The small-subunit polypeptide of methylamine dehydrogenase from Methylobacterium extorquens AM1 has an unusual leader sequence

Abstract

The nucleotide sequence for the N-terminal region of the small subunit of methylamine dehydrogenase from Methylobacterium extorquens AM1 has revealed a leader sequence that is unusual in both its length and composition. Gene Table 1, and all Escherichia coli DHSa (F-recAl lacZYA r-m+ thi-J gyrA66 supE44 endAl 4801acZAM15) and E. coli CC118 (F-araD139 lacX74 phoA galE galK thi-J rpsE rpoB recAl) strains containing plasmids were grown on L medium (16) with the addition of the appropriate antibiotics (100 ,ug of ampicillin or kanamycin per ml or 20 ,ug of tetracycline per ml). Plasmid pAYC152.1 was used for initial sequencing ( Fig. 1 concern that the sequence might have been generated artifactually by rearrangements occurring during the subcloning either of the original 5.2-kb BamHI-HindIII insert in pAYC147 (5) or of the insert in pAYC152.1. Therefore, sequence data were generated independently from plasmid pAYC142, a clone containing mauA that was isolated directly from an independent partial clone library (5). An oligonucleotide (5'-GTTGTCCTGCGGCTTCCACTT-3') complementary to the region encoding the mature portion of the polypeptide was synthesized and used as a primer for sequencing of pAYC142. The sequence obtained was identical to the sequence shown in Fig. 2.To obtain further evidence that the start codons identified were translated, translational fusions of the mauA open reading frame with lacZ were generated (Fig. 1). These were constructed from plasmid pAYC150 (Table 1). Plasmid pAYC150a (Fig. 1) was generated by deleting an AccI-AccI fragment of pAYC150, treating the AccI recessed ends with Klenow fragment, and religating. This leads to the loss of approximately one-third of the mauA downstream sequence and brings in frame the sequences of this portion of mauA (mauA') and the part of lacZ in pUC19 (lacZ') (5,26

The biosynthesis of periplasmic electron transport proteins in methylotrophic bacteria

“…As expected, the predicted gene products of mauA and mad3 include a signal peptide, but that for MauA is longer than normal (57 amino acids compared with the usual 20-28) and has two other unusual features (Fig. 2) (Chistoserdov & Lidstrom, 1991). Before the region of positively charged amino acids at the N-terminal end there are 13 residues which are mostly hydrophilic, and near the peptidase recognition site is a region of positively charged amino acids, which would be expected to slow down translocation.…”

Genetic organization of methylamine utilization genes from Methylobacterium extorquens AM1

“…P-Galactosidase activity in strains containing pTZ19TT derivatives is expressed from the lac promoter and is absent only in the cases in which transcriptional termination occurs within the cloned fragment. (13), so it seems likely that E. coli would produce the large form preferentially. However, it is also possible that the different amounts are observed because of different stabilities of the two forms.…”

“…Therefore, it seems likely that it is the mature form of the MADH small subunit. It is not surprising that this polypeptide might be poorly processed in E. coli, since it contains an unusual leader sequence (13).…”

“…3B, lane 6). Since the gene for the small subunit of MADH can encode polypeptides of 20 or 18.5 kDa, two polypeptides of about this size were expected, depending on which of the two possible start codons is used (13). The deletion in pAYC152.5 deprives the small-subunit gene of both possible start sites and leads as a result to loss of expression of these polypeptides.…”

Mutants of Methylobacterium extorquens and Paracoccus denitrificans deficient in c‐type cytochrome biogenesis synthesise the methylamine‐dehydrogenase polypeptides but cannot assemble the tryptophan‐tryptophylquinone group

“…denitrificans (Husain and Davidson, 1987). While this has not been demonstrated directly in M. extorquens [which does not release periplasmic proteins when either incubated with lysozyme or subjected to osmotic shock (Anthony, C., unpublished results), sequencing of the structural genes for the H and L subunits of the enzyme in this organism reveal that both proteins are synthesised with N-terminal signal peptides (Chistoserdov et al, 1990;Chistoserdov and Lidstrom, 1991 ;Lidstrom and Chistoserdov, 1993), indicating a periplasmic location for the mature enzyme. The molecular mass of the H subunit polypeptide accumulated by each of the M. extorquens mutants was indistinguishable from that of the H subunit polypeptide synthesised by the parental strain, suggesting the proteolytic removal of the H-subunit leader peptide and thus translocation of the Hsubunit polypeptide to the periplasm in each of the mutant strains (Fig.…”