Methylamine dehydrogenases of Methylomonas J and Pseudomonas AM1. Hybridization of light and heavy subunits and some properties of an isolated hybrid enzyme.

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 L3(MW: 13,000) and HA(MW: 40,000) but not with LA(MW: 13,000) and HJ(NW: 40,000). This active hybrid enzyme was isolated with a Sephadex G-150 column after incubation of LJ and HA. 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 microM for methylamine and 14 microM 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 LA and HJ, there being neither restoration of the enzymatic activity nor formation of cross-linked tetrameric proteins with dimethyl suberimidate. Hybridization of LJ and HA was further confirmed by the appearance of three enzymatic active bands on a gel after electrophoresis of an incubation mixture of LJ, HJ, and HA. The results are consistent with a previous observation; the two methylamine dehydrogenases from either obligate of facultative metaylotrophs are the alpha 2 beta 2-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.