Shapir Nir, Sadowsky Michael J, Wackett Lawrence P
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 1479 Gortner Avenue, St. Paul, Minnesota 55108, USA.
J Bacteriol. 2005 Jun;187(11):3731-8. doi: 10.1128/JB.187.11.3731-3738.2005.
AtzF, allophanate hydrolase, is a recently discovered member of the amidase signature family that catalyzes the terminal reaction during metabolism of s-triazine ring compounds by bacteria. In the present study, the atzF gene from Pseudomonas sp. strain ADP was cloned and expressed as a His-tagged protein, and the protein was purified and characterized. AtzF had a deduced subunit molecular mass of 66,223, based on the gene sequence, and an estimated holoenzyme molecular mass of 260,000. The active protein did not contain detectable metals or organic cofactors. Purified AtzF hydrolyzed allophanate with a k(cat)/K(m) of 1.1 x 10(4) s(-1) M(-1), and 2 mol of ammonia was released per mol allophanate. The substrate range of AtzF was very narrow. Urea, biuret, hydroxyurea, methylcarbamate, and other structurally analogous compounds were not substrates for AtzF. Only malonamate, which strongly inhibited allophanate hydrolysis, was an alternative substrate, with a greatly reduced k(cat)/K(m) of 21 s(-1) M(-1). Data suggested that the AtzF catalytic cycle proceeds through a covalent substrate-enzyme intermediate. AtzF reacts with malonamate and hydroxylamine to generate malonohydroxamate, potentially derived from hydroxylamine capture of an enzyme-tethered acyl group. Three putative catalytically important residues, one lysine and two serines, were altered by site-directed mutagenesis, each with complete loss of enzyme activity. The identity of a putative serine nucleophile was probed using phenyl phosphorodiamidate that was shown to be a time-dependent inhibitor of AtzF. Inhibition was due to phosphoroamidation of Ser189 as shown by liquid chromatography/matrix-assisted laser desorption ionization mass spectrometry. The modified residue corresponds in sequence alignments to the nucleophilic serine previously identified in other members of the amidase signature family. Thus, AtzF affects the cleavage of three carbon-to-nitrogen bonds via a mechanism similar to that of enzymes catalyzing single-amide-bond cleavage reactions. AtzF orthologs appear to be widespread among bacteria.
阿嗪水解酶（AtzF）是酰胺酶特征家族中最近发现的一个成员，它催化细菌代谢三嗪环化合物过程中的末端反应。在本研究中，克隆了来自假单胞菌属ADP菌株的atzF基因，并将其表达为带有His标签的蛋白，然后对该蛋白进行了纯化和表征。根据基因序列推断，AtzF的亚基分子量为66,223，全酶分子量估计为260,000。活性蛋白不含可检测到的金属或有机辅因子。纯化后的AtzF水解脲基甲酸，催化常数与米氏常数之比（k(cat)/K(m)）为1.1×10⁴ s⁻¹ M⁻¹，每摩尔脲基甲酸释放2摩尔氨。AtzF的底物范围非常窄。尿素、缩二脲、羟基脲、氨基甲酸甲酯和其他结构类似的化合物都不是AtzF的底物。只有丙二酸酰胺是一种替代底物，它强烈抑制脲基甲酸水解，其k(cat)/K(m)大幅降低至21 s⁻¹ M⁻¹。数据表明，AtzF催化循环通过共价底物 - 酶中间体进行。AtzF与丙二酸酰胺和羟胺反应生成丙二酸异羟肟酸，这可能源于羟胺捕获酶结合的酰基。通过定点诱变改变了三个推测具有催化重要性的残基，一个赖氨酸和两个丝氨酸，每个突变体的酶活性都完全丧失。使用苯基磷二酰胺探测推测的丝氨酸亲核试剂的身份，结果表明它是AtzF的时间依赖性抑制剂。液相色谱/基质辅助激光解吸电离质谱显示，抑制作用是由于Ser189的磷酰胺化。在序列比对中，修饰的残基与酰胺酶特征家族其他成员中先前鉴定的亲核丝氨酸相对应。因此，AtzF通过类似于催化单酰胺键裂解反应的酶的机制影响三个碳 - 氮键的裂解。AtzF直系同源物似乎在细菌中广泛存在。
