Active site residues of cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase from Comamonas testosteroni strain B-356.
Active site residues of cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase from Comamonas testosteroni strain B-356.
复制标题
DOI：
10.1021/bi992232k
复制
发表时间：
2000-05
期刊：
影响因子：
2.9
通讯作者：
M. Vedadi;D. Barriault;M. Sylvestre;J. Powlowski
中科院分区：
文献类型：
--
作者：
M. Vedadi;D. Barriault;M. Sylvestre;J. Powlowski
顺式-2,3-二氢-2,3-二羟基联苯脱氢酶（BphB）来自睾酮单胞菌B-356，是联苯/多氯联苯降解途径的第二个酶。基于相关BphB的晶体结构，三个保守残基Ser142、Tyr155和Lys159被认为与短链醇脱氢酶/还原酶（SDR）家族的其他成员一样具有“催化三联体”的功能。在本研究中，对BphB中每个三联体残基的替换进行了检测。pH为9.0时，相对于野生型酶的周转数为：Y155F， 0.1%；S142A, 1%;K159A占10%。虽然K159A和S142A对顺式-2,3-二氢-2,3-二羟基联苯的Michaelis常数增加了约20倍，但对二核苷酸的K(m)变化相对较小。K159A突变体在pH值为7时脱氢酶活性不高，但在pH值为9时脱氢酶活性达到野生型脱氢酶活性的25%。8. 因此，这三个残基对BphB的活性至关重要，正如晶体结构和与其他SDR家族成员的相似性所表明的那样。此外，BphB对NAD（+）比NADP（+）表现出强烈的偏好，其特异性常数(k(cat)/ k(m)高260倍。有证据表明，BphB对NADP（+）的低效利用可能部分是由于36位存在天冬氨酸残基。
cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB) from Comamonas testosteroni strain B-356 is the second enzyme of the biphenyl/polychlorinated biphenyl degradation pathway. Based on the crystal structure of a related BphB, three conserved residues, Ser142, Tyr155, and Lys159, have been suggested to function as a "catalytic triad" as for other members of the short-chain alcohol dehydrogenase/reductase (SDR) family. In this study, substitution of each triad residue was examined in BphB. At pH 9.0, turnover numbers relative to wild-type enzyme were as follows: Y155F, 0.1%; S142A, 1%; and K159A, 10%. Although the Michaelis constants of K159A and S142A for cis-2,3-dihydro-2,3-dihydroxybiphenyl increased about 20-fold, relatively little change was observed in the K(m) for dinucleotide. The K159A mutant, which showed little dehydrogenase activity at pH 7, was sharply activated by increasing the pH, reaching almost 25% of the activity of the wild-type enzyme at pH 9. 8. These three residues are therefore critical for BphB activity, as suggested by the crystal structure and similarity to other SDR family members. In addition, BphB showed a strong preference for NAD(+) over NADP(+), with a 260-fold higher specificity constant (k(cat)/K(m)). Evidence is presented that the inefficient use of NADP(+) by BphB might partly be due to the presence of an aspartate residue at position 36.