During aerobic growth, the Gram-positive facultative anaerobe and opportunistic human pathogen Streptococcus pneumoniae generates large amounts of hydrogen peroxide that can accumulate to millimolar concentrations. The mechanism by which this catalase-negative bacterium can withstand endogenous hydrogen peroxide is incompletely understood. The enzyme alkylhydroperoxidase D (AhpD) has been shown to contribute to pneumococcal virulence and oxidative stress responses in vivo We demonstrate here that SpAhpD exhibits weak thiol-dependent peroxidase activity and, unlike the previously reported Mycobacterium tuberculosis AhpC/D system, SpAhpD does not mediate electron transfer to SpAhpC. A 2.3-Å resolution crystal structure revealed several unusual structural features, including a three-cysteine active site architecture that is buried in a deep pocket, in contrast to the two-cysteine active site found in other AhpD enzymes. All single-cysteine SpAhpD variants remained partially active, and LC-MS/MS analyses revealed that the third cysteine, Cys-163, formed disulfide bonds with either of two cysteines in the canonical Cys-78-X-X-Cys-81 motif. We observed that SpAhpD formed a dimeric quaternary structure both in the crystal and in solution, and that the highly conserved Asn-76 of the AhpD core motif is important for SpAhpD folding. In summary, SpAhpD is a weak peroxidase and does not transfer electrons to AhpC, and therefore does not fit existing models of bacterial AhpD antioxidant defense mechanisms. We propose that it is unlikely that SpAhpD removes peroxides either directly or via AhpC, and that SpAhpD cysteine oxidation may act as a redox switch or mediate electron transfer with other thiol proteins.

中文翻译：

---

肺炎链球菌烷基氢过氧化物酶D的结构功能分析揭示了一个不寻常的三半胱氨酸活性位点结构。

在有氧生长期间，革兰氏阳性兼性厌氧菌和机会性人类病原体肺炎链球菌会产生大量的过氧化氢，这些过氧化氢可以累积到毫摩尔浓度。这种过氧化氢酶阴性细菌可以抵抗内源性过氧化氢的机理尚不完全清楚。已证明烷基氢过氧化物酶D（AhpD）有助于体内的肺炎球菌毒力和氧化应激反应。我们在此证明SpAhpD表现出弱的硫醇依赖性过氧化物酶活性，并且与先前报道的结核分枝杆菌AhpC / D系统不同，SpAhpD不介导电子转移到SpAhpC。2.3Å分辨率的晶体结构揭示了几个不寻常的结构特征，包括埋在深口袋中的三半胱氨酸活性位点结构，与其他AhpD酶中的两个半胱氨酸活性位点相反。所有单半胱氨酸SpAhpD变体均保持部分活性，并且LC-MS / MS分析显示，第三个半胱氨酸Cys-163与典型Cys-78-XX-Cys-81基序中的两个半胱氨酸之一形成了二硫键。我们观察到，SpAhpD在晶体和溶液中均形成了二聚体四元结构，并且高度保守的AhpD核心基序Asn-76对SpAhpD折叠很重要。总之，SpAhpD是一种弱的过氧化物酶，不会将电子转移到AhpC，因此不适合细菌AhpD抗氧化剂防御机制的现有模型。我们建议SpAhpD不太可能直接或通过AhpC去除过氧化物，