Kobylarz Marek J, Grigg Jason C, Liu Yunan, Lee Mathew S F, Heinrichs David E, Murphy Michael E P
Department of Microbiology and Immunology, Life Sciences Institute, The University of British Columbia , Vancouver, British Columbia, Canada V6T 1Z3.
Biochemistry. 2016 Feb 16;55(6):927-39. doi: 10.1021/acs.biochem.5b01045. Epub 2016 Feb 3.
Staphylococcus aureus assembles the siderophore, staphyloferrin B, from l-2,3-diaminopropionic acid (l-Dap), α-ketoglutarate, and citrate. Recently, SbnA and SbnB were shown to produce l-Dap and α-ketoglutarate from O-phospho-l-serine (OPS) and l-glutamate. SbnA is a pyridoxal 5'-phosphate (PLP)-dependent enzyme with homology to O-acetyl-l-serine sulfhydrylases; however, SbnA utilizes OPS instead of O-acetyl-l-serine (OAS), and l-glutamate serves as a nitrogen donor instead of a sulfide. In this work, we examined how SbnA dictates substrate specificity for OPS and l-glutamate using a combination of X-ray crystallography, enzyme kinetics, and site-directed mutagenesis. Analysis of SbnA crystals incubated with OPS revealed the structure of the PLP-α-aminoacrylate intermediate. Formation of the intermediate induced closure of the active site pocket by narrowing the channel leading to the active site and forming a second substrate binding pocket that likely binds l-glutamate. Three active site residues were identified: Arg132, Tyr152, Ser185 that were essential for OPS recognition and turnover. The Y152F/S185G SbnA double mutant was completely inactive, and its crystal structure revealed that the mutations induced a closed form of the enzyme in the absence of the α-aminoacrylate intermediate. Lastly, l-cysteine was shown to be a competitive inhibitor of SbnA by forming a nonproductive external aldimine with the PLP cofactor. These results suggest a regulatory link between siderophore and l-cysteine biosynthesis, revealing a potential mechanism to reduce iron uptake under oxidative stress.
金黄色葡萄球菌利用L-2,3-二氨基丙酸（L-Dap）、α-酮戊二酸和柠檬酸合成铁载体葡萄球菌铁载体B。最近的研究表明，SbnA和SbnB可从O-磷酸-L-丝氨酸（OPS）和L-谷氨酸生成L-Dap和α-酮戊二酸。SbnA是一种依赖于磷酸吡哆醛（PLP）的酶，与O-乙酰-L-丝氨酸巯基酶具有同源性；然而，SbnA利用OPS而非O-乙酰-L-丝氨酸（OAS），并且L-谷氨酸作为氮供体而非硫化物。在本研究中，我们结合X射线晶体学、酶动力学和定点诱变技术，研究了SbnA如何决定对OPS和L-谷氨酸的底物特异性。对与OPS孵育的SbnA晶体的分析揭示了PLP-α-氨基丙烯酸酯中间体的结构。中间体的形成通过缩小通向活性位点的通道并形成可能结合L-谷氨酸的第二个底物结合口袋，诱导活性位点口袋的闭合。鉴定出三个对OPS识别和周转至关重要的活性位点残基：Arg132、Tyr152、Ser185。Y152F/S185G SbnA双突变体完全无活性，其晶体结构表明，在没有α-氨基丙烯酸酯中间体的情况下，这些突变诱导了酶的闭合形式。最后，L-半胱氨酸通过与PLP辅因子形成无活性的外部醛亚胺，被证明是SbnA的竞争性抑制剂。这些结果表明铁载体和L-半胱氨酸生物合成之间存在调节联系，揭示了在氧化应激下减少铁摄取的潜在机制。
