克雷白氏肺炎桿菌CG43中磷酸甘露糖異構酶磷酸化的特性探討

Abstract

細菌酪胺酸激酶是近年來新建立的蛋白質家族，與真核生物之酪胺酸激酶有著 相似的功能。活體外磷酸化實驗證明克雷白氏肺炎菌 CG43 之酪胺酸激酶 Wzc 可以將多醣莢膜基因座上之基因產物包括 6-葡萄醣醛酸脫氫酶 Gnd、甘露糖-1- 磷酸鳥苷醯基轉移酶 ManC 及磷酸甘露糖異構酶 ManB 磷酸化，並增加 Gnd 之 酵素活性。本實驗建構克雷白氏肺炎菌 CG43 突變株 Δgnd、ΔmanC 與 ΔmanB 並分析基因缺失的影響，結果發現 ΔmanC 與 ΔmanB 之多醣莢膜合成量明顯下降而生物膜生成增加，為了證實 ManB 活性與酪胺酸殘基磷酸化的關係，我們藉由比對分析選出可能受磷酸化的酪胺酸殘基做定點突變，接著回補實驗分析發現 manB 基因剔除所造成的多醣莢膜含量下降可藉轉殖 ΔmanB 予表現質體 pRK415-manB、pRK415- manBY26F 或 pRK415-manB Y341F 而回補。然而 pRK415-manBY10F 僅能回復些微多醣莢膜含量，此暗示著 Tyr10 可能影響 ManB 之活性。而分析全菌裂解液之 ManB 酵素活性發現 Y10F、Y26F 及 Y341F 突變 會降低 ManB 酵素活性而以 Y10F 下降程度最大；另外，S98A 突變使 ManB 喪 失活性。進一步，在大腸桿菌中大量表現這些重組蛋白，以圓雙色光譜分析這些 純化的重組蛋白證實這些酪胺酸殘基突變並不影響 ManB 之二級結構，我們推測 Y10F 可能因降低 ManB 與受質的結合力而影響其催化活性，而 S98 的磷酸化決 定其酵素活性。最後，活體外磷酸化實驗顯示單株抗體和 Pro-Q Diamond 均可偵測 ManB 的磷酸化，ManB 的磷酸化些微提升其酵素活性；而 S98A 突變使 ManB 不被磷酸化且活性喪失，而其磷酸化不受酪胺酸激酶或絲胺酸-蘇胺酸激酶 的基因缺損影響。

Bacterial tyrosine kinases have been recently unified in a new enzyme family because of the similar structural and functional features with their eukaryotic counterparts. Via an in vitro phosphorylation assay, the protein-tyrosine kinase Wzc of Klebsiella pneumoniae CG43 was shown capable of phosphorylating the capsular polysaccharide biosynthesis operon cps gene products 6-phosphogluconate dehydrogenase (Gnd), GDP-mannose phosphorylase (ManC) and phosphomannomutase (ManB) and the phosphorylation leading to increase of the enzymatic activities. In this study, we generate CG43S3Δgnd, CG43S3ΔmanC and CG43S3ΔmanB mutants and the phenotypes analyzed and compared. The analysis revealed that deletion of manC or manB decreased the glucuronic acid production but increased the biofilm formation. Several of the ManB tyrosine residues were then selected for site directed mutagensis in order to determine which is the one that subjected to Wzc phosphorylation and if the phosphorylation influences the enzymztic activity. The subsequent complementation analysis revealed that the manB deleting effect could be complemented by introducing into the mutant with the ManB expression plasmid pRK415-manB or the plasmid pRK415-manBY26F and pRK415-manBY341F which carrying a point mutation of ManB.Whereas the bacteria CG43S3ΔmanB carrying pRK415-manBY10F exhibited a partially restored phenotype implying the phosphorylation on tyrosine residue 10 of ManB was critical for the enzymatic activity. In addition, the ManB activity was decreased in the bacteria CG43S3ΔmanB [pETQ33-manBY10F] or CG43S3ΔmanB [pETQ33-manBY341F] by comparing to that in CG43S3ΔmanB [pETQ33-manB ] or CG43S3ΔmanB [pETQ33- manBY26F]. These recombinant plasmids were then overexpressed in E. coli BL21[DE3] and the recombinant proteins purified. The circular dichroism spectra analysis of the purified proteins revealed that the site directed mutation had no effect on the secondary structure. Finally, the in virto phosphorylation assay showed that the monoclonal antibody and Pro-Q Diamond were able to detect the ManB phosphorylation form and ManB phosphorylation slightly enhanced the enzymatic activity. In addition, the S98 was found to be a critical phosphorylation residue which is required for the enzyme activity. However,the serine phosphorylation was not afftected by the deletion of wzc or stk.