Organic Chemistry II, Centre for Integrated Protein Science CIPSM, Institute of Advanced Studies, and ‡Biochemistry, Department of Chemistry, Technische Universität München , Lichtenbergstrasse 4, 85747 Garching, Germany.
J Am Chem Soc. 2014 Apr 2;136(13):4992-9. doi: 10.1021/ja411785r. Epub 2014 Mar 19.
Pyridoxal 5'-phosphate (PLP) is the active vitamer of vitamin B6 and acts as an essential cofactor in many aspects of amino acid and sugar metabolism. The virulence and survival of pathogenic bacteria such as Mycobacterium tuberculosis depend on PLP, and deficiencies in humans have also been associated with neurological disorders and inflammation. While PLP can be synthesized by a de novo pathway in bacteria and plants, most higher organisms rely on a salvage pathway that phosphorylates either pyridoxal (PL) or its related vitamers, pyridoxine (PN) and pyridoxamine (PM). PL kinases (PLKs) are essential for this phosphorylation step and are thus of major importance for cellular viability. We recently identified a pyridoxal kinase (SaPLK) as a target of the natural product antibiotic rugulactone (Ru) in Staphylococcus aureus. Surprisingly, Ru selectively modified SaPLK not at the active site cysteine, but on a remote cysteine residue. Based on structural and biochemical studies, we now provide insight into an unprecedented dual Cys charge relay network that is mandatory for PL phosphorylation. The key component is the reactive Cys 110 residue in the lid region that forms a hemithioactetal intermediate with the 4'-aldehyde of PL. This hemithioacetal, in concert with the catalytic Cys 214, increases the nucleophilicity of the PL 5'-OH group for the inline displacement reaction with the γ-phosphate of ATP. A closer inspection of related enzymes reveals that Cys 110 is conserved and thus serves as a characteristic mechanistic feature for a dual-function ribokinase subfamily herein termed CC-PLKs.
吡哆醛 5'-磷酸（PLP）是维生素 B6 的活性维生素，作为许多氨基酸和糖代谢方面的必需辅酶。结核分枝杆菌等病原体的毒力和生存取决于 PLP，而人类的缺乏也与神经紊乱和炎症有关。虽然 PLP 可以通过细菌和植物中的从头途径合成，但大多数高等生物都依赖于磷酸化吡哆醛（PL）或其相关维生素吡哆醇（PN）和吡哆胺（PM）的补救途径。PL 激酶（PLKs）对该磷酸化步骤至关重要，因此对细胞活力具有重要意义。我们最近发现一种吡哆醛激酶（SaPLK）是金黄色葡萄球菌中天然产物抗生素 Rugulactone（Ru）的靶标。令人惊讶的是，Ru 选择性地修饰 SaPLK 不是在活性位点半胱氨酸上，而是在远程半胱氨酸残基上。基于结构和生化研究，我们现在提供了对 PL 磷酸化必需的前所未有的双 Cys 电荷中继网络的深入了解。关键组成部分是盖区域中的反应性 Cys110 残基，它与 PL 的 4'-醛形成半硫缩醛中间体。该半硫缩醛与催化 Cys214 一起，增加了 PL 5'-OH 基团与 ATP 的γ-磷酸进行直线取代反应的亲核性。对相关酶的更仔细检查表明，Cys110 是保守的，因此是双功能核酮激酶亚家族的特征机制特征，在此将其称为 CC-PLKs。
