Biomedical subjects

Mariël G Pikkemaat

Publications and source records attributed to Mariël G Pikkemaat.

3 recordsLinked to original sources

PubMed2003

Steady-state and pre-steady-state kinetic analysis of halopropane conversion by a rhodococcus haloalkane dehalogenase.

Haloalkane dehalogenase from Rhodococcus rhodochrous NCIMB 13064 (DhaA) catalyzes the hydrolysis of carbon-halogen bonds in a wide range of haloalkanes. We examined the steady-state and pre-steady-state kinetics of halopropane conversion by DhaA to illuminate mechanistic details of the dehalogenation pathway. Steady-state kinetic analysis of DhaA with a range of halopropanes showed that bromopropanes had higher k(cat) and lower K(M) values than the chlorinated analogues. The kinetic mechanism of dehalogenation was further studied using rapid-quench-flow analysis of 1,3-dibromopropane conversion. This provided a direct measurement of the chemical steps in the reaction mechanism, i.e., cleavage of the carbon-halogen bond and hydrolysis of the covalent alkyl-enzyme intermediate. The results lead to a minimal mechanism consisting of four main steps. The occurrence of a pre-steady-state burst, both for bromide and 3-bromo-1-propanol, suggests that product release is rate-limiting under steady-state conditions. Combining pre-steady-state burst and single-turnover experiments indicated that the rate of carbon-bromine bond cleavage was indeed more than 100-fold higher than the steady-state k(cat). Product release occurred with a rate constant of 3.9 s(-1), a value close to the experimental k(cat) of 2.7 s(-1). Comparing the kinetic mechanism of DhaA with that of the corresponding enzyme from Xanthobacter autotrophicus GJ10 (DhlA) shows that the overall mechanisms are similar. However, whereas in DhlA the rate of halide release represents the slowest step in the catalytic cycle, our results suggest that in DhaA the release of 3-bromo-1-propanol is the slowest step during 1,3-dibromopropane conversion.

Binding Sites↗

PubMed2002

Generating segmental mutations in haloalkane dehalogenase: a novel part in the directed evolution toolbox.

Directed evolution techniques allow us to genuinely mimic molecular evolution in vitro. To enhance this imitation of natural evolutionary processes on a laboratory scale in even more detail, we developed an in vitro method for the generation of random deletions and repeats. The pairwise fusion of two fragments of the same gene that are truncated by exonuclease BAL-31 either at the 3' or 5' side results in a deletion or a repeat at the fusion point. Although in principle the method randomly covers the whole gene, it can also be limited to a predefined area in the sequence by controlling the level of the initial truncation. To test the procedure and to illustrate its potential, we used haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA) as a model enzyme, since the adaptation of this enzyme towards new substrates is known to occur via the generation of this type of mutation. The results show that the mutagenesis method presented here is an effective tool for accessing formerly unexplorable sequence space and can contribute to the success of future directed evolution experiments.

Amino Acid Sequence↗

PubMed2002

Molecular dynamics simulations as a tool for improving protein stability.

Haloalkane dehalogenase (DhlA) was used as a model protein to explore the possibility to use molecular dynamics (MD) simulations as a tool to identify flexible regions in proteins that can serve as a target for stability enhancement by introduction of a disulfide bond. DhlA consists of two domains: an alpha/beta-hydrolase fold main domain and a cap domain composed of five alpha-helices. MD simulations of DhlA showed high mobility in a helix-loop-helix region in the cap domain, involving residues 184-211. A disulfide cross-link was engineered between residue 201 of this flexible region and residue 16 of the main domain. The mutant enzyme showed substantial changes in both thermal and urea denaturation. The oxidized form of the mutant enzyme showed an increase of the apparent transition temperature from 47.5 to 52.5 degrees C, whereas the T(m,app) of the reduced mutant decreased by more than 8 degrees C compared to the wild-type enzyme. Urea denaturation results showed a similar trend. Measurement of the kinetic stability showed that the introduction of the disulfide bond caused a decrease in activation free energy of unfolding of 0.43 kcal mol(-1) compared to the wild-type enzyme and also indicated that the helix-loop-helix region was involved early in the unfolding process. The results show that MD simulations are capable of identifying mobile protein domains that can successfully be used as a target for stability enhancement by the introduction of a disulfide cross-link.

Computer Simulation↗