Pseudomonas aeruginosa C5-mannuronan epimerase: steady-state kinetics and characterization of the product.
Pseudomonas aeruginosa C5-mannuronan epimerase: steady-state kinetics and characterization of the product.
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DOI：
10.1021/bi051862l
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发表时间：
2006-01
期刊：
影响因子：
2.9
通讯作者：
Agoston Jerga;A. Raychaudhuri;P. Tipton
中科院分区：
文献类型：
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作者：
Agoston Jerga;A. Raychaudhuri;P. Tipton
藻酸盐是铜绿假单胞菌产生的成熟生物膜的主要成分。藻酸盐生物合成中的倒数第二步是在C5-甘露糖醛酸差向异构酶催化的反应中将聚合物底物聚甘露糖醛酸中的一些β-D-甘露糖醛酸残基转化为α-L-古洛糖醛酸残基。与大小分级的寡甘露糖醛酸进行的特异性研究表明，最小的底物含有9个单糖残基。随着底物大小从10个残基增加到20个残基，反应的最大速度从0.0018 s（-1）增加到0.0218 s（-1），并且对于长度高达100个残基的底物，没有观察到kcat的额外增加。Km从含有少于15个残基的底物的80 μ M降低到含有多于100个残基的底物的4 μ M。与已经在其他细菌物种中表征的C5-甘露糖醛酸差向异构酶相反，铜绿假单胞菌C5-甘露糖醛酸差向异构酶不需要Ca 2+来进行活性，并且Ca 2 +-藻酸盐复合物不是该酶的底物。通过电感耦合等离子体发射光谱法对纯化的活性酶进行分析，发现蛋白质中不存在金属。的pH依赖性的动力学参数表明，三个残基的酶都具有约7.6的pKa必须质子化的催化发生。通过1H NMR光谱分析差向异构酶反应的聚合产物的组成，其显示容易形成相邻古洛糖醛酸残基的束。当聚合物的古洛糖醛酸盐组成为75%时，反应达到表观平衡。
Alginate is a major constituent of mature biofilms produced by Pseudomonas aeruginosa. The penultimate step in the biosynthesis of alginate is the conversion of some beta-D-mannuronate residues in the polymeric substrate polymannuronan to alpha-L-guluronate residues in a reaction catalyzed by C5-mannuronan epimerase. Specificity studies conducted with size-fractionated oligomannuronates revealed that the minimal substrate contained nine monosaccharide residues. The maximum velocity of the reaction increased from 0.0018 to 0.0218 s(-1) as the substrate size increased from 10 to 20 residues, and no additional increase in kcat was observed for substrates up to 100 residues in length. The Km decreased from 80 microM for a substrate containing fewer than 15 residues to 4 microM for a substrate containing more than 100 residues. In contrast to C5-mannuronan epimerases that have been characterized in other bacterial species, P. aeruginosa C5-mannuronan epimerase does not require Ca2+ for activity, and the Ca2+-alginate complex is not a substrate for the enzyme. Analysis of the purified, active enzyme by inductively coupled plasma-emission spectroscopy revealed that no metals were present in the protein. The pH dependence of the kinetic parameters revealed that three residues on the enzyme which all have a pKa of approximately 7.6 must be protonated for catalysis to occur. The composition of the polymeric product of the epimerase reaction was analyzed by 1H NMR spectroscopy, which revealed that tracts of adjacent guluronate residues were readily formed. The reaction reached an apparent equilibrium when the guluronate composition of the polymer was 75%.