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首页> 外文期刊>Biochemistry >Biochemical Characterization of ThiT from Lactococcus lactis: A Thiamin Transporter with Picomolar Substrate Binding Affinity
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Biochemical Characterization of ThiT from Lactococcus lactis: A Thiamin Transporter with Picomolar Substrate Binding Affinity

机译：乳酸乳球菌ThiT的生化特性：具有皮摩尔底物结合亲和力的硫胺素转运蛋白。

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The putative thiamin transporter ThiT from Lactococcus lactis was overproduced in themembrane of lactococcal cells. In vivo transport assays using radiolabeled thiamin demonstrated that ThiTindeed was involved in thiamin transport. The protein was solubilized from the membranes and purified indetergent solution. Size exclusion chromatography coupled to static light scattering, refractive index, and UVabsorbance measurements (SEC-MALLS) showed that ThiT is a monomer of 22.7 kDa in detergent solution.When the cells overexpressing ThiT had been cultivated in complex growth medium, all binding sites of thepurified protein were occupied with substrate, which had copurified with the protein. MALDI-TOF massspectrometry analysis confirmed that the copurified substance was thiamin. Substrate-depleted ThiT wasobtained by expressing the protein in cells that were cultivated in chemically defined growth medium withoutthiamin. The intrinsic tryptophan fluorescence of substrate-depleted ThiT was strongly quenched uponthiamin binding. The quenching of the fluorescence was used to determine dissociation constants for thiaminand related compounds. ThiT had an unusually high affinity for thiamin (KID = 122 ± 13 pM) and bound thesubstrate with a 1:1 (proteindigand) stoichiometry. TPP, TMP, and pyrithiamin bound to ThiT withnanomolar affinity. A multiple sequence alignment of ThiT homologues revealed that well-conserved residueswere clustered in a tryptophan-rich stretch comprising the loop between the predicted membrane spanningsegments 5 and 6. Mutational analysis of the conserved residues in this region combined with binding assays ofthiamin and related compounds was used to build a model of the high-affinity binding site. The model wascompared with thiamin binding sites of other proteins and interpreted in terms of the transport mechanism.
机译：乳酸乳球菌的推定的硫胺素转运蛋白ThiT在乳球菌细胞膜中过量产生。使用放射性标记的硫胺素的体内转运试验表明，ThiTindeed参与了硫胺素的转运。从膜和纯化的去污剂溶液中溶解蛋白。尺寸排阻色谱结合静态光散射，折射率和紫外线吸收率测量（SEC-MALLS）表明，ThiT是洗涤剂溶液中的22.7 kDa单体。当在复杂的生长培养基中培养过表达ThiT的细胞时，所有ThiT的结合位点纯化的蛋白质被与蛋白质共纯化的底物占据。 MALDI-TOF质谱分析证实了共纯化的物质是硫胺素。通过在无硫胺素的化学成分确定的生长培养基中培养的细胞中表达蛋白质，从而获得了缺乏底物的ThiT。耗尽底物的ThiT的固有色氨酸荧光在硫胺素结合后被强烈淬灭。荧光的猝灭用于确定硫胺及相关化合物的解离常数。 ThiT对硫胺素具有异常高的亲和力（KID = 122±13 pM），并以1：1（蛋白质digand）化学计量比结合底物。 TPP，TMP和吡虫胺以纳摩尔亲和力与ThiT结合。 ThiT同源物的多序列比对表明，保守性好的残基聚集在一个富含色氨酸的片段中，该片段中包含预测的跨膜片段5和6之间的环。对该区域中的保守残基进行突变分析，并结合了硫胺素和相关化合物的结合测定。用于构建高亲和力结合位点的模型。该模型与其他蛋白质的硫胺素结合位点进行了比较，并根据转运机理进行了解释。

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