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 (C 1-C 10 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 ( S 20, 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.