Nikkomycin biosynthesis: formation of a 4-electron oxidation product during turnover of NikD with its physiological substrate

Nikkomycins are peptidyl nucleoside antibiotics that act as therapeutic antifungal agents in humans and easily degraded insecticides in agriculture. The nikkomycin peptidyl moiety contains a pyridyl residue derived from L-lysine. The first step in peptidyl biosynthesis is an aminotransferase-catalyzed reaction that converts L-lysine to Delta(1)- or Delta(2)-piperideine-2-carboxylate (P2C). Spectral, chromatographic, and kinetic analyses show that the aerobic reaction of nikD with P2C results in the stoichiometric formation of picolinate, accompanied by the reduction of 2 mol of oxygen to hydrogen peroxide. A high resolution HPLC method, capable of separating picolinate, nicotinate and isonicotinate, was developed and used in product identification. NikD contains 1 mol of covalently bound FAD and exists as a monomer in solution. Reductive and oxidative titrations with dithionite and potassium ferricyanide, respectively, show that FAD is the only redox-active group in nikD. Anaerobic reaction of nikD with 1 mol of P2C results in immediate reduction of enzyme-bound FAD. Because nikD is an obligate 2-electron acceptor, it is proposed that the observed 4-electron oxidation of P2C to picolinate occurs via a mechanism involving two successive nikD-catalyzed 2-electron oxidation steps. In addition to nikkomycins, a nikD-like reaction is implicated in the biosynthesis of an L-lysine-derived pyridyl moiety found in streptogramin group B antibiotics that are used as part of a last resort treatment for severe infections due to gram positive bacteria.     

A mobile tryptophan is the intrinsic charge transfer donor in a flavoenzyme essential for nikkomycin antibiotic biosynthesis

The flavoenzyme nikD is required for the biosynthesis of nikkomycin antibiotics. NikD exhibits an unusual long wavelength absorption band attributed to a charge transfer complex of FAD with an unknown charge transfer donor. NikD crystals contain an endogenous active site ligand. At least four different compounds are detected in nikD extracts, including variable amounts of two ADP derivatives that bind to the enzyme's dinucleotide binding motif in competition with FAD, picolinate (0.07 mol/mol of nikD) and an unknown picolinate-like compound. Picolinate, the product of the physiological catalytic reaction, matches the properties deduced for the active site ligand in nikD crystals. The charge transfer band is eliminated upon mixing nikD with excess picolinate but not by a reversible unfolding procedure that removes the picolinate-like compound, ruling out both compounds as the intrinsic charge transfer donor. Mutation of Trp355 to Phe eliminates the charge transfer band, accompanied by a 30-fold decrease in substrate binding affinity. The results provide definitive evidence for Trp355 as the intrinsic charge transfer donor. The indole ring of Trp355 is coplanar with or perpendicular to the flavin ring in "open" or "closed" crystalline forms of nikD, respectively. Importantly, a coplanar configuration is required for charge transfer interaction. Absorption in the long wavelength region therefore constitutes a valuable probe for monitoring conformational changes in solution that are likely to be important in nikD catalysis.     

Structure of the flavocoenzyme of two homologous amine oxidases: monomeric sarcosine oxidase and N-methyltryptophan oxidase

Monomeric sarcosine oxidase (MSOX) and N-methyltryptophan oxidase (MTOX) are homologous enzymes that catalyze the oxidative demethylation of sarcosine (N-methylglycine) and N-methyl-L-tryptophan, respectively. MSOX is induced in various bacteria upon growth on sarcosine. MTOX is an E. coli enzyme of unknown metabolic function. Both enzymes contain covalently bound flavin. The covalent flavin is at the FAD level as judged by electrospray mass spectrometry. The data provide the first evidence that MTOX is a flavoprotein. The following observations indicate that 8alpha-(S-cysteinyl)FAD is the covalent flavin in MSOX from Bacillus sp. B-0618 and MTOX. FMN-containing peptides, prepared by digestion of MSOX or MTOX with trypsin, chymotrypsin, and phosphodiesterase, exhibited absorption and fluorescence properties characteristic of an 8alpha-(S-cysteinyl)flavin and could be bound to apo-flavodoxin. The thioether link in the FMN-containing peptides was converted to the sulfone by performic acid oxidation, as judged by characteristic absorbance changes and an increase in flavin fluorescence. The sulfone underwent a predicted reductive cleavage reaction upon treatment with dithionite, releasing unmodified FMN. Cys315 was identified as the covalent FAD attachment site in MSOX from B. sp. B-0618, as judged by the sequence obtained for a flavin-containing tryptic peptide (GAVCMYT). Cys315 aligns with a conserved cysteine in MSOX from other bacteria, MTOX (Cys308) and pipecolate oxidase, a homologous mammalian enzyme known to contain covalently bound flavin. There is only one conserved cysteine found among these enzymes, suggesting that Cys308 is the covalent flavin attachment site in MTOX.     

Monomeric sarcosine oxidase: role of histidine 269 in catalysis

Conservative mutation of His269 (to Asn, Ala, or Gln) does not-significantly affect the expression of monomeric sarcosine oxidase (MSOX), covalent flavinylation, the physicochemical properties of bound FAD, or the overall protein structure. Turnover with sarcosine and the limiting rate of the reductive half-reaction with L-proline at pH 8.0 are, however, nearly 2 orders of magnitude slower than that with with wild-type MSOX. The crystal structure of the His269Asn complex with pyrrole-2-carboxylate shows that the pyrrole ring of the inhibitor is displaced as compared with wild-type MSOX. The His269 mutants all form charge-transfer complexes with pyrrole-2-carboxylate or methylthioacetate, but the charge-transfer bands are shifted to shorter wavelengths (higher energy) as compared with wild-type MSOX. Both wild-type MSOX and the His269Asn mutant bind the zwitterionic form of L-proline. The E(ox).L-proline complex formed with the His269Asn mutant or wild-type MSOX contains an ionizable group (pK(a) = 8.0) that is required for conversion of the zwitterionic L-proline to the reactive anionic form, indicating that His269 is not the active-site base. We propose that the change in ligand orientation observed upon mutation of His269 results in a less than optimal overlap of the highest occupied orbital of the ligand with the lowest unoccupied orbital of the flavin. The postulated effect on orbital overlap may account for the increased energy of charge-transfer bands and the slower rates of electron transfer observed for mutant enzyme complexes with charge-transfer ligands and substrates, respectively.     

Role of the covalent flavin linkage in monomeric sarcosine oxidase

Monomeric sarcosine oxidase (MSOX) is a prototypical member of a recently recognized family of amine-oxidizing enzymes that all contain covalently bound flavin. Mutation of the covalent flavin attachment site in MSOX produces a catalytically inactive apoprotein (apoCys315Ala) that forms an unstable complex with FAD (K(d) = 100 muM), similar to that observed with wild-type apoMSOX where the complex is formed as an intermediate during covalent flavin attachment. In situ reconstitution of sarcosine oxidase activity is achieved by assaying apoCys315Ala in the presence of FAD or 8-nor-8-chloroFAD, an analogue with an approximately 55 mV higher reduction potential. After correction for an estimated 65% reconstitutable apoprotein, the specific activity of apoCys315Ala in the presence of excess FAD or 8-nor-8-chloroFAD is 14% or 80%, respectively, of that observed with wild-type MSOX. Unlike oxidized flavin, apoCys315Ala exhibits a high affinity for reduced flavin, as judged by results obtained with reduced 5-deazaFAD (5-deazaFADH(2)) where the estimated binding stoichiometry is unaffected by dialysis. The Cys315Ala.5-deazaFADH(2) complex is also air-stable but is readily oxidized by sarcosine imine, a reaction accompanied by release of weakly bound oxidized 5-deazaFAD. The dramatic difference in the binding affinity of apoCys315Ala for oxidized and reduced flavin indicates that the protein environment must induce a sizable increase in the reduction potential of noncovalently bound flavin (DeltaE(m) approximately 120 mV). The covalent flavin linkage prevents loss of weakly bound oxidized FAD and also modulates the flavin reduction potential in conjunction with the protein environment.     

NikD, an unusual amino acid oxidase essential for nikkomycin biosynthesis: structures of closed and open forms at 1.15 and 1.90 A resolution

NikD is an unusual amino-acid-oxidizing enzyme that contains covalently bound FAD, catalyzes a 4-electron oxidation of piperideine-2-carboxylic acid to picolinate, and plays a critical role in the biosynthesis of nikkomycin antibiotics. Crystal structures of closed and open forms of nikD, a two-domain enzyme, have been determined to resolutions of 1.15 and 1.9 A, respectively. The two forms differ by an 11 degrees rotation of the catalytic domain with respect to the FAD-binding domain. The active site is inaccessible to solvent in the closed form; an endogenous ligand, believed to be picolinate, is bound close to and parallel with the flavin ring, an orientation compatible with redox catalysis. The active site is solvent accessible in the open form, but the picolinate ligand is approximately perpendicular to the flavin ring and a tryptophan is stacked above the flavin ring. NikD also contains a mobile cation binding loop.     

Tautomeric rearrangement of a dihydroflavin bound to monomeric sarcosine oxidase or N-methyltryptophan oxidase

Monomeric sarcosine oxidase (MSOX) and N-methyltryptophan oxidase (MTOX) are homologous bacterial flavoenzymes that contain covalently bound flavin [8alpha-(S-cysteinyl)FAD]. Reaction of MSOX or MTOX with a small excess of sodium borohydride results in immediate flavin reduction to a species that exhibits spectral properties (lambda(max) = 405 nm with a second broad peak at 332 nm) similar to those of 3,4-dihydroflavin. The borohydride-reduced enzymes retain full catalytic activity. Substrate reduction converts the 405 nm species to an air-sensitive tetrahydroflavin that reacts with oxygen to yield unmodified oxidized enzyme. Unexpectedly, the putative 3,4-dihydroflavin bound to MSOX or MTOX is unstable in the absence of substrate. An isosbestic conversion of the 405 nm species to yield unmodified, oxidized flavin is observed when the reaction is conducted under aerobic conditions (k(obs) = 4.9 x 10(-2) min(-1)). Under anaerobic conditions, an oxygen-sensitive species resembling 1,5-dihydroflavin is formed in an isosbestic reaction that occurs at a rate similar to that of the aerobic reaction (k(obs) = 5.3 x 10(-2) min(-1)). Possible reaction of the 3,4-dihydroflavin with a second molecule of borohydride to yield an air-sensitive tetrahydroflavin is unlikely since prior scavenging of residual borohydride with excess formaldehyde had no effect on the aerobic conversion to unmodified oxidized flavin. The observed instability is attributed to a tautomeric rearrangement of the 3,4-dihydroflavin to generate 1,5-dihydroflavin, a species that is also air-sensitive. Evidence in favor of an active site facilitated tautomerization reaction is provided by the fact that the stability of the 405 nm species formed with MSOX is enhanced 200-fold upon denaturation with urea or heat. The observed tautomeric rearrangement of 3,4-dihydroflavin may provide insight regarding a related flavin tautomerization reaction that has been proposed as a key step in the biosynthesis of covalent flavin linkages.     

Biosynthesis of covalently bound flavin: isolation and in vitro flavinylation of the monomeric sarcosine oxidase apoprotein

The covalently bound FAD in native monomeric sarcosine oxidase (MSOX) is attached to the protein by a thioether bond between the 8alpha-methyl group of the flavin and Cys315. Large amounts of soluble apoenzyme are produced by controlled expression in a riboflavin-dependent Escherichia coli strain. A time-dependent increase in catalytic activity is observed upon incubation of apoMSOX with FAD, accompanied by the covalent incorporation of FAD to approximately 80% of the level observed with the native enzyme. The spectral and catalytic properties of the reconstituted enzyme are otherwise indistinguishable from those of native MSOX. The reconstitution reaction exhibits apparent second-order kinetics (k = 139 M(-)(1) min(-)(1) at 23 degrees C) and is accompanied by the formation of a stoichiometric amount of hydrogen peroxide. A time-dependent reduction of FAD is observed when the reconstitution reaction is conducted under anaerobic conditions. The results provide definitive evidence for autoflavinylation in a reaction that proceeds via a reduced flavin intermediate and requires only apoMSOX and FAD. Flavinylation of apoMSOX is not observed with 5-deazaFAD or 1-deazaFAD, an outcome attributed to a decrease in the acidity of the 8alpha-methyl group protons. Covalent flavin attachment is observed with 8-nor-8-chloroFAD in an aromatic nucleophilic displacement reaction that proceeds via a quininoid intermediate but not a reduced flavin intermediate. The reconstituted enzyme contains a modified cysteine-flavin linkage (8-nor-8-S-cysteinyl) as compared with native MSOX (8alpha-S-cysteinyl), a difference that may account for its approximately 10-fold lower catalytic activity.     

Covalent flavinylation of monomeric sarcosine oxidase: identification of a residue essential for holoenzyme biosynthesis

FAD in monomeric sarcosine oxidase (MSOX) is covalently linked to the protein by a thioether linkage between its 8alpha-methyl group and Cys315. Covalent flavinylation of apoMSOX has been shown to proceed via an autocatalytic reaction that requires only FAD and is blocked by a mutation of Cys315. His45 and Arg49 are located just above the si-face of the flavin ring, near the site of covalent attachment. His45Ala and His45Asn mutants contain covalently bound FAD and exhibit catalytic properties similar to wild-type MSOX. The results rule out a significant role for His45 in covalent flavinylation or sarcosine oxidation. In contrast, Arg49Ala and Arg49Gln mutants are isolated as catalytically inactive apoproteins. ApoArg49Ala forms a stable noncovalent complex with reduced 5-deazaFAD that exhibits properties similar to those observed for the corresponding complex with apoCys315Ala. The results show that elimination of a basic residue at position 49 blocks covalent flavinylation but does not prevent noncovalent flavin binding. The Arg49Lys mutant contains covalently bound FAD, but its flavin content is approximately 4-fold lower than wild-type MSOX. However, most of the apoprotein in the Arg49Lys preparation is reconstitutable with FAD in a reaction that exhibits kinetic parameters similar to those observed for flavinylation of wild-type apoMSOX. Although covalent flavinylation is scarcely affected, the specific activity of the Arg49Lys mutant is only 4% of that observed with wild-type MSOX. The results show that a basic residue at position 49 is essential for covalent flavinylation of MSOX and suggest that Arg49 also plays an important role in sarcosine oxidation.     

Arginine 49 is a bifunctional residue important in catalysis and biosynthesis of monomeric sarcosine oxidase: a context-sensitive model for the electrostatic impact of arginine to lysine mutations

Monomeric sarcosine oxidase (MSOX) contains covalently bound FAD and catalyzes the oxidative demethylation of sarcosine ( N-methylglycine). The side chain of Arg49 is in van der Waals contact with the si face of the flavin ring; sarcosine binds just above the re face. Covalent flavin attachment requires a basic residue (Arg or Lys) at position 49. Although flavinylation is scarcely affected, mutation of Arg49 to Lys causes a 40-fold decrease in k cat and a 150-fold decrease in k cat/ K m sarcosine. The overall structure of the Arg49Lys mutant is very similar to wild-type MSOX; the side chain of Lys49 in the mutant is nearly congruent to that of Arg49 in the wild-type enzyme. The Arg49Lys mutant exhibits several features consistent with a less electropositive active site: (1) Charge transfer bands observed for mutant enzyme complexes with competitive inhibitors absorb at higher energy than the corresponding wild-type complexes. (2) The p K a for ionization at N(3)H of FAD is more than two pH units higher in the mutant than in wild-type MSOX. (3) The reduction potential of the oxidized/radical couple in the mutant is 100 mV lower than in the wild-type enzyme. The lower reduction potential is likely to be a major cause of the reduced catalytic activity of the mutant. Electrostatic interactions with Arg49 play an important role in catalysis and covalent flavinylation. A context-sensitive model for the electrostatic impact of an arginine to lysine mutation can account for the dramatically different consequences of the Arg49Lys mutation on MSOX catalysis and holoenzyme biosysnthesis.     

On the ligands in charge-transfer complexes of porcine kidney flavoenzyme D-amino acid oxidase in three redox states: a resonance Raman study.

To investigate the structural modulation of ligands and their interaction in the active-site nanospace when they form charge-transfer (CT) complexes with D-amino acid oxidase (DAO) in three redox states, we compared Raman bands of the ligands in complex with DAO with those of ligands free in solution. Isotope-labeled ligands were synthesized for assignments of observed bands. The COO(-) stretching of ligands observed around, 1,370 cm(-1) downshifted by about 17 cm(-1) upon complexation with oxidized, semiquinoid and reduced DAO, except for the case of reduced DAO-N-methylisonicotinate complex (8 cm(-1) downward shift); the interaction mode of the carboxylate group with the guanidino group of Arg283 and the hydroxy moiety of Tyr228 of DAO is similar in the three redox states. The C=N stretching mode (1,704 cm(-1)) of Delta(1)-piperideine-2-carboxylate (D1PC) downshifted to 1,675 and 1,681 cm(-1) upon complexation with reduced and semiquinoid DAO, respectively. The downward shifts indicate that the C=N bond is weakened upon the complexation. This is probably due mainly to charge-transfer (CT) interaction between D1PC and semiquinoid or reduced flavin, i.e., the partial electron donation from the highest occupied molecular orbital (HOMO) of reduced flavin or a singly occupied molecular orbital (SOMO) of semiquinoid flavin to the lowest unoccupied molecular orbital (LUMO), an antibonding orbital, of D1PC. This speculation was supported by the finding that the magnitude of the shift is smaller by 5 cm(-1) (observed at 1,680 cm(-1)) in the case of reduced DAO reconstituted with 7,8-Cl(2)-FAD, whose reduced form has lower electron-donating ability than natural reduced FAD. The amount of electron flow was estimated by applying the theory of Friedrich and Person [(1966) J. Chem. Phys. 44, 2166-2170] to these complexes; the amounts of charge transfer from reduced FAD and reduced 7,8-Cl(2)-FAD to D1PC were estimated to be about 10 and 8% of one electron, respectively, in the CT complexes of reduced DAO with D1PC.     

Monomeric sarcosine oxidase: 1. Flavin reactivity and active site binding determinants

Monomeric sarcosine oxidase (MSOX) is an inducible bacterial flavoenzyme that catalyzes the oxidative demethylation of sarcosine (N-methylglycine) and contains covalently bound FAD [8alpha-(S-cysteinyl)FAD]. This paper describes the spectroscopic and thermodynamic properties of MSOX as well as the X-ray crystallographic characterization of three new enzyme.inhibitor complexes. MSOX stabilizes the anionic form of the oxidized flavin (pK(a) = 8.3 versus 10.4 with free FAD), forms a thermodynamically stable flavin radical, and stabilizes the anionic form of the radical (pK(a) < 6 versus pK(a) = 8.3 with free FAD). MSOX forms a covalent flavin.sulfite complex, but there appears to be a significant kinetic barrier against complex formation. Active site binding determinants were probed in thermodynamic studies with various substrate analogues whose binding was found to perturb the flavin absorption spectrum and inhibit MSOX activity. The carboxyl group of sarcosine is essential for binding since none is observed with simple amines. The amino group of sarcosine is not essential, but binding affinity depends on the nature of the substitution (CH(3)XCH(2)CO(2)(-), X = CH(2) < O < S < Se < Te), an effect which has been attributed to differences in the strength of donor-pi interactions. MSOX probably binds the zwitterionic form of sarcosine, as judged by the spectrally similar complexes formed with dimethylthioacetate [(CH(3))(2)S(+)CH(2)CO(2)(-)] and dimethylglycine (K(d) = 20.5 and 17.4 mM, respectively) and by the crystal structure of the latter. The methyl group of sarcosine is not essential but does contribute to binding affinity. The methyl group contribution varied from -3.79 to -0.65 kcal/mol with CH(3)XCH(2)CO(2)(-) depending on the nature of the heteroatom (NH(2)(+) > O > S) and appeared to be inversely correlated with heteroatom electron density. Charge-transfer complexes are formed with MSOX and CH(3)XCH(2)CO(2)(-) when X = S, Se, or Te. An excellent linear correlation is observed between the energy of the charge transfer bands and the one-electron reduction potentials of the ligands. The presence of a sulfur, selenium, or telurium atom identically positioned with respect to the flavin ring is confirmed by X-ray crystallography, although the increased atomic radius of S < Se < Te appears to simultaneously favor an alternate binding position for the heavier atoms. Although L-proline is a poor substrate, aromatic heterocyclic carboxylates containing a five-membered ring and various heteroatoms (X = NH, O, S) are good ligands (K(d, X=NH) = 1.37 mM) and form charge-transfer complexes with MSOX. The energy of the charge-transfer bands (S > O > NH) is linearly correlated with the one-electron ionization potentials of the corresponding heterocyclic rings.     

Metabolism of pipecolic acid in a Pseudomonas species. V. Pipecolate oxidase and dehydrogenase

Oxidation of pipecolate to Delta(1)-piperideine-6-carboxylate is catalyzed by pipecolate oxidase, an inducible, membrane-bound dehydrogenase associated with the electron transport components of Pseudomonas putida P2. From the oxidase, we obtained a smaller particle containing flavine adenine dinucleotide (FAD) and cytochrome b, but no longer able to catalyze electron transfer to oxygen or to cytochrome c. Certain properties of this l-pipecolate dehydrogenase, an FAD-flavoprotein, are reported.     

Inactivation of monomeric sarcosine oxidase by reaction with N-(cyclopropyl)glycine

Monomeric sarcosine oxidase (MSOX) catalyzes the oxidative demethylation of sarcosine (N-methylglycine) and contains covalently bound flavin adenine dinucleotide (FAD). The present study demonstrates that N-(cyclopropyl)glycine (CPG) is a mechanism-based inhibitor. CPG forms a charge transfer complex with MSOX that reacts under aerobic conditions to yield a covalently modified, reduced flavin (lambda(max) = 422 nm, epsilon(422) = 3.9 mM(-1) cm(-1)), accompanied by a loss of enzyme activity. The CPG-modified flavin is converted at an 8-fold slower rate to 1,5-dihydro-FAD (EFADH(2)), which reacts rapidly with oxygen to regenerate unmodified, oxidized enzyme. As a result, CPG-modified MSOX reaches a CPG-dependent steady-state concentration under aerobic conditions and reverts back to unmodified enzyme upon removal of excess reagent. No loss of activity is observed under anaerobic conditions where EFADH(2) is formed in a reaction that goes to completion at low CPG concentrations. Aerobic denaturation of CPG-modified enzyme yields unmodified, oxidized flavin at a rate similar to the anaerobic denaturation reaction, which yields 1,5-dihydro-FAD. The CPG-modified flavin can be reduced with borohydride, a reaction that blocks conversion to unmodified flavin upon removal of excess CPG or enzyme denaturation. The possible chemical mechanism of inactivation and structure of the CPG-modified flavin are discussed.     

Analysis of the kinetic and redox properties of the NADH peroxidase R303M mutant: correlation with the crystal structure

The crystal structure of the flavoprotein NADH peroxidase shows that the Arg303 side chain forms a hydrogen bond with the active-site His10 imidazole and is therefore likely to influence the catalytic mechanism. Dithionite titration of an R303M mutant [E(FAD, Cys42-sulfenic acid)] yields a two-electron reduced intermediate (EH(2)) with enhanced flavin fluorescence and almost no charge-transfer absorbance at pH 7.0; the pK(a) for the nascent Cys42-SH is increased by over 3.5 units in comparison with the wild-type EH(2) pK(a) of Cys42-SOH. The crystal structure of the R303M peroxidase has been refined at 2.45 A resolution. In addition to eliminating the Arg303 interactions with His10 and Glu14, the mutant exhibits a significant change in the conformation of the Cys42-SOH side chain relative to FAD and His10 in particular. These and other results provide a detailed understanding of Arg303 and its role in the structure and mechanism of this unique flavoprotein peroxidase.     

Structure of the sodium borohydride-reduced N-(cyclopropyl)glycine adduct of the flavoenzyme monomeric sarcosine oxidase

Monomeric sarcosine oxidase (MSOX) is a flavoprotein that contains covalently bound FAD [8a-(S-cysteinyl)FAD] and catalyzes the oxidation of sarcosine (N-methylglycine) and other secondary amino acids, such as l-proline. Our previous studies showed that N-(cyclopropyl)glycine (CPG) acts as a mechanism-based inactivator of MSOX [Zhao, G., et al. (2000) Biochemistry 39, 14341-14347]. The reaction results in the formation of a modified reduced flavin that can be further reduced and stabilized by treatment with sodium borohydride. The borohydride-reduced CPG-modified enzyme exhibits a mass increase of 63 +/- 2 Da as compared with native MSOX. The crystal structure of the modified enzyme, solved at 1.85 A resolution, shows that FAD is the only site of modification. The modified FAD contains a fused five-membered ring, linking the C(4a) and N(5) atoms of the flavin ring, with an additional oxygen atom bound to the carbon atom attached to N(5) and a tetrahedral carbon atom at flavin C(4) with a hydroxyl group attached to C(4). On the basis of the crystal structure of the borohydride-stabilized adduct, we conclude that the labile CPG-modified flavin is a 4a,5-dihydroflavin derivative with a substituent derived from the cleavage of the cyclopropyl ring in CPG. The results are consistent with CPG-mediated inactivation in a reaction initiated by single electron transfer from the amine function in CPG to FAD in MSOX, followed by collapse of the radical pair to yield a covalently modified 4a,5-dihydroflavin.     

The X-ray structure of N-methyltryptophan oxidase reveals the structural determinants of substrate specificity

The X-ray structure of monomeric N-methyltryptophan oxidase from Escherichia coli (MTOX) has been solved at 3.2 A resolution by molecular replacement methods using Bacillus sp. sarcosine oxidase structure (MSOX, 43% sequence identity) as search model. The analysis of the substrate binding site highlights the structural determinants that favour the accommodation of the bulky N-methyltryptophan residue in MTOX. In fact, although the nature and geometry of the catalytic residues within the first contact shell of the FAD moiety appear to be virtually superposable in MTOX and MSOX, the presence of a Thr residue in position 239 in MTOX (Met245 in MSOX) located at the entrance of the active site appears to play a key role for the recognition of the amino acid substrate side chain. Accordingly, a 15 fold increase in k(cat) and 100 fold decrease in K(m) for sarcosine as substrate has been achieved in MTOX upon T239M mutation, with a concomitant three-fold decrease in activity towards N-methyltryptophan. These data provide clear evidence for the presence of a catalytic core, common to the members of the methylaminoacid oxidase subfamily, and of a side chain recognition pocket, located at the entrance of the active site, that can be adjusted to host diverse aminoacids in the different enzyme species. The site involved in the covalent attachment of flavin has also been addressed by screening degenerate mutants in the relevant positions around Cys308-FAD linkage. Lys341 appears to be the key residue involved in flavin incorporation and covalent linkage.     

Identification of the oxygen activation site in monomeric sarcosine oxidase: role of Lys265 in catalysis

Monomeric sarcosine oxidase (MSOX) catalyzes the oxidation of N-methylglycine and contains covalently bound FAD that is hydrogen bonded at position N(5) to Lys265 via a bridging water. Lys265 is absent in the homologous but oxygen-unreactive FAD site in heterotetrameric sarcosine oxidase. Isolated preparations of Lys265 mutants contain little or no flavin but can be covalently reconstituted with FAD. Mutation of Lys265 to a neutral residue (Ala, Gln, Met) causes a 6000- to 9000-fold decrease in apparent turnover rate whereas a 170-fold decrease is found with Lys265Arg. Substitution of Lys265 with Met or Arg causes only a modest decrease in the rate of sarcosine oxidation (9.0- or 3.8-fold, respectively), as judged by reductive half-reaction studies which show that the reactions proceed via an initial enzyme.sarcosine charge transfer complex and a novel spectral intermediate not detected with wild-type MSOX. Oxidation of reduced wild-type MSOX (k = 2.83 x 10(5) M(-1) s(-1)) is more than 1000-fold faster than observed for the reaction of oxygen with free reduced flavin. Mutation of Lys265 to a neutral residue causes a dramatic 8000-fold decrease in oxygen reactivity whereas a 250-fold decrease is observed with Lys265Arg. The results provide definitive evidence for Lys265 as the site of oxygen activation and show that a single positively charged amino acid residue is entirely responsible for the rate acceleration observed with wild-type enzyme. Significantly, the active sites for sarcosine oxidation and oxygen reduction are located on opposite faces of the flavin ring.     

Pipecolic acid biosynthesis in Rhizoctonia leguminicola. I. The lysine saccharopine, delta 1-piperideine-6-carboxylic acid pathway

The biosynthesis of pipecolic acid from L-lysine in the fungal parasite, Rhizoctonia leguminicola has been reinvestigated. Pipecolate is then utilized to form the toxic octahydroindolizine alkaloids, slaframine and swainsonine. Incorporation studies of L-versus D-[U-14C]lysine into R. leguminicola metabolites confirmed earlier findings that L-lysine is the predominant substrate for pipecolate formation and D-lysine for alpha-N-acetyllysine (concerned in lysine catabolism). However [alpha-15N]lysine, not [epsilon-15N]lysine as previously reported, labeled pipecolate. Such findings implied that delta 1-piperideine-6-carboxylate, not delta 1-piperideine-2-carboxylate, was formed from lysine and was the immediate precursor of pipecolate. Evidence from cell-free enzyme systems established the following biosynthetic events: L-lysine A----saccharopine B----delta 1-piperideine-6-carboxylate C----pipecolate. Products of reactions A and C were identified from biological and chemical considerations. Reaction B was carried out by a previously undescribed flavin enzyme termed saccharopine oxidase. The product of reaction B, which reacted with p-dimethylaminobenzaldehyde, was reduced with Na-CNB2H3. Its NMR spectrum was identical with that of deuteriated pipecolate prepared from authentic delta 1-piperideine-6-carboxylate, but not from authentic delta 1-piperideine-2-carboxylate. Reaction B represents a branching of primary lysine metabolism from saccharopine to a secondary pathway leading to pipecolate and to octahydroindolizine alkaloids in R. leguminicola.     

The putative malate/lactate dehydrogenase from Pseudomonas putida is an NADPH-dependent delta1-piperideine-2-carboxylate/delta1-pyrroline-2-carboxylate reductase involved in the catabolism of D-lysine and D-proline

A Pseudomonas putida ATCC12633 gene, dpkA, encoding a putative protein annotated as malate/L-lactate dehydrogenase in various sequence data bases was disrupted by homologous recombination. The resultant dpkA(-) mutant was deprived of the ability to use D-lysine and also D-proline as a sole carbon source. The dpkA gene was cloned and overexpressed in Escherichia coli, and the gene product was characterized. The enzyme showed neither malate dehydrogenase nor lactate dehydrogenase activity but catalyzed the NADPH-dependent reduction of such cyclic imines as Delta(1)-piperideine-2-carboxylate and Delta(1)-pyrroline-2-carboxylate to form L-pipecolate and L-proline, respectively. NADH also served as a hydrogen donor for both substrates, although the reaction rates were less than 1% of those with NADPH. The reverse reactions were also catalyzed by the enzyme but at much lower rates. Thus, the enzyme has dual metabolic functions, and we named the enzyme Delta(1)-piperideine-2-carboxylate/Delta(1)-pyrroline-2-carboxylate reductase, the first member of a novel subclass in a large family of NAD(P)-dependent oxidoreductases.