In off-line 2D-HPLC a continuous salt gradient is applied in the first separation dimension. This increases the number of identified proteins from complex samples significantly due to higher chromatographic resolution compared to stepwise elution. Achievement of optimal resolution requires the optimization of the two separation dimensions. The influence of LC elution gradients in the first and second dimensions, of analysis time, of stationary-phase material, and of column dimensions was systematically investigated in order to obtain information on the overall peak capacity of the separation system. Provided data indicate that for complex samples such as an E. coli cell extract, a shallow LC SCX gradient with a high number of collected fractions significantly increases the overall peak capacity while for lower complexity samples short gradients with few fractions were sufficient to obtain a maximum of identified peptides. In addition, column dimensions and materials exhibited a strong effect on the overall efficiency of the 2D HPLC separation. The outcome of these experiments could hence serve as a guideline for investigators to adapt their method for the separation of their specific proteome sample to achieve a maximum of peptide sequence information by 2D LC MS/MS analysis.

Today, 2D online or offline liquid chromatography/mass spectrometry is state of the art for the identification of proteins from complex proteome samples in many laboratories. Both 2D liquid chromatography methods use two orthogonal liquid chromatography separation techniques. The most commonly used techniques are strong cation exchange chromatography for the first dimension and reversed phase separation for the second dimension. In order to improve sensitivity the reversed phase separation is usually performed in the nanoflow scale and mass spectrometry is used as the final detection method. The high-performance liquid chromatography techniques complement the 2D-gel techniques supporting their weaknesses. This is especially true for the gel separation of hydrophobic membrane proteins, which play an important role in living cells as well as being important targets for future pharmaceutical drugs.

Muconate cycloisomerase (EC 5.5.1.1) and chloromuconate cycloisomerase (EC 5.5.1.7) were purified from extracts of Rhodococcus erythropolis 1CP cells grown with benzoate or 4-chlorophenol, respectively. Both enzymes discriminated between the two possible directions of 2-chloro-cis,cis-muconate cycloisomerization and converted this substrate to 5-chloromuconolactone as the only product. In contrast to chloromuconate cycloisomerases of gram-negative bacteria, the corresponding R. erythropolis enzyme is unable to catalyze elimination of chloride from (؉)-5-chloromuconolactone. Moreover, in being unable to convert (؉)-2-chloromuconolactone, the two cycloisomerases of R. erythropolis 1CP differ significantly from the known muconate and chloromuconate cycloisomerases of gram-negative strains. The catalytic properties indicate that efficient cycloisomerization of 3-chloro-and 2,4-dichloro-cis,cis-muconate might have evolved independently among gram-positive and gram-negative bacteria.Many chloroaromatic compounds are degraded by bacteria via chlorocatechols as central intermediates. Further catabolism involves ortho-cleavage of the chlorocatechols to chlorosubstituted cis,cis-muconates as well as cycloisomerization and dechlorination of the latter, yielding dienelactones (4-carboxymethylenebut-2-en-4-olides) which are hydrolyzed and finally funneled into the ubiquitous 3-oxoadipate pathway (Fig. 1). Despite much of the early work having been done with an Arthrobacter sp. (3,8,31) and despite many reports of transformation of halogenated aromatic compounds by gram-positive bacteria (recently reviewed in reference 35), the enzymology and genetics of the modified ortho-cleavage pathway outlined above have been elucidated almost exclusively in gram-negative strains. They usually contain separate sets of enzymes for catechol and chlorocatechol conversion, which differ from each other with respect to the affinities and turnover rates for chlorosubstituted catechols or the metabolites formed from them (6,21,25).The gram-positive strain Rhodococcus erythropolis 1CP has previously been reported to utilize 4-chlorophenol and 2,4-dichlorophenol as sole sources of carbon and energy (10). After some adaptation, it also grows slowly with 3-chlorophenol but not with 2-chlorophenol. Like many gram-negative strains, R. erythropolis 1CP possesses separate catechol and chlorocatechol catabolic enzymes (14, 16). The substrate preferences of the chlorocatechol 1,2-dioxygenase (15) and of the dienelactone hydrolase (16) of R. erythropolis 1CP suggest that, corresponding to the growth substrates, only a 4-chlorocatechol branch and a 3,5-dichlorocatechol branch are functional in strain 1CP, but there is no 3-chlorocatechol branch (Fig. 1). In this paper, we show that the substrate preference of the R. erythropolis 1CP chloromuconate cycloisomerase fits well with those of the dioxygenase and of the hydrolase. Moreover, 2-chloro-cis,cis-muconate was found to be converted to only one product, 5-chloromuconolactone, by both the muconate and the ...

The conversion of 2-chloro-cis,cis-muconate by muconate cycloisomerase from Pseudomonas putida PRS2000 yielded two products which by nuclear magnetic resonance spectroscopy were identified as 2-chloro-and 5-chloromuconolactone. High-pressure liquid chromatography analyses showed the same compounds to be formed also by muconate cycloisomerases from Acinetobacter calcoaceticus ADP1 and Pseudomonas sp. strain B13. During 2-chloro-cis,cis-muconate turnover by the enzyme from P. putida, 2-chloromuconolactone initially was the major product. After prolonged incubation, however, 5-chloromuconolactone dominated in the resulting equilibrium. In contrast to previous assumptions, both chloromuconolactones were found to be stable at physiological pH. Since the chloromuconate cycloisomerases of Pseudomonas sp. strain B13 and Akaligenes eutrophus JMP134 have been shown previously to produce the trans-dienelactone (trans4-carboxymethylenebut-2-en-4-olide) from 2-chloro-cis,cis-muconate, they must have evolved the capability to cleave the carbonchlorine bond during their divergence from normal muconate cycloisomerases.The bacterial mineralization of chloroaromatic compounds necessarily involves the cleavage of carbon-chlorine bonds, liberating inorganic chloride. In several cases, this is achieved prior to the opening of the aromatic ring by reductive, oxygenolytic, or hydrolytic reactions (for reviews, see references 12, 19, and 45). In many other cases, however, chloride elimination occurs only after ring cleavage has been accomplished. Corresponding catabolic pathways have been described mainly for mono-and dichlorosubstituted phenoxyacetates, phenols, benzoates, benzenes, anilines, salicylates, and metabolic precursors of them. Under aerobic conditions, all of these compounds are usually first converted to chlorocatechols as central intermediates. These are then subject to intradiol (ortho) ring cleavage, giving rise to chlorosubstituted cis,cismuconates. It has long been known (3,13,14,57) that chloride elimination on this pathway is related to the conversion of mono-and dichloromuconates to unsubstituted or chlorosubstituted dienelactones (4-carboxymethylenebut-2-en-4-olides) (Fig. 1). The dienelactones are then cleaved hydrolytically, and the products are finally funneled into the 3-oxoadipate pathway (10,13,14,24,48,52,57,60).Bacteria, employing the modified ortho cleavage pathway outlined above, usually do so by inducing a set of plasmidencoded enzymes which are specially adapted for the turnover of chlorocatechols or their respective metabolites (9,41,48). With the exception of maleylacetate reductase, these enzymes catalyze reactions analogous to those of the ordinary 3-oxoadipate pathway.Muconate cycloisomerase (EC 5.5.1.1) and chloromuconate cycloisomerase (EC 5.5.1.7) were first differentiated by Schmidt and Knackmuss (48) in the 3-chlorobenzoate-utilizing strain Pseudomonas sp. strain B13. The authors reported that both enzymes catalyze basically the same reactions and that they differ only with respect to their...