The reaction leading to the flavinylation of apo-6-hydroxy-~-nicotine oxidase was investigated in cell-free extracts of Eschericia coli carrying the 6-hydroxy-~-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-HDN0, no phosphorylated or pyruvylated apo-protein could be detected, however. In order to drive the reaction to completion, FAD and Ppyruvate had to be present simultaneously in the reaction mixture. When apo-6-HDN0, 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.Enzymes which are covalently modified by the attachment of their cofactor to the side chain of a specific amino acid residue represent a special category of post-translational modifications. The functional significance of these modifications and how they are accomplished is poorly understood. The covalent attachment of biotin to a specific lysine residue of carboxylases [l] and of heme to two specific cysteine residues of cytochrome c [2] are known to take place enzymatically. No corresponding holoenzyme synthetase, however, has been described so far for enzymes carrying covalently bound lipoic acid or covalently bound flavin. Both flavin cofactors, FAD as well as FMN, have been described as occurring in covalent linkage [3]. Biotin and lipoic-aciddependent enzymes always contain the cofactor bound to the polypeptide chain by a specific lysine residue [4]. Among the cytochromes, only cytochromes c andfcarry heme [5], always covalently bound by two essential cysteine residues. The situation with FAD, the primary flavin cofactor, is different. This cofactor is tightly but noncovalently bound in the majority of enzymes. In different covalently flavinylated enzymes, FAD is bound to the side chain of different amino acid residues. The 8a-methyl group of the FAD isoaloxazine ring is linked to the N1 or N3 of a histidine, to the sulfur of a cysteine or the oxygen of a tyrosine residue; 6-S-cysteinyl-FMN was also identified in some enzymes (reviewed in [3]). In addition to the differences in type of FAD linkage to the protein moiety, no amino acid sequence similarities surrounding the amino acid residue involved in FAD binding have been found thus far between covalently flavinylated enzymes. This situation is in contrast to information obtained from other examples of covalent modification of enzymes by cofactors where similar binding sequences are present [2, 3, 61. The question of whether the same mechanism accounts for €he covalent attachCorrespondence to R. Brandsch, Biochemisches Institut, Universit i t Freiburg, Hermann-Herder-StraBe 7, D-7800 Freiburg, Federal Republic of Germany Abbreviations. 6-HDNO, 6-hydroxy-~-nicotine oxidase; P-pyruvate, phosphoenolpyruvate.Enzyme. 6-Hydroxy-~-ni...

Modification by covalent FAD attachment to a histidine residue via an 8a-(N3-histidyl)-riboflavin linkage occurs in several flavoenzymes. Among them is 6-hydroxy-~-nicotine oxidase (6-HDNO) of Arthrohacter 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-HDN0, 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. 177,319-3251, 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. The first enzyme shown to contain covalently bound FAD was the flavoprotein subunit from mammalian succinate dehydrogenase with FAD covalently bound to a histidine residue via an 8a-(N3-histidyl)-riboflavin linkage [6]. The same type of linkage is found in the flavoprotein of the closely related fumarate reductase complex which permits bacteria to use fumarate as the terminal electron acceptor under anaerobic conditions [7]. It is also characteristic for 6-HDNO of Arthrobacter oxiduns [S]. No amino acid sequence similarities at the flavin attachment site exists among 6-HDNO these enzymes [4].Recently we showed that covalent flavinylation of apo-6-HDNO requires, besides FAD, phosphoenolpyruvate (Ppyruvate) [9, 101. However Abbreviations. 6-HDNO, 6-hydroxy-~-nicotine oxidase; P-pyruvate, phosphoenolpyruvate.Enzymes. 6-Hydroxy-~-nicotine oxidase (EC 1.5.3.6); fumarate I-eductase (EC I .3.99.1); succinate dehydrogenase (EC 1.3.99.1).in the flavinylation reaction could be detected and no requirement for a synthetase activity was found [lo].Thus, P-pyruvate might act as an allosteric effector and we investigated whether other glycolytic intermediates have the same effect. In addition, we show that flavinylation of the flavoprotein subunits of fumarate reductase and succinate dehydrogenase also require specific effectors represented by the citric acid cycle intermediates citrate, isocitrate, succinate and fumarate, the latter two being the substrates of these enzymes. MATERIALS AND METHODS ChemicalsATP and P-pyruvat...

The gene of 6-hydroxy-~-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 transcriptiontranslation 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 apod-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.The object of our study is related to the key reaction in a catabolic pathway which enables Arthrobacter oxidans to use nicotine as growth substrate [I]. This pathway consists of at least four flavoenzymes [2 -41 possibly arranged into one single operon [5] encoded on a 160-kb large plasmid [6]. Our aim is to elucidate the relationship between the genes coding for these flavoenzymes and the mechanism of their expression.A peculiar feature of this pathway is the structure of one of it enzymes, 6-hydroxy-~-nicotine oxidase (6-HDNO), which contains the cofactor FAD covalently bound [A via an 801-(N-3-histidyl)-riboflavin linkage. Expression of this enzyme is regulated differently from the other enzymes of the pathway [8] which are induced and expressed during the logarithmic phase of growth whereas 6-HDNO is expressed only when the bacterial culture reaches the stationary phase of growth.One possible explanation for the covalent modification of the 6-HDNO polypeptide would be the presence in the stationary phase of growth of an enzyme catalysing the flavinylation. Such a holoenzyme synthetase is known for biotin-dependent enzymes [9]. It catalyses the covalent binding of biotin to a lysine residue of the polypeptide chain. In a riboflavin-requiring A . oxidans mutant, however, the presence of a 6-HDNO apoenzyme could not be found under conditions of limiting riboflavin supply [lo]. Thus the question remained open that if apoenzyme was available could it be flavinylated in an enzymatically catalyzed reaction? We cloned the 6-HDNO gene in Escherichiu coli [ll] and were able to show that the 6-HDNO synthesized in vivo as well as

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.

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.6-Hydroxy-D-nicotine oxidase; Covalent flavinylation; Flavoenzyme; FAD