Covalent flavinylation of 6-hydroxy-D-nicotine oxidase analyzed by partial deletions of the gene

Abstract

The expression of the enzymatically active 6-hydroxy-u-nicotine oxidase (6-HDNO) from Arthrobucter oxidans requires the covalent attachment of FAD to the polypeptide chain. How this modification takes place and at what time during the synthesis of the polypeptide is not known. We investigated the possibility of cotranslational flavinylation by generating various deletions of the 6-HDNO gene carried on appropriate plasmid vectors. The polypeptides expressed from these plasmids were analyzed for their ability to incorporate [14C]FAD covalently in an Escherichiu coli-derived coupled transcription/translation system. The data show that removal of approximately 40% from the carboxy-terminal part of the 6-HDNO polypeptide did not inhibit the covalent flavinylation of the truncated protein. A fusion protein, consisting of the truncated 6-HDNO polypeptide and the B-lactamase of pBR322, was also covalently flavinylated.The amino acid sequence surrounding the histidine residue, assumed to bind FAD, was shown to be situated approximately 70 amino acid residues from the amino-terminal end of the 6-HDNO polypeptide. Removal of the first 30 amino acids did not abolish covalent flavinylation. Flavinylation could no longer be detected, however, if a short amino acid sequence, consisting of seven residues, replaced the amino acid sequence upstream of the histidine. These findings prove, in our opinion, that cotranslational flavinylation takes place in the synthesis of 6-HDNO.The gene encoding h-hydroxy-~-nicotine oxidase from Arthrobacter oxidans was cloned into Escherichia coli [l]. 6-HDNO belongs to a group of enzymes with covalently bound flavin [2]. In 6-HDNO the covalent bond is formed between the 8-methyl group of the isoalloxazine ring of FAD and the N-3 of histidine [3]. In previous work we have shown that in E. coli the cloned gene product is enzymatically active and covalently flavinylated [4]. In the absence of FAD, 6-HDNO apoenzyme is synthesized from the cloned gene in an E. coliderived transcription/translation system. Once released, the apoprotein could no longer be flavinylated [5] suggesting a cotranslational flavinylation process [6].The availability of the 6-HDNO gene enabled us to investigate the requirements for the covalent flavinylation of 6-HDNO in greater detail. Different deletions of the gene, carried on appropriate plasmid vectors, were performed and the corresponding polypeptides analyzed in vitro in an E. coliderived transcription/translation system. We show here that 40% can be removed from the carboxy-terminal part of the 6-HDNO polypeptide without inhibiting the covalent flavinylation of the truncated protein. When the remaining 60% was fused to the B-lactamase of pBR322, the resulting fusion protein was still covalently flavinylated.Correspondence to R. Brandsch,

Engineering disease resistance in plants

“…All other sequence similarities with Ha-CHOX ( Figure 2) were weak, with the highest region of identity associated with a conserved region of the sequence known to bind a flavin molecule in a range of oxidases (Brandsch et al, 1987;. Sequence similarity to other oxidases together with various enzyme assays allowed the putative classification of carbohydrate oxidase to be assigned.…”

Phosphoenolpyruvate‐dependent flavinylation of 6‐hydroxy‐D‐nicotine oxidase

“…Antibodies against the 6-HDNO polypeptide were raised iis described [15]. Monospecific antibody was obtained by coupling HPLC-purified 6-HDNO to CNBr-activated Sepharose.…”

The Biological Degradation of Nicotine by Nicotinophilic Microorganisms

“…However, the capacity for nicotine degradation can be reestablished by transfer of pA01 into the plasmid-deficient strains (2). In the mid-1980s Brandsch et aL succeeded in incorporating the 6-hydroxy-D-nicotine oxidase gene into the genome of Escherichia coli and expressing the genetic information in a cell-free coupled transcription-translation assay (5,9). The 6-hydroxy-D-nicotine oxidase derived gene from a 2,8 kb pA01 DNA fragment was already established by Brandsch et al in 1987 (11).…”

“…In the mid-1980s Brandsch et aL succeeded in incorporating the 6-hydroxy-D-nicotine oxidase gene into the genome of Escherichia coli and expressing the genetic information in a cell-free coupled transcription-translation assay (5,9). The 6-hydroxy-D-nicotine oxidase derived gene from a 2,8 kb pA01 DNA fragment was already established by Brandsch et al in 1987 (11). The promoter region of the gene exhibits two homologous palindromic sequences {IR 1 and IR 2) carrying the binding site for the DNA dependent RNA polymerase (2,28).…”