Title: Structural Basis of the Enhanced Pollutant-Degrading Capabilities of an Engineered Biphenyl Dioxygenase

Abstract

Biphenyl dioxygenase, the first enzyme of the biphenyl catabolic pathway, is a major determinant of which polychlorinated biphenyl (PCB) congeners are metabolized by a given bacterial strain. Ongoing efforts aim to engineer BphAE, the oxygenase component of the enzyme, to efficiently transform a wider range of congeners. BphAE_II9, a variant of BphAE_LB400 in which a seven-residue segment, ³³⁵TFNNIRI³⁴¹, has been replaced by the corresponding segment of BphAE_B356, ³³³GINTIRT³³⁹, transforms a broader range of PCB congeners than does either BphAE_LB400 or BphAE_B356, including 2,6-dichlorobiphenyl, 3,3'-dichlorobiphenyl, 4,4'-dichlorobiphenyl, and 2,3,4'-trichlorobiphenyl. To understand the structural basis of the enhanced activity of BphAE_II9, we have determined the three-dimensional structure of this variant in substrate-free and biphenyl-bound forms. Structural comparison with BphAE_LB400 reveals a flexible active-site mouth and a relaxed substrate binding pocket in BphAE_II9 that allow it to bind different congeners and which could be responsible for the enzyme's altered specificity. Biochemical experiments revealed that BphAE_II9 transformed 2,3,4'-trichlorobiphenyl and 2,2',5,5'-tetrachlorobiphenyl more efficiently than did BphAE_LB400 and BphAE_B356. BphAE_II9 also transformed the insecticide dichlorodiphenyltrichloroethane (DDT) more efficiently than did either parental enzyme (apparent k_cat/K_m of 2.2 ± 0.5 mM^–1 s^–1, versus 0.9 ± 0.5 mM^–1 s^–1 for BphAE_B356). Studies of docking of the enzymes with thesemore » three substrates provide insight into the structural basis of the different substrate selectivities and regiospecificities of the enzymes. Biphenyl dioxygenase is the first enzyme of the biphenyl degradation pathway that is involved in the degradation of polychlorinated biphenyls. Attempts have been made to identify the residues that influence the enzyme activity for the range of substrates among various species. In this study, we have done a structural study of one variant of this enzyme that was produced by family shuffling of genes from two different species. Comparison of the structure of this variant with those of the parent enzymes provided an important insight into the molecular basis for the broader substrate preference of this enzyme. Here, the structural and functional details gained in this study can be utilized to further engineer desired enzymatic activity, producing more potent enzymes.« less

Authors:

Dhindwal, Sonali ^[1]; Gomez-Gil, Leticia ^[2]; Neau, David B. ^[3]; Pham, Thi Thanh My ^[4]; Sylvestre, Michel ^[4]; Eltis, Lindsay D. ^[2]; Bolin, Jeffrey T. ^[5]; Kumar, Pravindra ^[1]

---

+ Show Author Affiliations

1. Indian Inst. of Technology, Uttarakhand (India)
2. Univ. of British Columbia, Vancouver, BC (Canada)
3. Argonne National Lab. (ANL), Argonne, IL (United States)
4. Inst. National de Recherche Scientifique (INRS-Inst. Armand-Frappier), Laval, QC (Canada)
5. Purdue Univ., West Lafayette, IN (United States)

Publication Date:

Mon Mar 07 00:00:00 EST 2016

Research Org.:

Argonne National Lab. (ANL), Argonne, IL (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities Division; National Inst. of General Medical Sciences (NIGMS); National Inst. of Health

OSTI Identifier:

1254512

Grant/Contract Number:  

AC02-06CH11357; R24GM111072; GM103403

Resource Type:

Accepted Manuscript

Journal Name:

Journal of Bacteriology

Additional Journal Information:

Journal Volume: 198; Journal Issue: 10; Journal ID: ISSN 0021-9193

Publisher:

American Society for Microbiology

Country of Publication:

United States

Language:

ENGLISH

Subject:

59 BASIC BIOLOGICAL SCIENCES