Cameron Stephan M, Durchschein Katharina, Richman Jack E, Sadowsky Michael J, Wackett Lawrence P
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, Minnesota 55108.
ACS Catal. 2011 Aug 1;2011(1):1075-1082. doi: 10.1021/cs200295n.
Biuret is an intermediate in the bacterial metabolism of s-triazine ring compounds and is occasionally used as a ruminant feed supplement. We used bioinformatics to identify a biuret hydrolase, an enzyme that has previously resisted efforts to stabilize, purify and characterize. This newly discovered enzyme is a member of the cysteine hydrolase superfamily, a family of enzymes previously not found to be involved in s-triazine metabolism. The gene from Rhizobium leguminosarum bv. viciae strain 3841 encoding biuret hydrolase was synthesized, transformed into Escherichia coli, and expressed. The enzyme was purified and found to be stable. Biuret hydrolase catalyzed the hydrolysis of biuret to allophanate and ammonia. The k(cat)/K(M) of 1.7 × 10(5) M(-1)s(-1) and the relatively low K(M) of 23 ± 4 μM together suggested that this enzyme acts uniquely on biuret physiologically. This is supported by the fact that of the 34 substrate analogs of biuret tested, only two demonstrated reactivity, both at less than 5% of the rate determined for biuret. Biuret hydrolase does not react with carboxybiuret, the product of the enzyme immediately preceding biuret hydrolase in the metabolic pathway for cyanuric acid. This suggests an unusual metabolic strategy of an enzymatically-produced intermediate undergoing non-enzymatic decarboxylation to produce the substrate for the next enzyme in the pathway.
缩二脲是细菌代谢三嗪环化合物过程中的一种中间产物，偶尔用作反刍动物饲料添加剂。我们利用生物信息学鉴定出一种缩二脲水解酶，这种酶此前一直难以实现稳定、纯化和表征。这种新发现的酶是半胱氨酸水解酶超家族的成员，该家族的酶此前未发现参与三嗪代谢。合成了来自豌豆根瘤菌蚕豆生物变种3841菌株的编码缩二脲水解酶的基因，将其转化到大肠杆菌中并进行表达。该酶经过纯化后发现很稳定。缩二脲水解酶催化缩二脲水解生成脲基甲酸酯和氨。1.7×10⁵ M⁻¹s⁻¹的k(cat)/K(M)以及相对较低的23±4 μM的K(M)共同表明，这种酶在生理条件下对缩二脲具有独特的作用。在所测试的34种缩二脲底物类似物中，只有两种表现出反应活性，且反应速率均低于缩二脲的5%，这一事实支持了上述观点。缩二脲水解酶不与羧基缩二脲反应，羧基缩二脲是氰尿酸代谢途径中在缩二脲水解酶之前的那种酶的产物。这表明了一种不同寻常的代谢策略，即一种酶促产生的中间产物进行非酶促脱羧反应，以产生该途径中下一种酶的底物。
