Cyanobacterial alkane biosynthesis is catalyzed by acyl-(acyl carrier protein (ACP)) reductase (AAR) and aldehyde-deformylating oxygenase (ADO) in a two-step reaction. AAR reduces acyl-ACPs to fatty aldehydes, which are then converted by ADO to alkanes, the main components of diesel fuel. Interaction between AAR and ADO allows AAR to efficiently deliver the aldehyde to ADO. However, this interaction is poorly understood. Here, using analytical size-exclusion chromatography (SEC), we show that electrostatic interactions play an important role in the binding of the two enzymes. Alanine-scanning mutagenesis at charged residues around the substrate entry site of ADO revealed that E201A mutation greatly reduced hydrocarbon production. SEC measurement of the mutant demonstrated that E201 of ADO is essential for the AAR-ADO interaction. Our results suggest that AAR binds to the substrate entrance gate of ADO and thereby facilitates the insertion of the reactive and relatively insoluble aldehyde into the hydrophobic channel of ADO.Abbreviations: AAR: acyl-ACP reductase; ACP: acyl carrier protein; ADO: aldehyde-deformylating oxygenase; ASA: solvent accessible surface area; BSA: bovine serum albumin; CD: circular dichroism; DMSO: dimethyl sulfoxide; DTT: dithiothreitol; GC-MS: gas chromatography-mass spectrometer; HPLC: high-performance liquid chromatography; IPTG: isopropyl-β-D-thiogalactoside; MRE: mean residue ellipticity; NpAAR: AAR from Nostoc punctiforme PCC 73102; NpADO: ADO from Nostoc punctiforme PCC 73102; PmADO: ADO from Prochlorococcus marinus MIT 9313; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SeAAR: AAR from Synechococcus elongatus PCC 7942; SeADO: ADO from Synechococcus elongatus PCC 7942; SEC: size-exclusion chromatography; TeAAR: AAR from Thermosynechococcus elongatus BP-1; TeADO: ADO from Thermosynechococcus elongatus BP-1; UV: ultraviolet.

中文翻译：

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两种酶界面上的静电相互作用对于蓝细菌中的两步烷烃生物合成至关重要。

酰基-（酰基载体蛋白（ACP））还原酶（AAR）和醛脱甲酰加氧酶（ADO）在两步反应中催化蓝藻烷烃的生物合成。AAR将酰基ACP还原为脂肪醛，然后由ADO将其转化为柴油的主要成分烷烃。AAR和ADO之间的相互作用使AAR可以将醛有效地传递到ADO。但是，这种相互作用了解甚少。在这里，我们使用分析尺寸排阻色谱法（SEC），表明静电相互作用在两种酶的结合中起着重要作用。在ADO底物进入位点周围带电残基的丙氨酸扫描诱变表明，E201A突变大大降低了碳氢化合物的产量。对该突变体的SEC测量表明，ADO的E201对于AAR-ADO相互作用是必不可少的。我们的结果表明，AAR与ADO的底物入口结合，从而有助于将反应性和相对不溶的醛插入ADO的疏水通道。ACP：酰基载体蛋白；ADO：醛脱甲酰加氧酶；ASA：溶剂可及的表面积；BSA：牛血清白蛋白；CD：圆二色性；DMSO：二甲基亚砜；DTT：二硫苏糖醇；GC-MS：气相色谱-质谱仪；HPLC：高效液相色谱法；IPTG：异丙基-β-D-硫代半乳糖苷；MRE：平均残留椭圆率；NpAAR：点状PCC 73102的AAR。NpADO：来自Nostoc点状PCC 73102的ADO。PmADO：来自海藻Prochlorococcus MIT MIT 9313的ADO。SDS-PAGE：十二烷基硫酸钠-聚丙烯酰胺凝胶电泳。SeAAR：来自延长的Synechococcus elongatus PCC 7942的AAR；SeADO：来自Synchococcus elongatus PCC 7942的ADO；参见SedADO。SEC：体积排阻色谱法；TeAAR：来自嗜热嗜热球菌BP-1的AAR。TeADO：来自嗜热嗜热球菌BP-1的ADO；参见下文。紫外线：紫外线。