Characterization of the Nit6803 nitrilase homolog from the cyanotroph Pseudomonas fluorescens NCIMB 11764.
Characterization of the Nit6803 nitrilase homolog from the cyanotroph Pseudomonas fluorescens NCIMB 11764.
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来自氰营养菌荧光假单胞菌NCIMB 11764的Nit6803腈水解酶同系物的表征。
DOI：
10.1016/j.bbrep.2020.100893
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发表时间：
2021-03
影响因子：
2.7
通讯作者：
Kunz DA
中科院分区：
文献类型：
--
作者：
Jones LB;Wang X;Gullapalli JS;Kunz DA
本文报道了一种腈水解酶（E.C. 3.5.5.1）（Nit 11764），其对于氰营养菌荧光假单胞菌NCIMB 11764同化作为唯一氮源的氰化物是必需的。Nit 11764是一个同源蛋白家族（nitrile_sll0784）的成员，其基因通常位于一个保守的七基因簇中，称为Nit 1C。Nit 11764的物理性质和底物特异性与Nit 6803相似，Nit 6803是该家族目前的参考蛋白，也是唯一一种已结晶的真正腈水解酶。两种酶的底物结合口袋将底物直接置于活性位点亲核体（C160）和保守催化三联体（Glu 44，Lys 126）附近。这两种酶表现出相似的底物谱，然而，对于Nit 11764，发现肉桂腈是比先前鉴定为Nit 6803的最佳底物的肉桂腈更好的底物。与肉桂腈（Km 8.57 mM）相比，肉桂腈（Km 1.27 mM）的亲和力更高，这与对接研究一致，该研究预测与衬在结合口袋中的疏水残基的相互作用更有利。相比之下，3，4-二甲氧基肉桂腈是较差的底物，取代的甲氧基基团明显阻碍进入结合口袋。原位1H NMR研究表明，在二腈中的两个腈取代基中只有一个--氰腈被攻击，产生反式-3-氰基丙烯酸酯（加上氨）作为产物。Nit 11764对氰营养的重要性仍然不确定，因为氰化物本身是一种不良的底物，即使是最好的腈底物（~5 × 103 M−1 s−1）的催化效率也低于恒星。腈水解酶Nit 11764与Nit 6803具有相同的性质，两者都来自于氰营养细菌中的保守基因簇。Nit 117674的最佳底物是肉桂腈，与有利的对接研究一致。二腈，三腈，是第二个最好的底物，3-氰基丙烯酸酯是由1 NMR确定的。虽然被注释为脂肪族腈水解酶，但Nit 11764的生理作用仍不清楚。
We report the purification and characterization of a nitrilase (E.C. 3.5.5.1) (Nit11764) essential for the assimilation of cyanide as the sole nitrogen source by the cyanotroph, Pseudomonas fluorescens NCIMB 11764. Nit11764, is a member of a family of homologous proteins (nitrile_sll0784) for which the genes typically reside in a conserved seven-gene cluster known as Nit1C. The physical properties and substrate specificity of Nit11764 resemble those of Nit6803, the current reference protein for the family, and the only true nitrilase that has been crystallized. The substrate binding pocket of the two enzymes places the substrate in direct proximity to the active site nucleophile (C160) and conserved catalytic triad (Glu44, Lys126). The two enzymes exhibit a similar substrate profile, however, for Nit11764, cinnamonitrile, was found to be an even better substrate than fumaronitrile the best substrate previously identified for Nit6803. A higher affinity for cinnamonitrile (Km 1.27 mM) compared to fumaronitrile (Km 8.57 mM) is consistent with docking studies predicting a more favorable interaction with hydrophobic residues lining the binding pocket. By comparison, 3,4-dimethoxycinnamonitrile was a poorer substrate the substituted methoxyl groups apparently hindering entry into the binding pocket. in situ1H NMR studies revealed that only one of the two nitrile substituents in the dinitrile, fumaronitrile, was attacked yielding trans-3-cyanoacrylate (plus ammonia) as a product. The essentiality of Nit11764 for cyanotrophy remains uncertain given that cyanide itself is a poor substrate and the catalytic efficiencies for even the best of nitrile substrates (~5 × 103 M−1 s−1) is less than stellar. The nitrilase, Nit11764, shares properties with Nit6803 both derived from a conserved gene cluster in cyanotrophic bacteria. The best substrate for Nit117674 was cinnamonitrile consistent with favorable docking studies. The dinitrile, fumaronitrile, was the second best substrate for which 3-cyanoacrylate was identified by 1NMR. While annotated as an aliphatic nitrilase the physiological role of Nit11764 remains unclear.
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影响因子：
2.8
作者：
Jones, Lauren B.;Ghosh, Pallab;Kunz, Daniel A.
通讯作者：
Kunz, Daniel A.
影响因子：
5.9
作者：
Mulelu, Andani E.;Kirykowicz, Angela M.;Woodward, Jeremy D.
通讯作者：
Woodward, Jeremy D.
DOI：
10.1046/j.1432-1327.2000.00983.x
发表时间：
2000-01-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
Nagasawa, T;Wieser, M;Gekko, K
通讯作者：
Gekko, K
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
5.3
作者：
Cutler, Sean R.;Somerville, Chris R.
通讯作者：
Somerville, Chris R.