Phenylalanine 664 of dipeptidyl peptidase (DPP) 7 and Phenylalanine 671 of DPP11 mediate preference for P2-position hydrophobic residues of a substrate.
Phenylalanine 664 of dipeptidyl peptidase (DPP) 7 and Phenylalanine 671 of DPP11 mediate preference for P2-position hydrophobic residues of a substrate.
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DOI：
10.1016/j.fob.2013.03.004
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发表时间：
2013
期刊：
影响因子：
2.6
通讯作者：
Nemoto TK
中科院分区：
文献类型：
--
作者：
Rouf SM;Ohara-Nemoto Y;Ono T;Shimoyama Y;Kimura S;Nemoto TK
牙龈卟啉单胞菌是一种革兰氏阴性厌氧菌，可引起慢性牙周炎，二肽基肽酶(DPP)对其能量代谢起着至关重要的作用。在该细菌中表达了3个DPP，分别是Pro的DPPIV、疏水残基的DPP7和P1位的Asp/Glu的DPP11。与DPP7类似，DPP11属于S46蛋白水解酶家族，序列同源性为38.7%。虽然DPP11优先于P2位的疏水残基，但已有报道DPP7在P2位没有偏好。在本研究中，我们定义了DPP7对P2底物的详细偏好，以及导致这种特异性的氨基酸残基。DPP7对P_1位携带疏水残基的甲基-亮氨酸-二肽-4-甲基香豆基-7-酰胺的水解率最高，kcat/Km为10.62±2.51μM−1 S−1，而它却意外地与亲水性残基(谷氨酰胺、天冬氨酸)或带电残基(天冬氨酸、精氨酸)切割底物。用21二肽MCA检测表明，DPP7-肽酶活性依赖于P2-和P1-位残基的疏水性，因此它与P1-和P2-氨基酸残基的疏水性指数之和相关性最好。P1和P2位的疏水性分别通过增加kcat和降低Km值确保了有效的酶催化。将S46 DPP7/DPP11家族中保守的疏水残基替换为ALA，发现DPP7的Phe664和DPP11的Phe671主要提供疏水P2偏好。模拟研究表明，DPP7的Phe664和Gly666以及DPP11的Phe671和Arg673分别与P2-和P1-位残基相关，位于催化Ser648/Ser655附近。本研究结果扩展了DPP7的底物谱系，确保了寡肽在无糖降解细菌中的高效降解。二肽基肽酶是牙龈卟啉单胞菌能量代谢的关键酶。牙龈假单胞菌DPP7偏好P2位疏水氨基酸残基。Met-Leu-Mca的DPP7kcat/kM为10.62±2.51μM−1 S−1。疏水的P1残基增强kcat，疏水的P2残基降低Km。DPP7的Phe664和DPP11的Phe671主要表现为疏水性的P2偏好。
Dipeptidyl peptidases (DPPs) are crucial for the energy metabolism in Porphyromonas gingivalis, a Gram-negative proteolytic and asaccharolytic anaerobic rod causing chronic periodontitis. Three DPPs, DPPIV specific for Pro, DPP7 for hydrophobic residues and DPP11 for Asp/Glu at the P1 position, are expressed in the bacterium. Like DPP7, DPP11 belongs to the S46 protease family, and they share 38.7% sequence identity. Although DPP11 is preferential for hydrophobic residues at the P2 position, it has been reported that DPP7 has no preference at the P2 position. In the present study, we defined the detailed P2 substrate preference of DPP7 and the amino acid residue responsible for the specificity. DPP7 most efficiently hydrolyzed Met-Leu-dipeptidyl-4-methylcoumaryl-7-amide (MCA) carrying hydrophobic residues at the P1 position with kcat/Km of 10.62 ± 2.51 μM−1 s−1, while it unexpectedly cleaved substrates with hydrophilic (Gln, Asn) or charged (Asp, Arg) residues. Examination with 21 dipeptidyl MCA demonstrated that DPP7-peptidase activity was dependent on hydrophobicity of the P2- as well as P1-position residue, thus it correlated best with the sum of the hydrophobicity index of P1- and P2-amino acid residues. Hydrophobicity of the P1 and P2 positions ensured efficient enzyme catalysis by increasing kcat and lowering Km values, respectively. Substitution of hydrophobic residues conserved in the S46 DPP7/DPP11 family to Ala revealed that Phe664 of DPP7 and Phe671 of DPP11 primarily afforded hydrophobic P2 preference. A modeling study suggested that Phe664 and Gly666 of DPP7 and Phe671 and Arg673 of DPP11 being associated with the P2- and P1-position residues, respectively, are located adjacent to the catalytic Ser648/Ser655. The present results expand the substrate repertoire of DPP7, which ensures efficient degradation of oligopeptides in asaccharolytic bacteria. Dipeptidyl peptidases are crucial for energy metabolism in Porphyromonas gingivalis. P. gingivalis DPP7 prefers a hydrophobic amino acid residue at P2 position. kcat/Km of DPP7 for Met-Leu-MCA is 10.62 ± 2.51 μM−1 s−1. A hydrophobic P1 residue enhances kcat and a hydrophobic P2 residue lowers Km. Phe664 of DPP7 and Phe671 of DPP11 primarily afford hydrophobic P2 preference.
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影响因子：
2.1
作者：
Monera, Oscar D.;Sereda, Terrance J.;Hodges, Robert S.
通讯作者：
Hodges, Robert S.
影响因子：
3.7
作者：
Bezerra, Gustavo Arruda;Dobrovetsky, Elena;Dhe-Paganon, Sirano
通讯作者：
Dhe-Paganon, Sirano
影响因子：
3
作者：
Kshirsagar, Abhijit V.;Offenbacher, Steven;Beck, James D.
通讯作者：
Beck, James D.
影响因子：
3.8
作者：
Ohara-Nemoto, Y;Ikeda, Y;Kimura, S
通讯作者：
Kimura, S
影响因子：
56.9
作者：
HOLT, SC;EBERSOLE, J;KORNMAN, KS
通讯作者：
KORNMAN, KS