Cysteine to serine replacements in 6-hydroxy-D-nicotine oxidase. Consequences for enzyme activity, cofactor incorporation, and formation of high molecular weight protein complexes with molecular chaperones (GroEL).

6-Hydroxy-D-nicotine oxidase, an enzyme with FAD covalently attached to the protein, contains 6 cysteine residues in positions 45 (Cys1), 59 (Cys2), 136 (Cys3), 173 (Cys4), 260 (Cys5) and 433 (Cys6). Cys2, 3, 5, and 6 were replaced with serine by site-directed mutagenesis. The effects of these exchanges on enzyme activity, the autocatalytic incorporation of the cofactor, and the interaction of the mutant proteins with molecular chaperones were analyzed. The flavinylation of 6-hydroxy-D-nicotine oxidase is dependent on the presence of allosteric effectors, e.g. glycerol 3-phosphate or other phosphorylated tricarbon compounds. Replacement of Cys2 or Cys5 abolished this dependence. Covalent incorporation of FAD was reduced to an undetectable level in the Cys3 and Cys5 mutants. Replacement of Cys6 by Ser had no significant effect on enzyme activity and cofactor attachment. Deletion of two amino acids, Phe and Arg, situated 12 and 11 amino acid residues, respectively, from the carboxyl terminus of the protein, resulted in an inactive enzyme with no covalently bound FAD. This result indicates that almost the entire protein chain has to be synthesized before the cofactor can be incorporated, making a cotranslational flavinylation step rather unlikely. The distribution of the 6-hydroxy-D-nicotine oxidase polypeptide between the high molecular weight complexes and the free soluble form was analyzed by gel filtration on Sephacryl S-200. The wild-type holoenzyme as well as the wild-type apoenzyme were recovered in the eluent fraction of the column while the mutant proteins were retained in high molecular weight complexes, predominantly in those associated with GroEL, as revealed by immunoprecipitation. The extent of complex formation with this molecular chaperone depended on the position of the mutated Cys residue within the protein. Complex formation was highest with protein from the mutants Cys2 and Cys3, less with the Cys5, and absent with the Cys6 mutant protein. Thus, alterations in the amino-terminal part of the 6-hydroxy-D-nicotine oxidase appear more important for the interaction with molecular chaperones than alterations situated in the carboxyl-terminal part of the protein.

GroE dependence of refolding and holoenzyme formation of 6-hydroxy-D-nicotine oxidase.

In Escherichia coli cells expressing 6-hydroxy-D-nicotine oxidase (6-HDNO), a flavoprotein with covalently bound FAD, approximately 40% of the polypeptide is in its apoform. We investigated whether in vivo holoenzyme formation was influenced by the association of the apoenzyme with cellular chaperones. Immunoprecipitation of apoenzyme-containing cell extract with protein-A-Sepharose-bound 6-HDNO- or GroEL-specific antibodies failed to reveal the formation of complexes between these proteins. The limiting factor in holoenzyme formation in vivo appeared to be the intracellular supply of phosphorylated tricarbon compounds (e.g. glycerol-3-P) acting as allosteric effectors in the flavinylation reaction. When holoenzyme formation from purified apo6-HDNO was investigated in vitro, addition of GroEL and GroES to the reaction assays increased the yield of holoenzyme formation. The observed increase in apoenzyme to holoenzyme transition was ATP independent, and the effect of GroE could be simulated by high concentrations of glycerol (40%). Apparently, a nonspecific protein-protein interaction between the GroE proteins and the apo6-HDNO favored holoenzyme formation. The refolding of guanidinium hydrochloride-unfolded holoenzyme, however, was catalyzed by GroEL and GroES in an ATP-dependent reaction. Recovery of the native, enzymatically active, conformation ranged from 30 to 40%. When apo6-HDNO was denatured and refolded, the same dependence on GroE and ATP was observed in the recovery of a conformation able to incorporate FAD and to holoenzyme. [14C] FAD in the refolding assay yielded radioactively labeled 6-HDNO demonstrating the autocatalytical covalent incorporation of FAD into the polypeptide during the folding process.

Autoflavinylation of apo6-hydroxy-D-nicotine oxidase.

6-Hydroxy-D-nicotine oxidase (6-HDNO) was expressed in Escherichia coli JM109 cells from the recombinant plasmid pAX-6-HDNO as a beta-galactosidase-6-HDNO fusion protein. Affinity chromatography of the fusion protein on p-aminobenzyl-1-thio-beta-galactopyranoside-agarose and subsequent digestion with protease Xa yielded highly purified apo6-HDNO. Incubation of the purified protein with [14C]FAD demonstrated that flavinylation of apo6-HDNO proceeds autocatalytically. Phosphorylated three-carbon compounds such as glycerol-3-P, which are known to stimulate the formation of the histidyl (N3)-(8 alpha) FAD between apo6-HDNO and FAD (Brandsch, R., and Bichler, V. (1989) Eur. J. Biochem. 182, 125-128), could be replaced in their action by high concentrations of glycerol (45%) or sucrose (20%). These substances apparently induced and stabilized a conformational state of the apoenzyme compatible with covalent attachment of FAD. In the absence of glycerol the apoenzyme readily lost the ability to form holoenzyme at temperatures above 30 degrees C. Holoenzyme formation protected the 6-HDNO polypeptide from this thermal denaturation. Autoflavinylation of 6-HDNO was inhibited by the sulfhydryl reagents dithionitrobenzoate or N-ethylmaleimide. Inhibition was prevented by mercaptoethanol or FAD, but not 6-hydroxy-D-nicotine, the substrate of the holoenzyme. A cysteine-thiol group may therefore be involved in reactions leading to the covalent attachment of FAD to apo6-HDNO. When flavinylation of apo6-HDNO proceeded under anaerobic conditions, the amount of incorporation of [14C]FAD into the polypeptide was indistinguishable from reactions performed in the presence of O2. None of the FAD-derivatives (8-demethyl-FAD, 8-chloro-FAD, and 5-deaza-FAD) could replace FAD in holoenzyme formation. The failure of covalent attachment of 5-deaza-FAD to apo6-HDNO is in agreement with the assumption that the quinone methide form of the isolloxazine ring is an intermediate in the flavinylation reaction.

Riboflavin-dependent expression of flavoenzymes of the nicotine regulon of Arthrobacter oxidans.

In cells of an Arthrobacter oxidans riboflavin-dependent mutant the specific activity of the DL-nicotine-inducible nicregulon enzymes nicotine dehydrogenase (NDH, EC 1.5.99.4), 6-hydroxy-L-nicotine oxidase (6-HLNO, EC 1.5.3.5) and 6-hydroxy-D-nicotine oxidase (6-HDNO, EC 1.5.3.6) was shown to be dependent on the supply of the vitamin in the growth medium. Experiments designed to identify at which level riboflavin directs the biosynthesis of these flavoenzymes revealed that the steady-state levels of enzyme protein analysed on Western blots correlated directly with riboflavin supply from the minimal concentration of 0.5 microns-riboflavin required for growth up to 8 microns-riboflavin. Mutant cells grown at the higher riboflavin concentration showed on dot-blots increased levels of RNA which hybridized to 32P-labelled probes derived from the nic-regulon genes. When cells grown at 2 microns-riboflavin were shifted to 8 microns-riboflavin, 6-HDNO expression increased as indicated by elevated enzyme and RNA levels. When the rates of synthesis of the 6-HDNO and 6-HLNO polypeptides after DL-nicotine induction was analysed in cells grown at 0.5 microns and 8 microns-riboflavin, only cells grown at the higher riboflavin concentration showed on Western blots an accumulation of the polypeptides. No 6-HDNO or 6-HLNO polypeptide was identified in cell extracts from cells grown on 0.5 microns-riboflavin. Pulse-chase experiments with [35S]methionine showed that 6-HDNO- and 6-HLNO synthesis was prevented in cells grown at the low riboflavin concentration. The absence of detectable enzyme levels seemed not to be caused by proteolytic breakdown. Incubation in vitro of apo-6HDNO with low- or high-riboflavin-grown-cell extracts showed no increased proteolytic activity in 0.5 microns-riboflavin-grown cells. From these results it is concluded that riboflavin supply co-regulates the expression of the nicregulon genes at the level of transcription and/or mRNA turnover.

Lysine can replace arginine 67 in the mediation of covalent attachment of FAD to histidine 71 of 6-hydroxy-D-nicotine oxidase.

The requirements for FAD-attachment to His71 of 6-hydroxy-D-nicotine oxidase (6-HDNO) were investigated by site-directed mutagenesis. The following amino acid replacements were introduced into the sequence Arg67-Ser68-Gly69-Gly70-His71 of the 6-HDNO-polypeptide: 1) Arg67 was replaced with Ala (A1 mutant); 2) Ser68 was replaced with Ala (A2 mutant); and 3) Arg67 was replaced with Lys (K mutant). The substitution in mutant A2 had no effect on flavinylation, measured as [14C]FAD incorporation into apo-6-HDNO. Replacement of Arg67 with Ala prevented, but replacement with Lys permitted the flavinylation of His71. Mutant A1 showed no 6-HDNO activity, whereas the replacement of Ser with Ala in mutant A2 had only a slight effect on 6-HDNO activity. The substitution of Lys for Arg67, however, reduced the specific 6-HDNO activity in extracts of Escherichia coli cells expressing the mutant polypeptide from 50.3 to 17.5 milliunits/mg protein. It is concluded that a basic amino acid residue (Arg67 or Lys67) is required to mediate the attachment of FAD to His71, and while Lys can substitute for Arg67 in this function, it can only partially replace Arg67 in the enzyme reaction mechanism 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.

A 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 sigma 70 and Bacillus subtilis sigma 43 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.(ABSTRACT TRUNCATED AT 250 WORDS)

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

In 6-hydroxy-D-nicotine oxidase (6-HDNO) FAD is covalently bound to His71 of the polypeptide chain by an 8 alpha-(N3-histidyl)-riboflavin linkage. The FAD-binding histidine was exchanged by site-directed mutagenesis to either a Cys- or Tyr-residue, two amino acids known to be involved in covalent binding of FAD in other enzymes, or to a Ser-residue. None of the amino acid replacements for His71 allowed covalent FAD incorporation into the 6-HDNO polypeptide. Thus, the amino acid residues involved in covalent FAD-binding require a specific polypeptide surrounding in order for this modification to proceed and cannot be replaced with each other. Enzyme activity was completely abolished with Tyr in place of His71. 6-HDNO activity with non-covalently bound FAD was found with 6-HDNO-Cys and to a lesser extent also with 6-HDNO-Ser. However, the Km values for 6-HDNO-Cys and 6-HDNO-Ser were increased approximately 20-fold as compared to 6-HDNO-His. Both mutant enzymes, in contrast to the wild-type enzyme, needed additional FAD in the enzymatic assay (50 microM for 6-HDNO-Ser and 10 microM for 6-HDNO-Cys) for maximal enzyme activity.

Covalent cofactor binding to flavoenzymes requires specific effectors.

Modification by covalent FAD attachment to a histidine residue via an 8 alpha-(N3-histidyl)-riboflavin linkage occurs in several flavoenzymes. Among them is 6-hydroxy-D-nicotine oxidase (6-HDNO) of Arthrobacter oxidans and the flavoprotein subunits of the fumarate reductase and succinate dehydrogenase complex of Escherichia coli and other bacterial and eukaryotic cells. We found that 6-HDNO holoenzyme formation from apo-6-HDNO, monitored by [14C]FAD incorporation and increase in enzyme activity, can be mediated not only by phosphoenolpyruvate [Nagursky, H., Bichler, V. and Brandsch, R. (1988) Eur. J. Biochem. 177, 319-325], but also by one of the glycolytic intermediates glyceraldehyde-3-P, glycerate-3-P, or the intermediate in glycerol utilization by bacteria, glycerol-3-P. Apoflavoprotein of fumarate reductase and succinate dehydrogenase was obtained in an E. coli riboflavin-requiring strain (E. coli RR28rf) overexpressing the frdABCD or the sdhCDAB operon from the recombinant plasmids pGS39 and pGS141, respectively. In extracts obtained from these cells, flavoprotein flavinylation, analyzed as covalent [14C]FAD incorporation into the apoflavoprotein polypeptide by polyacrylamide gel electrophoresis and fluorography, was stimulated severalfold by the citric acid cycle intermediates citrate, isocitrate, succinate and fumarate. Our results suggest that covalent modification and thus activation of these enzymes is dependent on specific metabolic intermediates which may act as allosteric effectors in the reaction.

Binding of FAD to 6-hydroxy-D-nicotine oxidase apoenzyme prevents degradation of the holoenzyme.

Expression of the 6-hydroxy-D-nicotine oxidase (6-HDNO) gene from Arthrobacter oxidans cloned into Escherichia coli showed a marked temperature-dependence. Transformed E. coli cells grown at 30 degrees C exhibited a several-fold higher 6-HDNO activity than did cells grown at 37 degrees C. This effect did not depend on the promoter used for expression of the cloned gene in E. coli, nor was it an effect of 6-HDNO mRNA instability at 37 degrees C. Studies performed in vivo and in vitro revealed that an increased susceptibility of apo-6-HDNO to proteolytic attack at 37 degrees C was responsible for the observed phenomenon. Extracts from cells grown at 37 degrees C showed on Western blots a decrease in immunologically detectable 6-HDNO polypeptide when compared with extracts from cells grown at 30 degrees C. The 6-HDNO polypeptide is covalently modified by attachment of the cofactor FAD to a histidine residue. It could be shown that covalent flavinylation of the apoenzyme in vitro, i.e. formation of holoenzyme, by incubation of cell extracts with FAD and phosphoenolpyruvate protected the 6-HDNO polypeptide from degradation at 37 degrees C. Of a variety of proteinase inhibitors tested only the cysteine-proteinase inhibitor L-3-trans-carboxyoxiran-2-carbonyl-L-leucylagmatine (E64) prevented degradation, by up to 70%, of the apoenzyme.

Phosphoenolpyruvate-dependent flavinylation of 6-hydroxy-D-nicotine oxidase.

The reaction leading to the flavinylation of apo-6-hydroxy-D-nicotine oxidase was investigated in cell-free extracts of Eschericia coli carrying the 6-hydroxy-D-nicotine oxidase (6-HDNO) gene on the expression plasmid pDB222. It was demonstrated that the reaction required phosphoenolpyruvate (P-pyruvate) in addition to FAD. When [32P]P-pyruvate or [14C]P-pyruvate were used in the reaction with apo-6-HDNO, no phosphorylated or pyruvylated apo-protein could be detected, however. In order to drive the reaction to completion, FAD and P-pyruvate had to be present simultaneously in the reaction mixture. When apo-6-HDNO, highly purified by affinity chromatography, was used in the reaction with P-pyruvate and FAD, no additional protein fraction was required. A possible reaction scheme for the formation of holoenzyme from 6-HDNO is discussed.

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.

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

The expression of the enzymatically active 6-hydroxy-D-nicotine oxidase (6-HDNO) from Arthrobacter 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 Escherichia 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 beta-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.

Studies in vitro on the flavinylation of 6-hydroxy-D-nicotine oxidase.

The gene of 6-hydroxy-D-nicotine oxidase (6-HDNO), a flavoenzyme from Arthrobacter oxidans with covalently bound FAD, was expressed with the aid of an expression vector in a cell-free coupled transcription-translation system derived from Escherichia coli MZ9. Ultraviolet irradiation of the E. coli extract did not affect synthesis of the 6-HDNO polypeptide nor total protein synthesis but enzymatic 6-HDNO activity could not be detected. Addition of FAD to the irradiated cell extract restored the capability of the transcription-translation assays to synthesize enzymatically active 6-HDNO. However, enzymatic activity could not be restored on addition of FAD plus cell-free extract to the ultraviolet-inactivated assays after completion of apo-6-HDNO synthesis (60 min) nor to immunoprecipitates thereof. Under similar conditions, addition of [14C]FAD did not increase the protein-bound radioactivity. These results indicate that under conditions of limited FAD supply in the in vitro system a flavinless apo-6-HDNO-polypeptide was synthesized. It was, however, not possible to bind the cofactor to the completed polypeptide chain. These findings argue for a cotranslational cofactor binding.

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 pKK223-3. 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-N3-8 alpha-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.