在多种微生物中发现的脲酶在尿素分解诱导的方解石沉淀(UICP)中起着至关重要的作用。然而,产脲酶菌株的基因组信息有限,镍配体残基对脲酶活性的调控等方面需要进一步深入研究。本研究通过采用生物矿化控制生长的遗传结构,阐明了新分离的菌株YX-3与镍配体残基偶联的脲酶活性,分子对接,分子动力学模拟(MDS),和定点诱变。全基因组测序显示存在脲酶基因簇,包含结构基因ureA,ureB,和ureC,除了辅助基因ureD,ureE,ureF,和ureG。RT-qPCR分析显示NiCl2的添加导致了ureC表达的显著上调。通过同源性建模和分子对接,进一步证明了UreC域中的His267,His294和Gly325与镍离子和尿素同时配位。和分子动力学模拟(MDS)表明,当这些残基分别突变为丙氨酸时,脲酶-尿素对接复合物表现出四个指标的递减结合稳定性,包括均方根偏差(RMSD)。蛋白质印迹显示H267A的突变,H294A,和G325A导致脲酶的相对表达减少,其中脲酶活性约为62%,45%,是野生型(WT)的20%,分别。过表达结果进一步证实了这些残基对于脲酶活性和CaCO3沉淀的重要性。这些结果将有助于在分子水平上加深对产脲酶菌株的认识,拓展调控脲酶活性的理论基础。
Urease found in a wide range of microorganisms plays a vital role in ureolytic induced calcite precipitation (UICP). However, the genomic information on urease-producing strains is limited, and there is a need for further in-depth studies on aspects such as the regulation of urease activity by nickel ligand residues. The present study delved into the elucidation of urease activity in a newly isolated strain YX-3 coupled with nickel-ligand residues by employing the genetic architecture of biomineralization-controlled growth, molecular docking, molecular dynamics simulation (MDS), and site-directed mutagenesis. Genome-wide sequencing showed the presence of urease gene clusters, comprising structural genes ureA, ureB, and ureC, alongside auxiliary genes ureD, ureE, ureF, and ureG. RT-qPCR analysis showed that the addition of NiCl2 resulted in a significant up-regulation of ureC expression. His267, His294, and Gly325 in the domain of UreC were further proved to coordinate with nickel ions and urea simultaneously through homology modeling and molecular docking, and molecular dynamics simulations (MDS) showed the urease-urea docking complexes exhibited degressive binding stability by four metrics including root mean square deviations (RMSD) when those residues were mutated into alanine respectively. Western blotting exhibited that mutations of H267A, H294A, and G325A led to a reduction in the relative expression of urease, wherein urease activity was about 62%, 45%, and 20% times that of the wild type (WT), respectively. The overexpression results further confirmed the importance of these residues for urease activity and CaCO3 precipitation. These results would help to deepen the understanding of urease-producing strains at a molecular level and expand the theoretical basis for modulating urease activity.