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Atomic resolution studies of haloalkane dehalogenases DhaA04, DhaA14 and DhaA15 with engineered access tunnels.

Stsiapanava A, Dohnalek J, Gavira JA, Kuty M, Koudelakova T, Damborsky J, Kuta Smatanova I
Acta Crystallogr D Biol Crystallogr 66 962-9 (2010)
Related entries: 3fwh, 3g9x

Cited: 8 times
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Abstract

The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 is a bacterial enzyme that shows catalytic activity for the hydrolytic degradation of the highly toxic industrial pollutant 1,2,3-trichloropropane (TCP). Mutagenesis focused on the access tunnels of DhaA produced protein variants with significantly improved activity towards TCP. Three mutants of DhaA named DhaA04 (C176Y), DhaA14 (I135F) and DhaA15 (C176Y + I135F) were constructed in order to study the functional relevance of the tunnels connecting the buried active site of the protein with the surrounding solvent. All three protein variants were crystallized using the sitting-drop vapour-diffusion technique. The crystals of DhaA04 belonged to the orthorhombic space group P2(1)2(1)2(1), while the crystals of DhaA14 and DhaA15 had triclinic symmetry in space group P1. The crystal structures of DhaA04, DhaA14 and DhaA15 with ligands present in the active site were solved and refined using diffraction data to 1.23, 0.95 and 1.22 A, resolution, respectively. Structural comparisons of the wild type and the three mutants suggest that the tunnels play a key role in the processes of ligand exchange between the buried active site and the surrounding solvent.

Articles - 3fbw mentioned but not cited (4)

  1. Structure of the Saccharomyces cerevisiae Cet1-Ceg1 mRNA capping apparatus. Gu M, Rajashankar KR, Lima CD. Structure 18 216-227 (2010)
  2. Crystallographic analysis of new psychrophilic haloalkane dehalogenases: DpcA from Psychrobacter cryohalolentis K5 and DmxA from Marinobacter sp. ELB17. Tratsiak K, Degtjarik O, Drienovska I, Chrast L, Rezacova P, Kuty M, Chaloupkova R, Damborsky J, Kuta Smatanova I. Acta Crystallogr Sect F Struct Biol Cryst Commun 69 683-688 (2013)
  3. Crystallization and crystallographic analysis of the Rhodococcus rhodochrous NCIMB 13064 DhaA mutant DhaA31 and its complex with 1,2,3-trichloropropane. Lahoda M, Chaloupkova R, Stsiapanava A, Damborsky J, Kuta Smatanova I. Acta Crystallogr Sect F Struct Biol Cryst Commun 67 397-400 (2011)
  4. Crystallization and preliminary X-ray diffraction analysis of the wild-type haloalkane dehalogenase DhaA and its variant DhaA13 complexed with different ligands. Stsiapanava A, Chaloupkova R, Fortova A, Brynda J, Weiss MS, Damborsky J, Smatanova IK. Acta Crystallogr Sect F Struct Biol Cryst Commun 67 253-257 (2011)


Articles citing this publication (4)

  1. Directed evolution strategies for enantiocomplementary haloalkane dehalogenases: from chemical waste to enantiopure building blocks. van Leeuwen JG, Wijma HJ, Floor RJ, van der Laan JM, Janssen DB. Chembiochem 13 137-148 (2012)
  2. Structure and activity of DmmA, a marine haloalkane dehalogenase. Gehret JJ, Gu L, Geders TW, Brown WC, Gerwick L, Gerwick WH, Sherman DH, Smith JL. Protein Sci 21 239-248 (2012)
  3. In silico design of potentially functional artificial metallo-haloalkane dehalogenase containing catalytic zinc. Ang TF, Salleh AB, Normi YM, Leow TC. 3 Biotech 8 314 (2018)
  4. Multimeric structure of a subfamily III haloalkane dehalogenase-like enzyme solved by combination of cryo-EM and x-ray crystallography. Chmelova K, Gao T, Polak M, Schenkmayerova A, Croll TI, Shaikh TR, Skarupova J, Chaloupkova R, Diederichs K, Read RJ, Damborsky J, Novacek J, Marek M. Protein Sci 32 e4751 (2023)


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