【作者】 沈裕虎；

【导师】 郑国锠；

【作者基本信息】 兰州大学 ， 细胞生物学， 2008， 博士

【摘要】 草酸脱羧酶（oxalate decarboxylases,OXDCs）降解草酸生成甲酸和二氧化碳。该酶属于cupin超家族bicupin亚族,在其氨基酸序列中,含有两个cupin结构域,每个cupin结构域由两个保守的基序:G（X）₅HXH（X）₁₁G和G（X）₅P（X）₄H（X）₃N（这里X指代任意氨基酸）组成。目前发现的草酸脱羧酶主要存在于一些腐生真菌和细菌中。一些腐生型真菌在向体外分泌草酸的同时,也能合成草酸脱羧酶分泌到胞外。这种分泌型草酸脱羧酶的存在被认为可以降低草酸对自身的毒性,保持细胞内外稳定的pH环境,保持草酸合成和降解的平衡。细菌来源的草酸脱羧酶主要存在于细胞质中,其功能可能和O.formigenes中的草酰-辅酶A一样,与细菌细胞的能量代谢相关:草酸与甲酸的反向转运造成细胞外高内低的质子浓度差,这种质子梯度又同磷酸化偶联,驱动ATP的合成。但是最近的研究发现,B.subtilis在酸诱导（外加肌醇六磷酸）条件下,草酸脱羧酶（OxdC）是其细胞壁主要成分,在细胞质中没有发现有OxdC的存在;在B.subtilis芽孢发育过程中,草酸脱羧酶（OxdD）能分泌到胞外,是芽孢外壳内层的组成成分。然而在这两个草酸脱羧酶中都没有发现分泌信号的存在,其分泌机制还不清楚。在全基因组测序已经完成的农杆菌C58菌株（Agrobacterium tumefaciens strain C58）的基因组中,发现存在编码产物和已知的草酸脱羧酶在氨基酸序列上表现高度同源的基因序列（定名为AtuOXDC）。我们用PCR方法从A.tumefaciens strain C58基因组中克隆到的AtuOXDC基因,全长1248bp,编码产物为415aa,理论分子量为44.7 kDa。氨基酸序列含两个cupin domain,属cupin超家族bicupin亚族蛋白。其中N-端和C-端cupin domain分别由第120～第175位、第301～第367位氨基酸残基组成,每个cupin domain中两个motif之间相隔（inter-motif region,IMR）20个aa。氨基酸序列分析发现,AtuOXDC N-端含有一段疏水跨膜区（第7～第29位氨基酸）,推测可能是一段分泌信号,预测剪切位点在第27和第28位氨基酸残基之间。序列比对和系统树分析表明,AtuOXDC同来自Bradyrhizobium japonicum和Burkholderia的草酸脱羧酶氨基酸序列一致性较高,被聚为一类。同来自真菌的草酸脱羧酶亲缘关系也较近。在大肠杆菌中成功实现了AtuOXDC的过量表达,并通过亲和层析纯化了表达产物,发现AtuOXDC在体外能够降解草酸,确实具有草酸脱羧酶活性。通过在A.tumefaciens strain C58中分别表达全长和截去信号肽序列的AtuOXDC,证明农杆菌草酸脱羧酶N-段确实存在一段分泌信号,并将该酶定位于细胞周质中;通过定点突变试验,用-KK-取代AtuOXDC信号肽序列中Tat信号特征性基序-RR-后,免疫印迹试验表明AtuOXDC向胞外的转运通过Tat途径进行。更多还原

【Abstract】 Oxalate decarboxylases （OXDCs） （EC4.1.1.2） are enzymes catalyzing the conversion of oxalate to formate and CO₂. These enzymes, which have been found in fungi and bacteria, belong to a functionally diverse protein superfamily known as the cupin that are characterized by their conserved motifs, most recently defined as G（X）₅HXH（X）₁₁G and G（X）₅P（X）₄H（X）₃N constituting a conserved six-strandedβ-barrel fold. Due to the presence of a duplication of this domain, OXDCs are members of the bicupin subclass and are thus thought to contain twoβ-barrels each comprising sixβ-strands.The best-characterized OXDCs are enzymes that have a wood-rotting fungal and bacterial origin. The fungal OXDCs are secreted enzymes, and a secretion signal has been found in the Flammulina velutipes oxalate decarboxylase that can mediate the secretion of heterologous proteins into the medium and periplasmic space in Schizosaccharomyces pombes. It is believed that oxalate synthesized by fungi contribute to lignin degradation, nutrient availability, pathogenesis, and competition. The oxalate-degrading enzymes secreted by these organisms are likely to be involved in pH regulation, and to help reduce toxicity caused by excess accumulations of oxalic acid in the microenvironment. In contrast, most bacterial OXDCs are localized in the cytosol. A decarboxylative phosphorylation mechanism has been described in the gram-negative bacterium Oxalobacter formigenes, in which the antiporting of oxalate and formate are coupled to oxalate decarboxylation by oxalyl-CoA decarboxylase, thereby generating a proton-motive gradient that drives ATP synthsis. It is possible that the bacterial Cytosolic OXDCs have a similar function in energy metabolism as oxalyl-CoA decarboxylase in O. formigenes.In A. tumfaciens C58 a locus for a putative oxalate decarboxylase （here denoted as AtuOXDC） is present. It contains a 1248 bp open reading frame （ORF） for a 415-amino acid polypeptide with a predicated molecular mass of 44.7 kDa. AtuOXDC has two conserved cupin domains shared by members of the cupin superfamily. The N- and C-terminus cupin domain was constituted of 120 tol75 and 301 to 367 amino acid residues respectively. The inter-motif region （IMR） was involved of 20 amino acid residues.Interestingly, analysis of the amino acids revealed a prominent hydrophobic segment of 23 amino acid residues, near the N-terminus of the AtuOXDC amino acid sequence, with a length characteristic of a secretion signal sequence. The most likely cleavage position is between position 27 and 28: AGA-AS. Multiple alignment and phylogeny tree indicate that AtuOXDC and putative OXDCs from Bradyrhizobium japonicum and Burkholderiashare were clustered into a subclass due to the more identity. In addition, the subclass was phylogeneticlly near with OXDCs from fungi.To confirm that the AtuOXDC does indeed encode an oxalate decarboxylase, the truncated AtuOXDC lacking the putative signal portion was expressed in E. coli, and its enzymatic activity was determined. Analysis of the sonicated cell extracts showed that the expression products were present either in the supernatants as soluble protein or in the pellets as inclusion bodies. Using Ni-NTA affinity chromatography, the homologous AtuOXDC was purified. The purified truncated AtuOXDC showed enzymatic activity at 6.567μmol formate/min·mg.A full-length version （AtuFOXDC） and a signal-peptideless version （AtuTOXDC） were cloned into the pTrc200 plasmid which can function as an expression vector in both E. coli and A. tumefaciens. The results of expression support the prediction that a secretion signal is present in the AtuOXDC protein and the protein was targeted in the periplasm of A. tumefaciens C58.When the -RR- motif of the AtuOXDC signal peptide was replaced with -KK- using site-directed mutagenesis, the expression of resulting mutant version （AtuMOXDC） of the full-length AtuOXDC protein in A. tumefaciens strain C58 and subsequent immune-blotting analysis showed that OXDC translocation of the mutant version was completely blocked, implying that the AtuOXDC protein is translocated by the TAT pathway.更多还原