Cloning, sequencing and expression studies of the genes encoding amicyanin and the β‐subunit of methylamine dehydrogenase from Thiobacillus versutus

Abstract

The genes encoding amicyanin and the P-subunit of methylamine dehydrogenase (MADH) from Thiohacillus versutus have been cloned and sequenced. The organization of these genes makes it likely that they are coordinately expressed and it supports earlier findings that the blue copper protein amicyanin is involved in electron transport from methylamine to oxygen. The amino acid sequence deduced from the nucleotide sequence of the amicyaninencoding gene is in agreement with the published protein sequence. The gene codes for a pre-protein with a 25-amino-acid-long signal peptide. The amicyanin gene could be expressed efficiently in Escherichia coli. The protein was extracted with the periplasmic fraction, indicating that pre-amicyanin is translocated across the inner membrane of E. coli. Sequence studies on the purified P-subunit of MADH confirm the amino acid sequence deduced from the nucleotide sequence of the corresponding gene. The latter codes for a pre-protein with an unusually long (56 amino acids) leader peptide. The sequencing results strongly suggest that pyrroloquinoline quinone (PQQ) or pro-PQQ is not the co-factor of MADH.When Thiobacillus versutus is offered methylamine as its sole source of carbon, nitrogen and energy, it synthesises a redox chain which converts the amine into the aldehyde and which transports the electrons generated in the methylamine conversion eventually to oxygen as the final electron acceptor [1]. Because the redox chain is short and the constituting proteins are relatively easy to manipulate, there is currently strong interest in the physiology, enzymology and mode of operation of this chain [l -91. Although the composition of this chain has not yet been established with absolute certainty, there is agreement [l, 3, 5, 10, 111 that the first two proteins in the redox path are: (a) methylamine dehydrogenase (MADH), an a2P2 dimer with a molecular mass of 123 kDa and a quinone compound as a prosthetic group [12, 131 and (b) amicyanin, a type I copper protein with a molecular mass of 11.7 kDa [ 14, 151. Crystals of MADH have been analysed with X-ray diffraction techniques but the,interpretation of the

N‐terminal heterogeneity of methylamine dehydrogenase from Thiobacillus versutus

“…As expected, we obtained both the sequence Thr-GluPro-Ala-Glu-Pro-Glu-Ala-Pro from the cleaved a subunit as the sequence Ala-Gly-Pro-Ala-Glu-Gly-ValAsp-Pro [6] from the B subunit (quantitative data not shown). On these grounds, we unambiguously confirm that the polypeptide of the 01 subunit of MADH consists of 395 residues and that it covers the region Gln-32-Ser-426 of the coding gene.…”

“…The primary structure of the smaller fi subunit has already been determined after cloning of the corresponding gene [6]. The cloning and sequencing of the gene coding for the d subunit has recently been finished [7].…”

“…Since the sequence of the B subunit was already available both from the gene and from peptides covering most of this sequence, the a subunit was first separated from the /3 subunit following the procedure described before [6]. Whereas the /I subunit is stable when isolated separately, the a subunit, on the contrary, appears to undergo a particular and reproducible degradation pattern which can be made visible by SDS-PAGE ( Fig.…”

“…In situ treatment of the electroblotteda subunit with pyroglutamate aminopeptidase After SDS-polyacrylamide gel electrophoresis of the a subunit, prepared as in [6], the excised band of the electroblotted components was cut into two parts and treated at room temperature with 100~1 ofO.Z% PVP-40 (Sigma, USA) for 20 min. To this solution, 100 JLI of water was added and incubated for 30 min at 30°C.…”

Quantum study of the structure of the active site of methylamine dehydrogenase

“…Two classes of MADH have been proposed based on biochemical characterization and sequence comparison 4. The first class includes MADH from Methylobacterium extorquens (MADH‐AM) 5, Thiobacillus versutus (MADH‐TV) 6, and Paracoccus denitrificans (MADH‐PD) 7. In this class, the substrate‐reduced enzyme interacts with the type I blue copper protein Amicyanin, which mediates electron transfer from MADH to c‐type cytochromes.…”