Mutational Analysis of Mau Genes Involved in Methylamine Metabolism in Paracoccus Denitrificans

Abstract

A chromosomal fragment containing DNA downstream from mauC was isolated from Paracoccus denitrificans. Sequence analysis of this fragment revealed the presence of four open reading frames, all transcribed in the same direction. The products of the putative genes were found to be highly similar to MauJ, MauG, MauM and MauN of Methylobacterium extorquens AM1. Using these four mau genes, 11 mau genes have been cloned from P. denitrificans to date. The gene order is mauRFBEDACJGMN, which is similar to that in M. extorquens AM1. mauL, present in M. extorquens AM1, seems to be absent in P. denitrificans. MauJ is predicted to be a cytoplasmic protein, and MauG a periplasmic protein. The latter protein contains two putative heme-binding sites, and has some sequence resemblance to the cytochrome c peroxidase from Pseudomonas aeruginosa. MauM is also predicted to be located in the periplasm, but MauN appears to be membrane associated. Both resemble ferredoxin-like proteins and contain four and two motifs, respectively, characteristic for [4Fe-4S] clusters. Inactivation of mauA, mauJ, mauG, mauM and mauN was carried out by introduction of unmarked mutations in the chromosomal copies of these genes. mauA and mauG mutant strains were unable to grow on methylamine. The mauJ mutant strain had an impaired growth rate and showed a lower dye-linked methylamine dehydrogenase (MADH) activity than the parent strain. Mutations in mauM and mauN had no effect on methylamine metabolism. The mauA mutant strain specifically lacked the beta subunit of MADH, but the alpha subunit and amicyanin, the natural electron acceptors of MADH, were still produced. The mauG mutant strain synthesized the alpha and beta subunits of MADH as well as amicyanin. However, no dye-linked MADH activity was found in this mutant strain. In addition, as the wild-type enzyme displays a characteristic fluorescence emission spectrum upon addition of methylamine, this property was lost in the mauG mutant strain. These results clearly show that MauG is essential for the maturation of the beta subunit of MADH, presumably via a step in the biosynthesis of tryptophan tryptophylquinone, the cofactor of MADH. The mau gene cluster mauRFBEDACJGMN was cloned on the broad-host vector pEG400. Transfer of this construct to mutant strains which were unable to grow on methylamine fully restored their ability to grow on this compound. A similar result was achieved for the closely related bacterium Thiosphaera pantotropha, which is unable to utilize methylamine as the sole sources of carbon and energy.

Posttranslational Biosynthesis of the Protein-Derived Cofactor Tryptophan Tryptophylquinone

“…The genes encoding the α and β subunits of MADH are located in the methylamine utilization ( mau ) gene cluster (22). The mau cluster of P. denitrificans has 11 genes with a gene order of mauRFBEDACJGMN (Figure 3) (23). The α and β subunits of MADH are encoded by mauB and mauA , respectively, and mauC (24) encodes amicyanin.…”

“…Four other genes were shown to be essential for MADH biogenesis in P. denitrificans . Deletions of either mauF (25), mauD (26), mauE (26) or mauG (23) result in loss of MADH activity and the ability of the bacterium to grow on methylamine. In the first three deletions, no MADH β subunit could be detected in cell extracts, and the levels of MADH α subunit were significantly decreased.…”

“…In the first three deletions, no MADH β subunit could be detected in cell extracts, and the levels of MADH α subunit were significantly decreased. However, while cells with the mauG deletion are lacking in MADH activity and unable to grow on methylamine, it was shown by Western blot analysis that near wild-type levels of the MADH α and β subunits are expressed (23). This suggested that MauG might play a role in TTQ formation.…”

“…For the other genes assignment of structure or function is based on sequence similarity to other genes of known function. The information in this figure is based on data presented in references (23, 24, 26). …”

Tryptophan tryptophylquinone biosynthesis: A radical approach to posttranslational modification

“…The genes encoding the α and β subunits of MADH are located in the methylamine utilization ( mau ) gene cluster [10]. The mau cluster of P. denitrificans has 11 genes with a gene order of mauRFBEDACJGMN [11]. The α and β subunits of MADH are encoded by mauB and mauA , respectively, and mauC [12] encodes the electron acceptor for MADH, amicyanin.…”