Structure and denaturation of 4-chlorobenzoyl coenzyme A dehalogenase from Arthrobacter sp. strain TM-1

The secondary structure of the trimeric protein 4-chlorobenzoyl coenzyme A dehalogenase from Arthrobacter sp. strain TM-1, the second of three enzymes involved in the dechlorination of 4-chlorobenzoate to form 4-hydroxybenzoate, has been examined. E(mM) for the enzyme was 12.59. Analysis by circular dichroism spectrometry in the far uv indicated that 4-chlorobenzoyl coenzyme A dehalogenase was composed mostly of alpha-helix (56%) with lesser amounts of random coil (21%), beta-turn (13%) and beta-sheet (9%). These data are in close agreement with a computational prediction of secondary structure from the primary amino acid sequence, which indicated 55.8% alpha-helix, 33.7% random coil and 10.5% beta-sheet; the enzyme is, therefore, similar to the 4-chlorobenzoyl coenzyme A dehalogenase from Pseudomonas sp. CBS-3. The three-dimensional structure, including that of the presumed active site, predicted by computational analysis, is also closely similar to that of the Pseudomonas dehalogenase. Study of the stability and physicochemical properties revealed that at room temperature, the enzyme was stable for 24 h but was completely inactivated by heating to 60 degrees C for 5 min; thereafter by cooling at 1 degrees C min(-1) to 45 degrees C, 20.6% of the activity could be recovered. Mildly acidic (pH 5.2) or alkaline (pH 10.1) conditions caused complete inactivation, but activity was fully recovered on returning the enzyme to pH 7.4. Circular dichroism studies also indicated that secondary structure was little altered by heating to 60 degrees C, or by changing the pH from 7.4 to 6.0 or 9.2. Complete, irreversible destruction of, and maximal decrease in the fluorescence yield of the protein at 330-350 nm were brought about by 4.5 M urea or 1.1 M guanidinium chloride. Evidence was obtained to support the hypothetical three-dimensional model, that residues W140 and W167 are buried in a non-polar environment, whereas W182 appears at or close to the surface of the protein. At least one of the enzymes of the dehalogenase system (the combined 4-chlorobenzoate:CoA ligase, the dehalogenase and 4-hydroxybenzoyl coenzyme A thioesterase) appears to be capable of association with the cell membrane.

Prediction of reinforcement corrosion in concrete and its effects on concrete cracking and strength reduction

Based on extensive research on reinforcing steel corrosion in concrete in the past decades, it is now possible to estimate the effect of the progression of reinforcement corrosion in concrete infrastructure on its structural performance. There are still areas of considerable uncertainty in the models and in the data available, however This paper uses a recently developed model for reinforcement corrosion in concrete to improve the estimation process and to indicate the practical implications. In particular stochastic models are used to estimate the time likely to elapse for each phase of the whole corrosion process: initiation, corrosion-induced concrete cracking, and structural strength reduction. It was found that, for practical flexural structures subject to chloride attacks, corrosion initiation may start quite early in their service life. It was also found that, once the structure is considered to be unserviceable due to corrosion-induced cracking, there is considerable remaining service life before the structure can be considered to have become unsafe. The procedure proposed in the paper has the potential to serve as a rational tool for practitioners, operators, and asset managers to make decisions about the optimal timing of repairs, strengthening, and/or rehabilitation of corrosion-affected concrete infrastructure. Timely intervention has the potential to prolong the service life of infrastructure.