Heterologous expression of correctly assembled methylamine dehydrogenase in Rhodobacter sphaeroides.
The biosynthesis of methylamine dehydrogenase (MADH) from Paracoccus denitrificans requires four genes in addition to those that encode the two structural protein subunits, mauB and mauA. The accessory gene products appear to be required for proper export of the protein to the periplasm, synthesis of the tryptophan tryptophylquinone (TTQ) prosthetic group, and formation of several structural disulfide bonds. To accomplish the heterologous expression of correctly assembled MADH, eight genes from the methylamine utilization gene cluster of P. denitrificans, mauFBEDACJG, were placed under the regulatory control of the coxII promoter of Rhodobacter sphaeroides and introduced into R. sphaeroides by using a broad-host-range vector. The heterologous expression of MADH was constitutive with respect to carbon source, whereas the native mau promoter allows induction only when cells are grown in the presence of methylamine as a sole carbon source and is repressed by other carbon sources. The recombinant MADH was localized exclusively in the periplasm, and its physical, spectroscopic, kinetic and redox properties were indistinguishable from those of the enzyme isolated from P. denitrificans. These results indicate that mauM and mauN are not required for MADH or TTQ biosynthesis and that mauFBEDACJG are sufficient for TTQ biosynthesis, since R. sphaeroides cannot synthesize TTQ. A similar construct introduced into Escherichia coli did not produce detectable MADH activity or accumulation of the mauB and mauA gene products but did lead to synthesizes of amicyanin, the mauC gene product. This finding suggests that active recombinant MADH is not expressed in E. coli because one of the accessory gene products is not functionally expressed. This study illustrates the potential utility of R. sphaeroides and the coxII promoter for heterologous expression of complex enzymes such as MADH which cannot be expressed in E. coli. These results also provide the foundation for future studies on the molecular mechanisms of MADH and TTQ biosynthesis, as well as a system for performing site-directed mutagenesis of the MADH gene and other mau genes.
Department of Biochemistry, The University of Mississippi Medical Center, Jackson, Mississippi 39216-4505, USA.
M E
ME
Graichen
L H
LH
Jones
B V
BV
Sharma
R J
RJ
van Spanning
J P
JP
Hosler
V L
VL
Davidson
D001426:Bacterial Proteins / Q000378:metabolism; D003227:Conjugation, Genetic; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D010231:Paracoccus denitrificans / Q000201:enzymology / Q000235:genetics*; D019897:Periplasm / Q000201:enzymology; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics*; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D011994:Recombinant Proteins; C017079:mauC protein, Methylobacterium extorquens; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors