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首页> 外文学位 >Biochemical characterization and structure determination of a prolyl 4-hydroxylase-like protein from Bacillus anthracis.
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Biochemical characterization and structure determination of a prolyl 4-hydroxylase-like protein from Bacillus anthracis.

机译：炭疽芽孢杆菌脯氨酰4-羟化酶样蛋白的生化特性和结构测定。

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摘要

Procollagen prolyl-4-hydroxylase (P4H) catalyzes the conversion of peptidyl proline to trans-4-hydroxyproline (Hyp). P4H is a member of the alpha-ketoglutarate-dependent mononuclear non-heme iron oxygenase (alphaKG/Fe(II)-oxygenase) family of enzymes, which catalyze a wide variety of reactions. The genome of Bacillus anthracis (B. anthracis), the causative agent of anthrax, contains a gene that is annotated as a p4h based on the predicted amino acid sequence. Recently the immunodominant protein of the B. anthracis exosporium has been identified as BclA. BclA has collagen-like repeat sequences and has a triple helical structure similar to that of animal collagens. We have detected Hyp from the protein extracts of spores of B. anthracis and this is the first report for Hyp detection in bacteria. These results strongly support that B. anthracis contains a P4H-like protein, anthrax-P4H.;We have expressed and purified a recombinant form of the putative anthrax-P4H and found that it is a homodimer of 24.3 kDa subunit. The anthrax-P4H exhibits activity and UV-vis spectroscopic properties characteristic of an alphaKG/Fe(II)-oxygenase, and it can bind a collagen-like substrate, (Gly-Pro-Pro)10, with an affinity comparable to that of human type (I) collagen-P4H (human-P4H-1). This is the first report of any P4H-like protein from a bacterial source. We propose that anthrax-P4H can serve as a model for human-P4H-1 from its substrate specificity and kinetic parameters comparable to human-P4H-1.;We also report the crystal structure of anthrax-P4H to 1.40 A. The structure reveals the double stranded beta-helix core fold (jellyroll) motif, characteristic of Fe(II)/alphaKG oxygenases. The oligomerization of the protein is a homodimeric, alpha2, structure, which makes the anthrax-P4H a new member to the P4H family of enzymes. A putative peptide-binding groove has been proposed based on that identified in the structure of P4H from Chlamydomonas reinhardtii (green alga) (Cr-P4H-1). The anthrax-P4H structure provides insight into the dimeric structure of the alpha-subunits of human-P4H-1, as well as to help understand the mode of substrate recognition that may aide in the development of selective inhibitors.
机译：前胶原脯氨酰-4-羟化酶（P4H）催化肽基脯氨酸向反式-4-羟脯氨酸（Hyp）的转化。 P4H是依赖α-酮戊二酸的单核非血红素铁加氧酶（alphaKG / Fe（II）-加氧酶）酶的成员，可催化多种反应。炭疽杆菌（炭疽杆菌）的基因组是炭疽的病原体，它的基因基于预测的氨基酸序列标注为p4h。最近，炭疽芽孢杆菌外孢子的免疫优势蛋白已被鉴定为BclA。 BclA具有类似胶原蛋白的重复序列，并具有类似于动物胶原蛋白的三螺旋结构。我们已经从炭疽芽孢杆菌的孢子蛋白提取物中检测到Hyp，这是细菌中检测Hyp的第一份报告。这些结果强烈支持炭疽芽孢杆菌含有P4H样蛋白炭疽-P4H。我们已经表达并纯化了推定的炭疽-P4H的重组形式，发现它是24.3 kDa亚基的同型二聚体。炭疽-P4H具有alphaKG / Fe（II）加氧酶的活性和紫外可见光谱特性，它可以结合胶原蛋白样底物（Gly-Pro-Pro）10，其亲和力可与人型（I）胶原蛋白P4H（人P4H-1）。这是细菌来源的任何P4H样蛋白的首次报道。我们提出炭疽-P4H可以作为人类-P4H-1的模型，因为它的底物特异性和动力学参数可与人类-P4H-1媲美。我们还报道了炭疽-P4H的晶体结构为1.40A。 Fe（II）/ alphaKG加氧酶的特征性双链β-螺旋核心折叠（果冻卷）基序。蛋白质的低聚是一种同型二聚体α2结构，这使炭疽P4H成为P4H酶家族的新成员。基于在莱茵衣藻（绿藻）（Cr-P4H-1）的P4H的结构中鉴定出的肽结合槽，已经提出了推测的肽结合槽。炭疽-P4H结构提供了对人-P4H-1α-亚基二聚体结构的深入了解，并有助于理解可能有助于选择性抑制剂发展的底物识别模式。

著录项

  • 作者

    Culpepper, Megen A.;

  • 作者单位

    University of Kansas.;

  • 授予单位 University of Kansas.;
  • 学科 Chemistry Analytical.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 210 p.
  • 总页数 210
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 化学;
  • 关键词

  • 入库时间 2022-08-17 11:38:28

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