Veronika Bichler

Site-directed mutagenesis of the FAD-binding histidine of 6-hydroxy-D-nicotine oxidase: Consequences on flavinylation and enzyme activity

6‐Hydroxy‐D‐nicotine oxidase; Site‐directed mutagenesis; Covalant flavinylation; Flavoenzyme; Covalent modification; Protein modification

In vivo and in vitro expression of the 6-hydroxy-D-nicotine oxidase gene of Arthrobacter oxidans, cloned into Escherichia coli, as an enzymatically active, covalently flavinylated polypeptide

The 6‐hydroxy‐D‐nicotine oxidase gene of Arthrobacter oxidans was cloned into E.coli with the aid of the expression vector pKK‐223‐3. This enzyme, as well as the E.coli enzymes succinate dehydrogenase and fumarate reductase, bears the cofactor FAD covalently attached to the polypeptide through a His‐N3‐8α‐linkage. The amino acid sequence surrounding the histidine residue involved in FAD binding in 6‐hydroxy‐D‐nicotine oxidase and the two E.coli enzymes, however, show no homology. Nevertheless, 6‐hydroxy‐D‐nicotine oxidase is expressed in E.coli in vivo and in an E.coli‐derived coupled transcription‐translation system as a covalently flavinylated, enzymatically active polypeptide.

Covalent flavinylation of 6-hydroxy-D-nicotine oxidase involves an energy-requiring process

E. coli cells harbouring the recombinant plasmid pDB222 with the 6‐HDNO gene under the control of the tac‐promotor were induced with IPTG to synthesize a high amount of 6‐HDNO protein. Part of this protein was present as 6‐HDNO apoenzyme. The proportion of 6‐HDNO apoenzyme formed could be increased when the induction of 6‐HDNO synthesis by IPTG was performed in the presence of the inhibitor diphenyleneiodonium. The 6‐HDNO apoenzyme thus formed could be transformed into enzymatically active holoenzyme in the presence of FAD by a process requiring an energy‐generating system consisting of ATP, phosphoenolpyruvate and pyruvate kinase. This finding suggests that an enzymatic step(s) is (are) involved in the covalent flavinylation of 6‐HDNO.

Functional analysis of the 5′ regulatory region and the UUG translation initiation codon of the Arthrobacter oxidans 6-hydroxy-d-nicotine oxidase gene

SummaryA functional analysis of the Arthrobacter oxidans 6-hydroxy-d-nicotine oxidase (6-HDNO) gene promoter (−35 region TTGACA and −10 region TATCAAT) and the UUG translation start codon was performed using site-directed mutagenesis. Deletion of the C residue from the −10 promoter region or mutations introduced upstream of the −10 region resulted in an increased 6-HDNO expression in Escherichia coli cells in vivo and in both E. coli and A. oxidans coupled transcription-translation systems in vitro. From the identical behaviour of 6-HDNO promoter mutants in the heterologous and homologous systems, it is concluded that A. oxidans harbours an RNA polymerase functionally homologous to the E. coli σ70 and Bacillus subtilis σ43 polymerases. Replacement of the TTG codon (UUG translation initiation codon) with ATG led to a 3.7-fold increase in 6-HDNO expression in E. coli. This effect was less pronounced at higher promoter strengths, from 3.7 in the case of the 6-HDNO wild-type promoter, to 2.5 in the case of the consensus −10 region and to 1.7 in the case of the tac promoter. A double point mutation introduced close to the ribosome binding site resulted in almost the same increase in 6-HDNO expression (3.1-fold) as the TTG-to-ATG exchange. The failure of cAMP to stimulate 6-HDNO expression in the A. oxidans system indicated that expression of this gene in stationary phase cells is not regulated by cAMP-catabolite repressore protein-mediated mechanism of catabolite repression. ppGpp, a nucleotide involved in the initiation of morphological and physiological differentiation in stationary cells, did not significantly affect 6-HDNO expression in the in vitro transcription-translation system.