Research School of Chemistry, Australian National University, Canberra, Australian Capital Territory 2601, Australia.
ARC Centre of Excellence in Synthetic Biology, Australian National University, Canberra, Australian Capital Territory 2601, Australia.
Biochemistry. 2024 May 21;63(10):1322-1334. doi: 10.1021/acs.biochem.4c00096. Epub 2024 May 2.
Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP from sp. A09 (Ami_SBP) and show that Ami_SBP binds l-amino acid amides but not the corresponding l-amino acids. An X-ray crystal structure of Ami_SBP bound to l-serinamide highlights the residues that impart distinct specificity for l-amino acid amides and reveals a structural Ca binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca binding are conserved among the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake, and metabolism of l-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose that Ami_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase-glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions with l-amino acid amides and bacterial nitrogen utilization.
周质溶质结合蛋白（SBPs）是细菌 ATP 结合盒（ABC）转运蛋白的关键配体识别组成部分，使细菌能够从环境中摄取营养物质和代谢前体。周质 SBPs 由一个庞大而多样化的蛋白质家族组成，其中只有少数已被经验证。在这项工作中，我们在 SBP_bac_5 家族中鉴定了一组 610 个独特的未表征蛋白质，这些蛋白质存在于保守操纵子中，这些操纵子包含编码（i）ABC 转运系统和（ii）来自 FmdA_AmdA 家族的假定酰胺酶的基因。从这些未表征的 SBP_bac_5 蛋白中，我们表征了 sp. A09 的一个代表性周质 SBP（Ami_SBP），并表明 Ami_SBP 结合 l-氨基酸酰胺但不结合相应的 l-氨基酸。Ami_SBP 与 l-丝氨酸酰胺结合的 X 射线晶体结构突出了赋予 l-氨基酸酰胺独特特异性的残基，并揭示了蛋白质一个叶中的一个结构 Ca 结合位点。我们表明，参与配体和 Ca 结合的残基在来自实验上未表征的 FmdA_AmdA 酰胺酶相关 ABC 转运系统的 610 个 SBP 中保守，这表明这些同源系统也可能参与许多革兰氏阴性固氮土壤细菌中 l-氨基酸酰胺的感应、摄取和代谢。我们提出 Ami_SBP 参与了这些溶质的摄取，以补充柠檬酸循环和谷氨酰胺合成酶-谷氨酸合酶途径等途径。这项工作扩展了我们目前对微生物与 l-氨基酸酰胺相互作用和细菌氮利用的有限理解。
