Active-site engineering of biphenyl dioxygenase: effect of substituted amino acids on substrate specificity and regiospecificity
Active-site engineering of biphenyl dioxygenase: effect of substituted amino acids on substrate specificity and regiospecificity
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DOI：
10.1007/s00253-005-0135-2
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发表时间：
2006-06
影响因子：
5
通讯作者：
H. Suenaga;M. Goto;K. Furukawa
中科院分区：
文献类型：
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作者：
H. Suenaga;M. Goto;K. Furukawa
联苯双加氧酶（Bph Dox）催化联苯代谢中的初始双加氧步骤。Bph Dox的大亚基（BphA 1）在确定联苯相关化合物（包括多氯联苯（PCBs））的底物特异性方面起着至关重要的作用。在此之前，Asn在Thr-376附近的活性位点铁在BphA 1 ofPseudomonaspseudoalcaligenesKF 707的取代扩大了氧化范围，并改变了多氯联苯的BphDox的区域特异性。在本研究中，我们用Gly、Ser、Gln、Tyr、瓦尔、Phe、Asp和Lys取代Thr-376，并在大肠杆菌中表达这些酶。Thr 376 Asn、Thr 376 Val、Thr 376 Phe和Thr 376 Lys的Bph Dox突变体显示出对二苯并呋喃的新的降解活性，二苯并呋喃是KF 707 Bph Dox的不良底物。所有活性Bph Dox突变体对2，2 ′-二氯联苯和2，5，4 ′-三氯联苯的区域特异性都发生了改变。Thr 376 Gly、Thr 376 Val、Thr 376 Phe和Thr 376 Asp Bph Dox突变体在2，2 ′-二氯联苯的2，3位引入分子氧，形成2-氯-2 ′，3 ′-二羟基联苯并伴随脱氯。Bph Dox突变体Thr 376 Gly、Thr 376 Ser、Thr 376 Asp和Thr 376 Lys通过2′，3 ′-和3，4-双加氧活性攻击2，5，4 ′-三氯联苯。特别地，Thr 376 Phe Bph Dox突变体对所有测试的化合物表现出增强和扩大的降解活性。进一步的定点突变使KF 707 Bph Dox的氧化特性变为Burkholderia xenovorans LB 400的Bph Dox，通过在活性位点附近的两个氨基酸Ile 335 Phe和Thr 376 Asn的取代。
Biphenyl dioxygenase (Bph Dox) catalyzes the initial dioxygenation step in the metabolism of biphenyl. The large subunit (BphA1) of Bph Dox plays a crucial role in the determination of the substrate specificity of biphenyl-related compounds including polychlorinated biphenyls (PCBs). Previously, the substitution of Asn at Thr-376 near the active-site iron in the BphA1 ofPseudomonas pseudoalcaligenesKF707 expanded the oxidation range and altered the regiospecificity of Bph Dox for PCBs. In this study, we replaced Thr-376 with Gly, Ser, Gln, Tyr, Val, Phe, Asp, and Lys and expressed these enzymes inEscherichia coli. Bph Dox mutants of Thr376Asn, Thr376Val, Thr376Phe, and Thr376Lys showed novel degradation activity for dibenzofuran, which is a poor substrate for KF707 Bph Dox. All active Bph Dox mutants showed altered regiospecificity with 2,2′-dichlorobiphenyl and 2,5,4′-trichlorobiphenyl. The Thr376Gly, Thr376Val, Thr376Phe, and Thr376Asp Bph Dox mutants introduced molecular oxygen at the 2,3 position of 2,2′-dichlorobiphenyl, forming 2-chloro-2′,3′-dihydroxybiphenyl with concomitant dechlorination. The Bph Dox mutants of Thr376Gly, Thr376Ser, Thr376Asp, and Thr376Lys attacked 2,5,4′-trichlorobiphenyl via both 2′,3′- and 3,4-dioxygenation activities. In particular, the Thr376Phe Bph Dox mutant exhibited enhanced and expanded degradation activities toward all of the compounds tested. Further site-directed mutation was induced to change the oxidizing character of KF707 Bph Dox to that of the Bph Dox ofBurkholderia xenovoransLB400 by the substitution of two amino acids, Ile335Phe and Thr376Asn, near the active-site.