Structure, Function and Regulation of a Second Pyruvate Kinase Isozyme in Pseudomonas aeruginosa.
Structure, Function and Regulation of a Second Pyruvate Kinase Isozyme in Pseudomonas aeruginosa.
复制标题
DOI：
10.3389/fmicb.2021.790742
复制
发表时间：
2021
影响因子：
5.2
通讯作者：
Welch M
中科院分区：
文献类型：
--
作者：
Abdelhamid Y;Wang M;Parkhill SL;Brear P;Chee X;Rahman T;Welch M
铜绿假单胞菌（PA）依靠enterner - doudoroff途径（EDP）进行糖酵解。EDP下半部分的主要酶调节因子是丙酮酸激酶。PA含有编码丙酮酸激酶两种异构体的基因，分别为PykAPA和PykFPA。在其他含有两种丙酮酸激酶同工异构体的生物中（如大肠杆菌），每种同工酶受到不同的调节。PykFPA同工酶的结构、功能和调控已经被详细地描述，因此在这项工作中，我们开始评估PykFPA同工酶的生化和结构特性。我们发现，在尿囊素和尿尿素存在的情况下，pykFPA的表达被诱导。尽管它们的氨基酸序列同源性相对较低，但PykAPA和PykFPA表现出广泛相似的动力学参数，并由一组非常相似的代谢物进行变构调节。然而，PykFPA的x射线晶体结构与PykAPA相比有显著差异。值得注意的是，尽管PykFPA的主要变构调节剂结合位点是空的，但覆盖该位点的“环”呈部分封闭的构象。环侧脯氨酸残基的定点突变产生明显的“锁定”和“锁定”变构激活表型，这取决于突变的残基。PykFPA原聚体间相互作用的分析支持一种模型，即伴随变构激活的构象转变涉及酶的a和B结构域的重新定向以及随后活性位点的关闭。
Pseudomonas aeruginosa (PA) depends on the Entner-Doudoroff pathway (EDP) for glycolysis. The main enzymatic regulator in the lower half of the EDP is pyruvate kinase. PA contains genes that encode two isoforms of pyruvate kinase, denoted PykAPA and PykFPA. In other well-characterized organisms containing two pyruvate kinase isoforms (such as Escherichia coli) each isozyme is differentially regulated. The structure, function and regulation of PykAPA has been previously characterized in detail, so in this work, we set out to assess the biochemical and structural properties of the PykFPA isozyme. We show that pykFPA expression is induced in the presence of the diureide, allantoin. In spite of their relatively low amino acid sequence identity, PykAPA and PykFPA display broadly comparable kinetic parameters, and are allosterically regulated by a very similar set of metabolites. However, the x-ray crystal structure of PykFPA revealed significant differences compared with PykAPA. Notably, although the main allosteric regulator binding-site of PykFPA was empty, the “ring loop” covering the site adopted a partially closed conformation. Site-directed mutation of the proline residues flanking the ring loop yielded apparent “locked on” and “locked off” allosteric activation phenotypes, depending on the residue mutated. Analysis of PykFPA inter-protomer interactions supports a model in which the conformational transition(s) accompanying allosteric activation involve re-orientation of the A and B domains of the enzyme and subsequent closure of the active site.
登录
查看更多内容
影响因子：
14.9
作者：
Goujon M;McWilliam H;Li W;Valentin F;Squizzato S;Paern J;Lopez R
通讯作者：
Lopez R
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH
影响因子：
5.2
作者：
Kumar, Sheemal Shanista;Penesyan, Anahit;Paulsen, Ian T.
通讯作者：
Paulsen, Ian T.
影响因子：
4.8
作者：
Nikel, Pablo I.;Chavarria, Max;de Lorenzo, Victor
通讯作者：
de Lorenzo, Victor
影响因子：
4.8
作者：
Buecker, Rene;Heroven, Ann Kathrin;Wittmann, Christoph
通讯作者：
Wittmann, Christoph