In bacteria, the biosynthesis of the cofactor flavin adenine dinucleotide (FAD), important in many physiological responses, is catalyzed by the bifunctional enzyme FAD synthase (FADSyn) which converts riboflavin into FAD by both kinase and adenylylation activity. The in silico 3D structure of a putative FADSyn from Mycoplasma hyopneumoniae (MhpFADSyn), the etiological agent of enzootic pneumonia was already reported, nevertheless, the in vitro functional characterization was not yet demonstrated. Our phylogenetic analysis revealed that MhpFADSyn is close related to the bifunctional FADSyn from Corynebacterium ammoniagenes. However, only the domain related to adenylylation was assigned by InterPro database. The activity of MhpFADSyn was evaluated through in vitro enzymatic assays using cell extracts from IPTG-inducible heterologous expression of MhpFADSyn in Escherichia coli. The flavoproteins were analyzed by HPLC and results showed that IPTG-induced cell lysate resulted in the formation of twofold increased amounts of FAD if compared to non IPTG-induced cells. Consumption of riboflavin substrate was also threefold greater in IPTG-induced lysate compared to non IPTG-induced cell extract. Thus, the recombinant MhpFADSyn protein could be associated to FAD biosynthesis. These findings contribute to expand the range of potential drug targets in diseases control and unveil metabolic pathways that could be attribute to mycoplasmas.

中文翻译：

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来自猪肺炎支原体的推定 FAD 合酶的功能表征

在细菌中，辅助因子黄素腺嘌呤二核苷酸 (FAD) 的生物合成在许多生理反应中很重要，它由双功能酶 FAD 合酶 (FADSyn) 催化，该酶通过激酶和腺苷酸化活性将核黄素转化为 FAD。的，在硅片从推定FADSyn的3D结构猪肺炎支原体（MhpFADSyn），地方性肺炎的病原体已经报道，尽管如此，体外功能特性尚未证实。我们的系统发育分析表明，MhpFADSyn 与产氨棒状杆菌的双功能 FADSyn 密切相关. 然而，InterPro 数据库仅分配了与腺苷酸化相关的域。MhpFADSyn 的活性通过体外酶促测定评估，使用来自 IPTG 诱导的MhpFADSyn在大肠杆菌中的异源表达的细胞提取物. 通过 HPLC 分析黄素蛋白，结果表明，与非 IPTG 诱导的细胞相比，IPTG 诱导的细胞裂解物导致形成的 FAD 量增加了两倍。与非 IPTG 诱导的细胞提取物相比，IPTG 诱导的裂解物中核黄素底物的消耗量也高出三倍。因此，重组 MhpFADSyn 蛋白可能与 FAD 生物合成有关。这些发现有助于扩大疾病控制中潜在药物靶点的范围，并揭示可能归因于支原体的代谢途径。