D7A Mutagenesis and In Silico Analysis on Haloacid Dehalogenase Gene from Pseudomonas aeruginosa ITB1 in pET/ paed‑d Recombinant Clone

Abstract

Organohalogens, including haloalkanoic acids, are organic compounds synthesized in large quantities for various purposes in the agriculture, pharmaceutics, textile, and chemical industries. Unfortunately, organohalogen waste is persistent in the environment and toxic to many organisms, including humans. In this respect, bioremediation is considered the safest and most ecofriendly technique to reduce this class of xenobiotics. Pseudomonas aeruginosa ITB1 local strain has been identified to produce haloacid dehalogenase, a hydrolytic enzyme that catalyzes the breakage of carbon-halogen covalent bonds in haloalkanoic acids. Our previous studies have successfully cloned the haloacid dehalogenase gene from this bacterium into pGEM-T Easy in E. coli TOP10, sequenced, and then subcloned into pET-30a-(+) for expression in E. coli BL21-(DE3). The gene was named paed-d, and its enzyme was called Paed-d. In silico analyses suggested that the D7 residue plays an essential role in catalysis. This study was aimed to evaluate the hypothesis by performing mutagenesis of D7A on the cloned gene using a PCR approach. The obtained amplicon was used to transform E. coli TOP10 to confirm the mutation (paed-d D7A) by sequencing and to transform E. coli BL21-(DE3) for expression analyses. Wet lab analysis demonstrated that the Paed-d D7A mutant enzyme was revealed to have 2.5-fold lowered specific activity compared to the wild type. This result proved the hypothesis that D7 in haloacid dehalogenase plays an important role in the catalysis process.

1

Secondary structure of wild-type Paed-d and the mutant Paed-d D7A compared to the 3umc.1.A pdb model. The arrow box represents the helix structure, the cylindrical box represents the sheet structure, and the amino acid without the arrow box and the cylindrical box represents the coil structure.

2

Superimposed structure of the wild-type Paed-d and mutant Paed-d D7A proteins by VMD. Red structure indicates aspartate at 7th position in wild-type Paed-d, and blue structure indicates alanine at 7th position in the mutant Paed-d protein.

3

SAS prediction of the secondary structure of wild type (a) and D7A Paed-d (b). The α-helices are represented by red helix, β-sheets are represented by red arrow, and coils are represented by red line. Active site residues are marked as green triangles, and catalytic residues are marked as red boxes.

4

Reaction mechanisms of MCA dehalogenation catalyzed by Paed-d.

5

Schematic interaction of the MCA ligand with amino acid residues in the active sites of wild-type Paed-d (a) and D7A Paed-d mutant (b).

6

Electropherogram for paed-d gene confirmation by restriction analysis (a) and re-PCR (b). M = molecular DNA marker; (1) uncut pET-paed-d plasmid; (2) double-digested pET-paed-d plasmid using EcoRI and HindIII; (3) amplicon from re-PCR using Fr1 and Rv1 primers.

7

Part of the paed-d gene sequence in the mutated area (a) and position of the mutated primers to create D7A mutation (b) by changing 5′-GAT-3′ and 3′-CTA-5′ into 5′-GCT-3′ and 3′-CGA-5′, respectively.

8

Schematic diagram of site-directed mutagenesis.

9

Electropherogram of amplicon obtained from mutagenesis by long-range PCR. M = molecular DNA marker; (1) pET-30a­(+) and (2) pET-paed-d D7A.

10

Nucleotide and amino acid sequences of wild-type and mutations of paed-d.

11

Expression of haloacid dehalogenase in E. coli BL21­(DE) cells. (M, protein marker PM2700; 1, uninduced E. coli BL21­(DE3); 2, uninduced wild-type Paed-d; 3, induced wild-type Paed-d; 4, uninduced D7A Paed-d mutant; 5, induced D7A Paed-d mutant).