Carbon monoxide dehydrogenase inhibitor in cell extracts of Pseudomonas carboxydovorans

Abstract

Extracts of heterotrophically grown cells ofPseudomonas carboxydovorans were found to contain an inhibitor of carbon monoxide dehydrogenase (CO-DH). The inhibitor activity was not detected in CO-autotrophically grown cells. The inhibitor was extremely stable to heat treatment based on the extent of inhibition of CO-DH activity. The extent of inhibition was proportional to the amount of cell extract added to the reaction mixture.The inhibition was independent of a prior incubation period of the extracts with CO-DH. The inhibitor was precipitable with ammonium sulfate, phenol, and trichloroacetic acid. It was passed through benzoylated dialysis tubing and Amicon ultrafiltration membrane YM2. Denaturing and nondenturing polyacrylamide gel electrophoresis of CO-DH inactivated by inhibitor revealed that the mobilities of native enzyme and subunits were identical to those of active CO-DH. The inhibitor-treated CO-DH retained its original antigenic sites and exhibited enzyme activity upon activity staining. The CO-DH inhibitor of P. carboxydovorans was also active on CO-DHs from Pseudomonas carboxydohydrogena, Acinetobacter sp. strain JC1, and Pseudomonas carboxydoflava.Carbon monoxide dehydrogenase (CO-DH) is the key enzyme in the utilization of CO as the sole energy and carbon source in several aerobic carboxydobacteria (1,5,6,(15)(16)(17)22). The enzyme has generally been considered as an inducible enzyme, with the exception of the CO-DH in Pseudomonas carboxydoflava (1,2,5,15,17). Biochemical and immunological studies revealed that the enzymes from several carboxydobacteria share similar properties, with few exceptions (3,5,(15)(16)(17).We have recently found that Pseudomonas carboxydovorans growing under heterotrophic condition contains an inhibitor of CO-DH from this bacterium. Since this was the first observation of the presence of CO-DH inhibitor in carboxydobacteria and since nothing is known about the molecule, it was of interest to study the inhibitor in cell extracts. The present work was undertaken to study the physiological and biochemical properties of CO-DH inhibitor in cell extracts of P. carboxydovorans.

Growth of Mycobacteria on Carbon Monoxide and Methanol

“…strain JC1 (20). Identical results were obtained with extracts prepared from cells grown in 7H9-CO, except that the CO-DH activity was not detectable by enzyme assay (data not shown), possibly due to the presence of a certain molecule(s) inhibiting the CO-DH assay when extracts prepared from cells grown in the presence of organic material were used, as suggested previously (11,37). CO-DHs in the mycobacteria tested were also found to be expressed in cells grown in SMB-MeOH or 7H9 medium supplemented with 0.2% (wt/vol) glucose (data not shown), indicating that the enzymes in mycobacteria may be constitutively expressed, like that of Mycobacterium sp.…”