Structural and Biochemical Characterization of SbnC as a Representative Type B Siderophore Synthetase

Staphyloferrin B
is a key siderophore secreted by Staphylococcus
aureus to acquire ferric ions from a host during infection,
and its biosynthetic pathway has been validated to develop efficient
antibacterial agents. Herein, we report the crystal structure of AMP-bound
SbnC from S. aureus (SaSbnC) as
the first representative structure of type B synthetases in the biosynthesis
of α-hydroxycarboxylate siderophores. While type B synthetases
specifically use α-ketoglutarate (α-KG) as their carboxylic
acid substrate, SaSbnC showed unique structural features
in the substrate pocket compared with the type A and C synthetases.
Screening of α-KG analogues suggested that the hydrogen-bonding
interaction between the α-carbonyl group of α-KG and residue
Lys552 is a key determinant for the substrate selectivity of type
B synthetases. Interestingly, citrate, the product of the tricarboxylic
acid cycle and the substrate of type A synthetases, was found to inhibit
the activity of SaSbnC with an IC50 value
of 83 μM by mimicking α-KG binding, suggesting a potential
regulatory role of the tricarboxylic acid cycle, whose activity is
under the control of the intracellular iron concentration, to SaSbnC and other type B synthetases. These results provide
critical new information to understand the structure, function, and
regulation of type B synthetases in the siderophore-based iron acquisition
system employed by a large number of pathogenic microbes.

Staphyloferrin B，作为一种关键的铁载体，由金黄色葡萄球菌分泌，用以在感染过程中从宿主体内获取铁离子。其生物合成途径已被验证，可用于开发高效的抗菌剂。本研究报告了金黄色葡萄球菌中AMP结合的SbnC（SaSbnC）的晶体结构，该结构作为α-羟基羧酸铁载体生物合成中B型合成酶的第一代表性结构。尽管B型合成酶特异性地以α-酮戊二酸（α-KG）作为其羧酸底物，但与A型和C型合成酶相比，SaSbnC在底物口袋中展现出独特的结构特征。对α-KG类似物的筛选表明，α-KG的α-羰基与残基Lys552之间的氢键相互作用是决定B型合成酶底物选择性的关键因素。有趣的是，柠檬酸，作为三羧酸循环的产物和A型合成酶的底物，被发现通过模拟α-KG的结合抑制了SaSbnC的活性，IC50值为83 μM，这暗示了三羧酸循环在SaSbnC及其他B型合成酶中的作用可能具有潜在的调节功能。这一作用受细胞内铁浓度的控制。这些研究结果为理解大量病原微生物所采用的基于铁载体的铁获取系统中B型合成酶的结构、功能及调控提供了关键的新信息。