Seah Stephen Y K, Ke Jiyuan, Denis Geoffroy, Horsman Geoff P, Fortin Pascal D, Whiting Cheryl J, Eltis Lindsay D
Department of Biochemistry, Université Laval, Canada.
J Bacteriol. 2007 Jun;189(11):4038-45. doi: 10.1128/JB.01950-06. Epub 2007 Apr 6.
Sphingomonas wittichii RW1 degrades chlorinated dibenzofurans and dibenzo-p-dioxins via meta cleavage. We used inverse PCR to amplify dxnB2, a gene encoding one of three meta-cleavage product (MCP) hydrolases identified in the organism that are homologues of BphD involved in biphenyl catabolism. Purified DxnB2 catalyzed the hydrolysis of 8-OH 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) approximately six times faster than for HOPDA at saturating substrate concentrations. Moreover, the specificity of DxnB2 for HOPDA (k(cat)/K(m) = 1.2 x 10(7) M(-1) s(-1)) was about half that of the BphDs of Burkholderia xenovorans LB400 and Rhodococcus globerulus P6, two potent polychlorinated biphenyl (PCB)-degrading strains. Interestingly, DxnB2 transformed 3-Cl and 4-OH HOPDAs, compounds that inhibit the BphDs and limit PCB degradation. DxnB2 had a higher specificity for 9-Cl HOPDA than for HOPDA but a lower specificity for 8-Cl HOPDA (k(cat)/K(m) = 1.7 x 10(6) M(-1) s(-1)), the chlorinated analog of 8-OH HOPDA produced during dibenzofuran catabolism. Phylogenetic analyses based on structure-guided sequence alignment revealed that DxnB2 belongs to a previously unrecognized class of MCP hydrolases, evolutionarily divergent from the BphDs although the physiological substrates of both enzyme types are HOPDAs. However, both classes of enzymes have mainly small hydrophobic residues lining the subsite that binds the C-6 phenyl of HOPDA, in contrast to the bulky hydrophobic residues (Phe106, Phe135, Trp150, and Phe197) found in the class II enzymes that prefer substrates possessing a C-6 alkyl. Thr196 and/or Asn203 appears to be an important determinant of specificity for DxnB2, potentially forming hydrogen bonds with the 8-OH substituent. This study demonstrates that the substrate specificities of evolutionarily divergent hydrolases may be useful for degrading mixtures of pollutants, such as PCBs.
维氏鞘氨醇单胞菌RW1通过间位裂解途径降解氯化二苯并呋喃和二苯并 - p - 二恶英。我们利用反向PCR扩增了dxnB2基因，该基因编码在该生物体中鉴定出的三种间位裂解产物（MCP）水解酶之一，它们是参与联苯分解代谢的BphD的同源物。在底物浓度饱和时，纯化的DxnB2催化8 - OH 2 - 羟基 - 6 - 氧代 - 6 - 苯基己 - 2,4 - 二烯酸（HOPDA）的水解速度比催化HOPDA快约六倍。此外，DxnB2对HOPDA的特异性（k(cat)/K(m)=1.2×10(7) M(-1) s(-1)）约为伯克霍尔德氏菌LB400和球形红球菌P6的BphD的一半，这两种菌株是高效的多氯联苯（PCB）降解菌株。有趣的是，DxnB2能转化3 - Cl和4 - OH HOPDAs，这两种化合物会抑制BphD并限制PCB的降解。DxnB2对9 - Cl HOPDA的特异性高于对HOPDA的特异性，但对8 - Cl HOPDA的特异性较低（k(cat)/K(m)=1.7×10(6) M(-1) s(-1)），8 - Cl HOPDA是二苯并呋喃分解代谢过程中产生的8 - OH HOPDA的氯化类似物。基于结构引导序列比对的系统发育分析表明，DxnB2属于一类先前未被识别的MCP水解酶，尽管这两种酶的生理底物都是HOPDAs，但它在进化上与BphD不同。然而，与偏好具有C - 6烷基底物的II类酶中发现的大的疏水残基（Phe106、Phe135、Trp150和Phe197）相比，这两类酶在结合HOPDA的C - 6苯基的亚位点主要都有小的疏水残基。Thr196和/或Asn203似乎是DxnB2特异性的重要决定因素，可能与8 - OH取代基形成氢键。这项研究表明，进化上不同的水解酶的底物特异性可能有助于降解污染物混合物，如多氯联苯。
