Protein Identification And Activation Mechanism Of Microbial Urease In Rumen Of Dairy Cows

The utilization of endogenous and exogenous urea-N in the rumen of ruminants would be benefit for saving dietary protein,increasing microbial protein,and decreasing nitrogen emission.The hydrolysis of urea is carried out by microbial urease,and the high urease activity easily leads to the low urea-N utilization.To regulate the urease activity,microbial ureases from the rumen of dairy cows were identified using meta-proteomics.Targeting the rumen microbial urease,we cloned,overexpressed and purified accessory proteins UreG and UreE in vitro,revealed the properties of UreE-UreG interaction as well as the key UreG residues involved in the urease activation.Furthermore,natural compounds targeting UreG were screeded,which provides potential urease inhibitors to regulate ruminal microbial urease activity.The main results were as follows:1.To make the meta-proteomics technology better application in the identification of rumen microbial urease,protein extraction and protein digestion in the metaproteomic analysis were optimized.The results showed that cryomilling in liquid nitrogen for 4 min maximized urease activity among the six different methods of protein extraction(ultrasonication,bead beating at room temperature,cryomilling in liquid nitrogen,high-pressure press,freeze thawing and protein extraction kit),and trypsin digestion of in-gel proteins outperformed other three digestion methods(trypsin digestion of in-solution proteins,Glu-C/Lys C digestion of in-solution proteins,and Glu-C/Lys-C digestion of in-gel proteins),which yielded the greatest number of identifications with superior peptide performance.2.To improve the accuracy of protein identification,the extracted DNA of samples were directly sequenced by meta-genomics,and PCR amplification products of their urease genes were also sequenced.The sequencing results were processed to construct the protein database of the samples themselves.To increase the purity of active urease,the proteins extracted by cryomilling were purified through gel filtration chromatography,and the fraction with the highest urease activity was analyzed using mass spectrometry,in which protein digestion was carried out by trypsin digestion of in-gel proteins,and data searching was carried out by constructed protein database of the samples themselves.The results indicated that rumen microbial urease had high homology with that of from Fibrobacter sp.and Treponema sp.3.Urease is composed of structure proteins and accessory proteins,and its accessory proteins(especially UreE and UreG)play important roles in urease maturation.Isothermal titration calorimetry and analytical ultracentrifugation analyses showed that the reaction of identified UreE with UreG was endothermic,and was dominated by a hydrophobic interaction,in which each UreE dimer bound 2 M equivalents of UreG monomer to form a UreE₂-2UreG complex.Mutagenesis analyses showed that the UreG residues Glu66,Cys70,His72 and Asp78 not only prevented interactions with UreE,but also prevented nickel binding.4.UreG is thought to act as a bridge in the process of urease activation,given the importance and conservation of UreG,targeting UreG to screen urease inhibitor is a new strategy to regulate urease activity.Effects of natural compounds on the GTPase activity of UreG were first investigated,showing that berberine chloride,epiberberine and chelerythrine chloride had the potential to be urease inhibitors.The mechanism of these three compounds inhibit UreG was further evaluated,found that epiberberine was located the nearby CPH metal-binding motif of UreG,and affected the binding of UreG to nickel significantly.In addition,the inhibitory effect of epiberberine on rumen crude enzyme solution and jack bean was significantly stronger than that of acetohydroxamic acid.Based on those findings,epiberberine will be a great potential urease inhibitor from the natural compound.