Cloning, sequencing and expression studies of the genes encoding amicyanin and the beta-subunit of methylamine dehydrogenase from Thiobacillus versutus.
The genes encoding amicyanin and the beta-subunit of methylamine dehydrogenase (MADH) from Thiobacillus 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 amicyanin-encoding 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 beta-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.
Ubbink
M
M
Gorlaeus Laboratories, Leiden University, The Netherlands.
van Kleef
M A
MA
Kleinjan
D J
DJ
Hoitink
C W
CW
Huitema
F
F
Beintema
J J
JJ
Duine
J A
JA
Canters
G W
GW
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002918:Chymotrypsin; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics* / Q000302:isolation & purification; D010449:Peptide Mapping; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013855:Thiobacillus / Q000201:enzymology / Q000235:genetics*; D014357:Trypsin
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; C017079:mauC protein, Methylobacterium extorquens; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors; D002918:Chymotrypsin; D014357:Trypsin