Allosteric Site at the Biotin Carboxylase Dimer Interface Mediates Activation and Inhibition in Staphylococcus aureus Pyruvate Carboxylase,Biochemistry

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Allosteric Site at the Biotin Carboxylase Dimer Interface Mediates Activation and Inhibition in Staphylococcus aureus Pyruvate Carboxylase
Biochemistry ( IF 2.9 ) Pub Date : 2023-08-21 , DOI: 10.1021/acs.biochem.3c00280
Amanda J Laseke ₁ , Trevor J Boram ₂ , Nicholas O Schneider ₁ , Jeremy R Lohman ₂ , Martin St Maurice ₁

Affiliation

Allosteric regulation of the essential anaplerotic enzyme, pyruvate carboxylase (PC), is vital for metabolic homeostasis. PC catalyzes the bicarbonate- and ATP-dependent carboxylation of pyruvate to form oxaloacetate. Dysregulation of PC activity can impact glucose and redox metabolism, which contributes to the pathogenicity of many diseases. To maintain homeostasis, PC is allosterically activated by acetyl-CoA and allosterically inhibited by l-aspartate. In this study, we further characterize the molecular basis of allosteric regulation in Staphylococcus aureus PC (SaPC) using slowly/nonhydrolyzable dethia analogues of acetyl-CoA and site-directed mutagenesis of residues at the biotin carboxylase homodimer interface. The dethia analogues fully activate SaPC but demonstrate significantly reduced binding affinities relative to acetyl-CoA. Residues Arg₂₁, Lys₄₆, and Glu₄₁₈ of SaPC are located at the biotin carboxylase dimer interface and play a critical role in both allosteric activation and inhibition. A structure of R21A SaPC in complex with acetyl-CoA reveals an intact molecule of acetyl-CoA bound at the allosteric site, offering new molecular insights into the acetyl-CoA binding site. This study demonstrates that the biotin carboxylase domain dimer interface is a critical allosteric site in PC, serving as a convergence point for allosteric activation by acetyl-CoA and inhibition by l-aspartate.

中文翻译：

生物素羧化酶二聚体界面的变构位点介导金黄色葡萄球菌丙酮酸羧化酶的激活和抑制

必需的回补酶丙酮酸羧化酶 (PC) 的变构调节对于代谢稳态至关重要。 PC 催化丙酮酸的碳酸氢盐和 ATP 依赖性羧化形成草酰乙酸。 PC 活性失调会影响葡萄糖和氧化还原代谢，从而导致许多疾病的致病性。为了维持体内平衡，PC 被乙酰辅酶 A 变构激活，并被L-天冬氨酸变构抑制。在这项研究中，我们利用乙酰辅酶A的缓慢/不可水解的脱硫类似物和生物素羧化酶同二聚体界面残基的定点诱变进一步表征了金黄色葡萄球菌PC（ Sa PC）变构调节的分子基础。脱硫类似物完全激活Sa PC，但与乙酰辅酶 A 相比，其结合亲和力显着降低。 Sa PC 的残基 Arg ₂₁ 、Lys ₄₆和 Glu ₄₁₈位于生物素羧化酶二聚体界面，在变构激活和抑制中发挥关键作用。 R21A Sa PC 与乙酰辅酶 A 复合的结构揭示了在变构位点结合的完整乙酰辅酶 A 分子，为乙酰辅酶 A 结合位点提供了新的分子见解。这项研究表明，生物素羧化酶结构域二聚体界面是 PC 中的一个关键变构位点，是乙酰辅酶 A 变构激活和L -天冬氨酸抑制的汇聚点。

更新日期：2023-08-21

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