Methylamine Dehydrogenases of Methylomonas J and Pseudomonas AM1

Hybridization of Light and Heavy Subunits and Some Properties of an Isolated Hybrid Enzyme

Abstract

Hybridization reactions were carried out in vitro between the light and heavy subunits of two methylamine dehydrogenases obtained from Methylomonas J (formerly Pseudomonas sp. J) and Pseudomonas AM1. Enzymatic activity was restored with a combination of L_J (MW: 13, 000) and H_A (MW: 40, 000) but not with L_A (MW: 13, 000) and H_J (MW: 40, 000). This active hybrid enzyme was isolated with a Sephadex G-150 column after incubation of L, and H_A. Its molecular weight was nearly identical to that of the two native enzymes (MW: 105, 000). The electrophoretic pattern of proteins cross-linked by dimethyl suberimidate showed the formation of a tetrameric structure. Its absorption spectrum was similar to that of the native enzymes. The specific activity was 4.3 and the substrate specificity resembled that of the Methylomonas J enzyme rather than that of the Pseudomonas AM1 enzyme. Michaelis constants were 290 μM for methylamine and 14 μM for phenazine methosulfate. The circular dichroism spectrum and thermal stability resembled those of the Pseudomonas AM1 enzyme. No appreciable amount of an inactive hybrid enzyme was formed in the reaction between L_A and H_J, there being neither restoration of the enzymatic activity, nor formation of cross-linked tetrameric proteins with dimethyl suberimidate. Hybridization of L_J and H_A was further confirmed by the appearance of three enzymatic active bands on a gel after electrophoresis of an incubation mixture of L_J, H_J, and H_A. The results are consistent with a previous observation; the two methylamine dehydrogenases from either obligate or facultative methylotrophs are the α₂β₂-type and homologous enzymes. It is also suggested that substrate specificity and catalytic activity are directed by the light subunit, and binding affinity of the substrate to the active site, electrophoretic mobility, and thermal stability of the enzyme are endowed by the heavy subunit.