克雷白氏肺炎桿菌CG43中尿嘧啶雙磷酸葡萄糖去氫酶之酪胺酸磷酸化的角色

Abstract

克雷白氏肺炎桿菌為一伺機性感染的格蘭氏陰性菌，可藉其外部包覆的厚重多醣體莢膜躲避細胞的吞噬作用及避免被血清因子所毒殺。我們先前的研究證實克雷白氏肺炎桿菌蛋白質酪胺酸激酶(Wzc)可藉由磷酸化尿嘧啶雙磷酸葡萄糖去氫酶(UDP-glucose dehydrogenase, Ugd)來提升Ugd酵素活性，進而調控莢膜多醣體的生成；而在Ugd上的17個酪胺酸殘基，已被選取作定點突變的9個酪胺酸殘基的改變並未影響Ugd的磷酸化。為了確認Ugd上被磷酸化的酪胺酸殘基，本研究將其他8個酪胺酸殘基，分別在53、71、76、85、203、252、302和380的位置，經點突變技術換成苯丙胺酸。再將這幾個定點突變後的蛋白質表現純化後，利用西方墨點法發現這些突變株仍能在大腸桿菌體內被磷酸化，此結果顯示克雷白氏肺炎桿菌不同於已報導的大腸桿菌與枯草桿菌，可能具有不只一個可受磷酸化的酪胺酸殘基。同時，酵素活性與動力學特性的分析結果得知，Tyr71、Tyr85、Tyr252和Tyr380的突變明顯影響了Ugd酵素活性，而這些點突變蛋白經由(circular dichroism, CD)圖譜分析，確認其二級結構組成與野生型Ugd無顯著差異，暗示著這些酪胺酸殘基的改變可能因無法被磷酸化而降低Ugd活性。

Klebsiella pneumoniae, an opportunistic gram-negative bacterial phathogen, is mostly encapsulated by a thick capsular polysaccharide (CPS) which acting to protect the bacterium from phagocytosis and prevent from damage by serum factors. Via an in vitro phosphorylation assay, we have previously demonstrated the protein-tyrosine kinase, Wzc, was capable of phosphorylating the enzyme UDP-glucose dehydrogenase (Ugd) to increase the enzymatic activity. The Ugd phosphorylation led to increase of the synthesis of CPS. Nine of the 17 tyrosine residues on KpUgd have been substituted individually with phenylalanine by site-directed mutagenesis. However, in vitro phosphorylation assay revealed that none of the changes affected the Ugd phosphorylation. Here, we generate specific mutation on the rest of the tyrosine residues on Ugd. Interestingly, all of the mutant proteins, Y53F, Y71F, Y76F, Y85F, Y203F, Y252F, Y302F, and Y380F, isolated from E. coli appeared to be phosphorylated. This suggested that KpUgd carried an additional tyrosine phosphorylation residue except the one reported for EcUgd-Y71 and BsUgd-Y70. Nevertheless, the enzyme kinetics analysis revealed that UgdY71F, UgdY85F, UgdY252F, and UgdY380F exhibit much lower activity than wild type Ugd. Circular dichroism analysis of the mutant Ugd indicated that the reduced activity was not due to structural alteration, implying the change of Y85, Y252, or Y380 impaired the subjection to phosphorylation leading to the decreased activity.