A. Felix

Purification and properties of a heme-containing aldehyde dehydrogenase from Methylosinus trichosporium

Abstract Crude extracts of various methylotrophic bacteria contained a soluble phenazine methosulfate-linked aldehyde dehydrogenase. Procedures for the purification of an aldehyde dehydrogenase from extracts of the obligate methane-utilizing bacterium Methylosinus trichosporium are described. The purified enzyme is homogeneous as judged from polyacrylamide gel electrophoresis. The purified enzyme catalyzes the oxidation of straight-chain aldehydes (C1-C10 tested), aromatic aldehydes (benzaldehyde, salicylaldehyde), glyoxylate, and glyceraldehyde. Biological electron acceptors such as NAD+, NADP+, FAD, FMN, pyridoxal phosphate, and cytochrome c do not act as electron carriers. Sulfhydryl agents [p-chloromercuribenzoate, N-ethylmaleimide, 5,5-dithiobis (2-nitrobenzoic acid), and thioacetamide], cuprous chloride, cupric sulfate, and thiourea inhibited enzyme activity. The molecular weight of the enzyme as estimated by gel filtration is approximately 43,000 and as estimated by sedimentation equilibrium analysis, 50,000. The sedimentation constant (S20, w) is 2.8. The subunit size determined by sodium dodecyl sulfate-gel electrophoresis is approximately 22,000. The purified enzyme is light brown and has an absorption peak at 410 nm. Reduction of the enzyme with sodium dithionite resulted in the appearance of peaks at 523 and 552 nm and a shift in the Soret peak from 410 to 412 nm was observed. These results suggest that the enzyme is a hemoprotein. There was no evidence that flavins were present as a prosthetic group. The amino acid composition of the enzyme is also presented. Antisera prepared against the purified enzyme are nonspecific; they cross-reacted with isofunctional enzyme from other methylotrophic bacteria on Ouchterlony double-diffusion plates.

Microbial oxidation of methane and methanol: purification and properties of a heme-containing aldehyde dehydrogenase from Methylomonas methylovora.

Procedures for the purification of an aldehyde dehydrogenase from extracts of the obligate methylotroph, Methylomonas methylovora are described. The purified enzyme is homogeneous as judged from polyacrylamide gel electrophoresis. In the presence of an artificial electron acceptor (phenazine methosulfate), the purified enzyme catalyzes the oxidation of straight chain aldehydes (C1-C10 tested), aromatic aldehydes (benzaldehyde, salicylaldehyde), glyoxylate, and glyceraldehyde. Biological electron acceptors such as NAD+, NADP+, FAD, FMN, pyridoxal phosphate, and cytochrome c cannot act as electron carriers. The activity of the enzyme is inhibited by sulfhydryl agents [p-chloromercuribenzoate, N-ethylmaleimide and 5,5-dithiobis (2-nitrobenzoic acid)], cuprous chloride, and ferrour nitrate. The molecular weight of the enzyme as estimated by gel filtration is approximately 45000 and the subunit size determined by sodium dodecyl sulfate-gel electrophoresis is approximately 23000. The purified enzyme is light brown and has an absorption peak at 410 nm. Reduction of enzyme with sodium dithionite or aldehyde substrate resulted in the appearance of peaks at 523 nm and 552 nm. These results suggest that the enzyme is a hemoprotein. There was no evidence that flavins were present as prosthetic group. The amino acid composition of the enzyme is also presented.

Circular dichroism of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus

Abstract Circular dichroism (CD) spectra of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus exhibit three positive ellipticity bands between 240 and 300 nm (250, 283, and 292 nm), two negative bands at 327 and 480 nm, and a low-intensity positive band at 390 nm. The fractions of helix β-form, and unordered form of the enzyme are 8, 38, and 54%, respectively. The circular dichroic bands at 327 and 480 nm and a part of the positive bands at 292 and 390 nm are associated with enzyme activity. Significant changes in absorption and CD spectra of the enzyme were observed when the temperature of the enzyme preparation was increased to 47°C, coinciding with the sharp decrease in enzyme activity observed at this temperature.