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.

Condition assessment of a 63-year old reinforced concrete structure exposed to the North Sea

This paper describes the condition of a reinforced concrete balustrade consisting of some 1000 individual beam elements all exposed similarly to the hostile marine environment of the North Sea at Arbroath, Scotland since 1943. A comparison is made of the condition of the original construction with the condition of repairs carried out in 1968 and in 1993. It is shown that the 1943 construction shows very little corrosion-induced cracking and little rust staining even though it does not appear to be of high construction quality. Only a very low percentage of the balustrade beams have been replaced. In contrast the beam installed in 1968 and later in 1993 show very considerable and large concrete cracks directly attributable to the corrosion of the longitudinal reinforcement, even though the concrete is of a higher quality and density. A detailed condition survey and statistics of crack sizes are presented in the paper. It is found that the higher corrosion resistance of the 1943 concrete is generally consistent with the concrete electrical resistivity measurements but the degree of corrosion of the reinforcing bars is inconsistent with chloride penetration measurements. The results are compared with the very few observations available in the literature for ageing concrete structures in marine environments. The results cast doubt on the conventional wisdom that chloride content at the reinforcement correlates well with reinforcement corrosion.