the presence and absence of O(2) and (ii) (18)O(2) and H(2)(18)O isotope-tracer experiments with direct mass-spectrometric detection of the HCO(2)(-) product. For multiple combinations of the AD orthologue (Np and Pm), reducing system (protein-based and chemical), and substrate (n-heptanal and n-octadecanal), our preparations strictly require O(2) for activity and do not support detectable hydrolytic formate production, despite having catalytic activities similar to or greater than those reported by Marsh and co-workers. Our results, especially of the (18)O-tracer experiments, suggest that the activity observed by Marsh and co-workers could have arisen from contaminating O(2) in their assays. The definitive reaffirmation of the oxygenative nature of the reaction implies that the enzyme, initially designated as aldehyde decarbonylase when the C1-derived coproduct was thought to be carbon monoxide rather than formate, should be redesignated as aldehyde-deformylating oxygenase (ADO).
蓝细菌醛脱羰基酶（AD）催化 C(n) 脂肪酸醛转化为甲酸盐（HCO(2)(-))和相应的 C(n-1) 链烯（烷/烯）。先前对在大肠杆菌（Ec）中产生的鱼腥藻（Np）AD 的研究表明，这种明显的水解反应实际上是一种隐匿的氧化还原过程，其中一个氧原子从 O(2)掺入甲酸盐中，而一个基于蛋白质的还原系统（NADPH、铁氧还蛋白和铁氧还蛋白还原酶；N/F/FR）提供完全还原 O(2)所需的四个电子。Marsh 及其同事的两篇后续出版物[Das 等人，（2011 年）Angew. Chem. Int. Ed. 50，7148-7152；Eser 等人，（2011 年）Biochemistry 50，10743-10750]报道，他们在 Ec 中表达的 Np 和聚球藻（Pm）AD 制剂通过一种不依赖 O(2)的真正水解过程更快地将醛转化为相同产物，他们认为该过程通过与还原系统（他们使用了一种化学系统，NADH 和吩嗪甲硫酸盐；N/PMS）的强制底物还原进行。为了解决这一差异，我们通过（i）在存在和不存在 O(2)的情况下进行活性测定，以及（ii）用（18）O 和 H(2)(18)O 同位素示踪实验并用直接质谱检测 HCO(2)(-)产物，重新检查了两种 AD 同系物的制剂。对于 AD 同系物（Np 和 Pm）、还原系统（基于蛋白质和化学）和底物（正庚醛和正十八醛）的多种组合，我们的制剂严格需要 O(2)才能发挥活性，并且不支持可检测的水解甲酸盐生成，尽管它们的催化活性与 Marsh 及其同事报道的活性相似或更高。我们的结果，特别是（18）O 示踪实验的结果表明，Marsh 及其同事观察到的活性可能是由于他们的测定中存在污染的 O(2)。该反应的氧化性的明确再次证实，最初被指定为醛脱羰基酶的酶，当认为 C1 衍生的副产物是一氧化碳而不是甲酸盐时，应该重新指定为醛脱甲酰化氧化酶（ADO）。
