Protection of radical intermediates at the active site of adenosylcobalamin-dependent methylmalonyl-CoA mutase

Adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the interconversion of methylmalonyl-CoA and succinyl-CoA via radical intermediates generated by substrate-induced homolysis of the coenzyme carbon-cobalt bond. From the structure of methylmalonyl-CoA mutase it is evident that the deeply buried active site is completely shielded from solvent with only a few polar contacts made between the protein and the substrate. Site-directed mutants of amino acid His244, a residue close to the inferred site of radical chemistry, were engineered to investigate its role in catalysis. Two mutants, His244Ala and His244Gln, were characterized using kinetic and spectroscopic techniques. These results confirmed that His244 is not an essential residue. However, compared with that of the wild type, k(cat) was lowered by 10(2)- and 10(3)-fold for the His244Gln and His244Ala mutants, respectively, while the K(m) for succinyl-CoA was essentially unchanged in both cases. The primary kinetic tritium isotope effect (k(H)/k(T)) for the His244Gln mutant was 1.5 +/- 0.3, and tritium partitioning was now found to be dependent on the substrate used to initiate the reaction, indicating that the rearrangement of the substrate radical to the product radical was extremely slow. The His244Ala mutant underwent inactivation under aerobic conditions at a rate between 1 and 10% of the initial rate of turnover. The crystal structure of the His244Ala mutant, determined at 2.6 A resolution, indicated that the mutant enzyme is unaltered except for a cavity in the active site which is occupied by an ordered water molecule. Molecular oxygen reaching this cavity may lead directly to inactivation. These results indicate that His244 assists directly in the unusual carbon skeleton rearrangement and that alterations in this residue substantially lower the protection of reactive radical intermediates during catalysis.     

Evidence for a 1,2 Shift of a Hydrogen Atom in a Radical Intermediate of the Methylmalonyl-CoA Mutase Reaction

An excellent substrate of methylmalonyl-CoA mutase, methylmalonyl-carba-(dethia) coenzyme A (methylmalonyl-CH(2)-CoA), was synthesized by a chemoenzymatic method and its alpha-proton was exchanged with deuterium by long-term incubation in deuterium oxide at pH 6.9. After addition of highly purified epimerase-free methylmalonyl-CoA mutase the enzymatic rearrangement was monitored by 1H NMR spectroscopy. Already in the initial phases of the reaction only 72% of the produced succinyl-CH(2)-CoA was monodeuterated, while unlabeled and geminally dideuterated species, 14% of each, were also formed. After the addition of more enzyme the equilibrium (methylmalonyl-CoA:succinyl-CoA = 1:20) was quickly established, while the proportion of unlabeled succinyl-CH(2)-CoA rose to 30% and the geminally dideuterated species were slowly transformed to vicinally dideuterated ones. After 19 h of incubation the ratio of the unlabeled, monodeuterated, and dideuterated species was roughly 1:1:1 while no appreciable deuterium incorporation from the solvent occurred. The unexpected disproportionation of deuterium can be best explained by a 1,2 shift of a hydrogen atom in the succinyl-CH(2)-CoA radical intermediate competing with the hydrogen transfer from 5'-deoxyadenosine. A precedence for such a hydrogen shift in a radical was previously observed only in the mass spectrometer and was supported by ab initio calculations. Copyright 2000 Academic Press.     

When a spectator turns killer: suicidal electron transfer from cobalamin in methylmalonyl-CoA mutase

Methylmalonyl-CoA mutase belongs to the class of adenosylcobalamin (AdoCbl)-dependent carbon skeleton isomerases and catalyzes the rearrangement of methylmalonyl-CoA to succinyl-CoA. In this study, we have evaluated the contribution of the active site residue, R207, in the methylmalonyl-CoA mutase-catalyzed reaction. The R207Q mutation results in a 10(4)-fold decrease in k(cat) and >30-fold increase in the K(M) for the substrate, methylmalonyl-CoA. R207 and the active site residue, Y89, are within hydrogen bonding distance to the carboxylate of the substrate. In the closely related isomerase, isobutyryl-CoA mutase the homologous residues are F80 and Q198, respectively. We therefore characterized the ability of the double mutant (Y89F/R207Q) of methylmalonyl-CoA mutase as well as of the single mutants (Y89F and R207Q) to catalyze the rearrangement of n-butyryl-CoA to isobutyryl-CoA. While none of the mutant enzymes is capable of isomerizing these substrates, the R207Q (single and double) mutants exhibited irreversible inactivation upon incubation with either n-butyryl-CoA or isobutyryl-CoA. The two products observed during inactivation under both aerobic and strictly anaerobic conditions were 5'-deoxyadenosine and hydroxocobalamin, which suggested internal electron transfer from cob(II)alamin to the substrate or the 5'-deoxyadenosyl radical. Deuterium transfer from substrate to deoxyadenosine demonstrated that the substrate radical is formed and is presumably the acceptor in the electron-transfer reaction from cob(II)alamin. These studies provide evidence for the critical role of active site residues in controlling radical reactivity and thereby suppressing inactivating side reactions.     

Importance of the histidine ligand to coenzyme B12 in the reaction catalyzed by methylmalonyl-CoA mutase

Methylmalonyl-CoA mutase is an adenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the rearrangement of methylmalonyl-CoA to succinyl-CoA. The crystal structure of this protein revealed that binding of the cofactor is accompanied by a significant conformational change in which dimethylbenzimidazole, the lower axial ligand to the cobalt in solution, is replaced by His-610 donated by the active site. The contribution of the lower axial base to the approximately 10(12)-fold rate acceleration of the homolytic cleavage of the upper axial cobalt-carbon bond has been the subject of intense scrutiny in the model inorganic literature. In contrast, trans ligand effects in methylmalonyl-CoA mutase and indeed the significance of the ligand replacement are poorly understood. In this study, we have used site-directed mutagenesis to create the H610A and H610N variants of methylmalonyl-CoA mutase and report that both mutations exhibit both diminished activity (5,000- and 40,000-fold, respectively) and profoundly weakened affinity for the native cofactor, AdoCbl. In contrast, binding of the truncated cofactor analog, adenosylcobinamide, lacking the nucleotide tail, is less impaired. The catalytic failure of the His-610 mutants is in marked contrast to the phenotype of the adenosylcobinamide-GDP reconstituted wild type enzyme that exhibits only a 4-fold decrease in activity, although His-610 fails to coordinate when this cofactor analog is bound. Together, these studies suggest that His-610 may: (i) play a structural role in organizing a high affinity cofactor binding site possibly via electrostatic interactions with Asp-608 and Lys-604, as suggested by the crystal structure and (ii) play a role in catalyzing the displacement of dimethylbenzimidazole thereby facilitating the conformational change that must precede cofactor docking to the mutase active site.     

Crystallographic, kinetic, and spectroscopic study of the first ligninolytic peroxidase presenting a catalytic tyrosine

Trametes cervina lignin peroxidase (LiP) is a unique enzyme lacking the catalytic tryptophan strictly conserved in all other LiPs and versatile peroxidases (more than 30 sequences available). Recombinant T. cervina LiP and site-directed variants were investigated by crystallographic, kinetic, and spectroscopic techniques. The crystal structure shows three substrate oxidation site candidates involving His-170, Asp-146, and Tyr-181. Steady-state kinetics for oxidation of veratryl alcohol (the typical LiP substrate) by variants at the above three residues reveals a crucial role of Tyr-181 in LiP activity. Moreover, assays with ferrocytochrome c show that its ability to oxidize large molecules (a requisite property for oxidation of the lignin polymer) originates in Tyr-181. This residue is also involved in the oxidation of 1,4-dimethoxybenzene, a reaction initiated by the one-electron abstraction with formation of substrate cation radical, as described for the well known Phanerochaete chrysosporium LiP. Detailed spectroscopic and kinetic investigations, including low temperature EPR, show that the porphyrin radical in the two-electron activated T. cervina LiP is unstable and rapidly receives one electron from Tyr-181, forming a catalytic protein radical, which is identified as an H-bonded neutral tyrosyl radical. The crystal structure reveals a partially exposed location of Tyr-181, compatible with its catalytic role, and several neighbor residues probably contributing to catalysis: (i) by enabling substrate recognition by aromatic interactions; (ii) by acting as proton acceptor/donor from Tyr-181 or H-bonding the radical form; and (iii) by providing the acidic environment that would facilitate oxidation. This is the first structure-function study of the only ligninolytic peroxidase described to date that has a catalytic tyrosine.     

Stabilization of the imidazole ring of His-195 at the active site of chloramphenicol acetyltransferase

The imidazole of His-195 plays an essential role in the proposed general base mechanism of chloramphenicol acetyltransferase (CAT). The structure of the binary complex of CATIII and chloramphenicol suggests that two unusual interactions might determine the conformation of the side chain of His-195: (i) an intraresidue hydrogen bond between its main chain carbonyl and the protonated N delta 1 of the imidazole ring and (ii) face-to-face van der Waals contact between the His-195 imidazole group and the aromatic side chain of Tyr-25. Tyr-25 also makes a hydrogen bond, via its phenolic hydroxyl, to the carbonyl oxygen of the substrate chloramphenicol. Replacement of Tyr-25 of CATIII by phenylalanine results in a modest increase in the Km for chloramphenicol (from 11.6 to 14.6 microM) and a 2-fold fall in kcat (599 to 258 s-1), indicative of a free energy contribution to transition state binding of 0.6 kcal mol-1 for the hydrogen bond between Tyr-25 and chloramphenicol. In contrast, substitution of Tyr-25 by alanine yields an enzyme that is dramatically impaired in its ability to bind chloramphenicol (Km = 173 microM). As kcat for Ala-25 CAT is also reduced (130 s-1), the loss of the aryl group results in a 69-fold decrease in kcat/Km, corresponding to a free energy contribution to binding and catalysis of 2.5 kcal mol-1. In addition to the loss of the hydrogen bond between Tyr-25 and chloramphenicol, the loss of substrate affinity in Ala-25 CAT may be a direct consequence of reduced hydrophobicity of the chloramphenicol-binding site and/or the loss of critical constraints on the precise conformation of the catalytic imidazole. However, as with wild type CAT, inactivation of Ala-25 CAT by the affinity reagent 3-(bromoacetyl) chloramphenicol is accompanied by modification solely at N epsilon 2 of His-195. Hence, the results demonstrate that tautomeric stabilization of the imidazole ring persists in the absence of van der Waals interactions with the side chain of Tyr-25, probably as a consequence of hydrogen bonding between the protonated N delta 1 and the carbonyl oxygen of His-195.     

Human peroxisomal multifunctional enzyme type 2. Site-directed mutagenesis studies show the importance of two protic residues for 2-enoyl-CoA hydratase 2 activity

Beta-oxidation of acyl-CoAs in mammalian peroxisomes can occur via either multifunctional enzyme type 1 (MFE-1) or type 2 (MFE-2), both of which catalyze the hydration of trans-2-enoyl-CoA and the dehydrogenation of 3-hydroxyacyl-CoA, but with opposite chiral specificity. Amino acid sequence alignment of the 2-enoyl-CoA hydratase 2 domain in human MFE-2 with other MFE-2s reveals conserved protic residues: Tyr-347, Glu-366, Asp-370, His-406, Glu-408, Tyr-410, Asp-490, Tyr-505, Asp-510, His-515, Asp-517, and His-532. To investigate their potential roles in catalysis, each residue was replaced by alanine in site-directed mutagenesis, and the resulting constructs were tested for complementation in a yeast. After additional screening, the wild type and noncomplementing E366A and D510A variants were expressed and characterized. The purified proteins have similar secondary structural elements, with the same subunit composition. The E366A variant had a k(cat)/K(m) value 100 times lower than that of the wild type MFE-2 at pH 5, whereas the D510A variant was inactive. Asp-510 was imbedded in a novel hydratase 2 motif found in the hydratase 2 proteins. The data show that the hydratase 2 reaction catalyzed by MFE-2 requires two protic residues, Glu-366 and Asp-510, suggesting that their catalytic role may be equivalent to that of the two catalytic residues of hydratase 1.     

Identification of the stable free radical tyrosine residue in ribonucleotide reductase. A sequence comparison

The small subunit of ribonucleoside diphosphate reductase contains a unique tyrosine radical and a binuclear iron center. An alignment of different primary structures of the small subunit in Escherichia coli, the marine mollusc Spisula solidissima, Epstein Barr and Herpes simplex viruses shows that regions comprising residues 115-122, 204-212 and 234-241 (in E.coli numbering) are strikingly similar and are likely to be recognized as functionally important. Two of 16 tyrosine residues and 2 of 8 histidine residues are conserved. We propose that Tyr-122 is responsible for radical stabilization and that His-118 and His-241 together with Glu-115 and Asp-237 or Glu-238 are ligands of the iron center.     

Site-specific mutagenesis of lysine-204, tyrosine-224, tyrosine-228, and histidine-307 of porcine kidney D-amino acid oxidase and the implications as to its catalytic function

In order to evaluate the possible contributions of Lys-204, Tyr-224, Tyr-228, and His-307 in porcine kidney D-amino acid oxidase [EC 1.4.3.3] (DAO) to its catalytic function, we constructed four point mutant cDNAs encoding enzymes possessing Glu-204, Phe-224, Phe-228, and Leu-307 by oligonucleotide-directed in vitro mutagenesis. The four mutant cDNAs and the wild type cDNA could be expressed in vitro with similar efficiencies and about 200 ng of each enzyme protein was produced from 5 micrograms of the respective capped RNA. The electrophoretic mobilities of the in vitro synthesized mutant enzymes on SDS-polyacrylamide gel were almost identical with that of the wild type DAO, and the molecular weight was calculated to be 38,000. The Glu-204 and Phe-224 mutant DAOs showed comparable enzyme activities to that of the wild type enzyme, and were inhibited strongly by sodium benzoate, a potent competitive inhibitor of DAO. The kinetic parameters of the two mutant DAOs were also comparable to those of the wild type DAO. On the other hand, the Phe-228 and Leu-307 mutant DAOs showed no detectable activity. The results indicate that Tyr-228 and His-307 play important roles as to the constitution of the active site or participate in the reaction directly, while Lys-204 and Tyr-224 are not essential in the enzyme reaction.     

Crystal structure of the Y52F/Y73F double mutant of phospholipase A2: increased hydrophobic interactions of the phenyl groups compensate for the disrupted hydrogen bonds of the tyrosines.

The enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 ester bond of membrane phospholipids. The highly conserved Tyr residues 52 and 73 in the enzyme form hydrogen bonds to the carboxylate group of the catalytic Asp-99. These hydrogen bonds were initially regarded as essential for the interfacial recognition and the stability of the overall catalytic network. The elimination of the hydrogen bonds involving the phenolic hydroxyl groups of the Tyr-52 and -73 by changing them to Phe lowered the stability but did not significantly affect the catalytic activity of the enzyme. The X-ray crystal structure of the double mutant Y52F/Y73F has been determined at 1.93 A resolution to study the effect of the mutation on the structure. The crystals are trigonal, space group P3(1)21, with cell parameters a = b = 46.3 A and c = 102.95 A. Intensity data were collected on a Siemens area detector, 8,024 reflections were unique with an R(sym) of 4.5% out of a total of 27,203. The structure was refined using all the unique reflections by XPLOR to a final R-factor of 18.6% for 955 protein atoms, 91 water molecules, and 1 calcium ion. The root mean square deviation for the alpha-carbon atoms between the double mutant and wild type was 0.56 A. The crystal structure revealed that four hydrogen bonds were lost in the catalytic network; three involving the tyrosines and one involving Pro-68. However, the hydrogen bonds of the catalytic triad, His-48, Asp-99, and the catalytic water, are retained. There is no additional solvent molecule at the active site to replace the missing hydroxyl groups; instead, the replacement of the phenolic OH groups by H atoms draws the Phe residues closer to the neighboring residues compared to wild type; Phe-52 moves toward His-48 and Asp-99 of the catalytic diad, and Phe-73 moves toward Met-8, both by about 0.5 A. The closing of the voids left by the OH groups increases the hydrophobic interactions compensating for the lost hydrogen bonds. The conservation of the triad hydrogen bonds and the stabilization of the active site by the increased hydrophobic interactions could explain why the double mutant has activity similar to wild type. The results indicate that the aspartyl carboxylate group of the catalytic triad can function alone without additional support from the hydrogen bonds of the two Tyr residues.     

Assignment of the aromatic 1H-NMR resonances of myotoxin a isolated from the venom of Crotalus viridis viridis

The 400 MHz 1H-NMR spectrum of myotoxin a from the venom of Crotalus viridis viridis is described. The identification of spin systems in the aromatic region corresponding to the six aromatic residues of myotoxin a was completed using both one- and two-dimensional NMR spectroscopy and the pH dependence of chemical shifts. Assignments of these spin systems to specific residues was possible for the singly occurring amino acids Tyr-1 and Phe-12. Resonances from Tyr-1, His-5 and His-10 were shifted significantly from their random coil values in a pH-dependent manner. These shift perturbations were deemed evidence of a helical arrangement of the amino terminal region which placed these residues in close proximity to each other.     

A proton nuclear magnetic resonance study on the solution structure of crotamine

Proton nuclear magnetic resonance (NMR) spectra of crotamine, a myotoxic protein from a Brazilian rattlesnake (Crotalus durissus terrificus), have been analyzed. All the aromatic proton resonances have been assigned to amino acid types, and those from Tyr-1, Phe-12, and Phe-25 to the individual residues. ThepH dependence of the chemical shifts of the aromatic proton resonances indicates that Tyr-1 and one of the two histidines (His-5 or His-10) are in close proximity. A conformational transition takes place at acidicpH, together with immobilization of Met-28 and His-5 or His-10. Two sets of proton resonances have been observed for He-17 and His-5 or His-10, which suggests the presence of two structural states for the crotamine molecule in solution.     

Quantum catalysis in B12-dependent methylmalonyl-CoA mutase: experimental and computational insights

B12-dependent methylmalonyl-CoA mutase catalyses the interchange of a hydrogen atom and the carbonyl-CoA group on adjacent carbons of methylmalonyl-CoA to give the rearranged product, succinyl-CoA. The first step in this reaction involves the transient generation of cofactor radicals by homolytic rupture of the cobalt-carbon bond to generate the deoxyadenosyl radical and cob(II)alamin. This step exhibits a curious sensitivity to isotopic substitution in the substrate, methylmalonyl-CoA, which has been interpreted as evidence for kinetic coupling. The magnitude of the isotopic discrimination is large and a deuterium isotope effect ranging from 35.6 at 20 degrees C to 49.9 at 5 degrees C has been recorded. Arrhenius analysis of the temperature dependence of this isotope effect provides evidence for quantum tunnelling in this hydrogen transfer step. The mechanistic complexity of the observed rate constant for cobalt-carbon bond homolysis together with the spectroscopically silent nature of many of the component steps limits the insights that can be derived by experimental approaches alone. Computational studies using a newly developed geometry optimization scheme that allows determination of the transition state in the full quantum mechanical/molecular mechanical coordinate space have yielded novel insights into the strategy deployed for labilizing the cobalt-carbon bond and poising the resulting deoxyadenosyl radical for subsequent hydrogen atom abstraction.     

The kinetic analysis of recognition of the damaged nucleotides by mutant forms of the 8-oxoguanine DNA glycosylase hOGG1

We have investigated the role of Tyr-203, His-270, and Lys-249 amino acid residues from the 8-oxoguanine glycosylase (hOGG1) active site in the process of recognition of 7,8-dihydro-8-oxoguanine (oxoG) damaged nucleotide and in the catalytic stages of enzymatic reaction. The pre-steady state kinetic analysis of conformational transitions of mutant forms of the enzyme and model DNA substrates during the enzymatic process revealed that the studied amino acid residues are involved in the specific binding of DNA substrates. The Tyr-203 is responsible for recognition of the damaged nucleotide; interaction between His-270 and DNA is necessary for the formation of the catalytically active complex with the oxoG-containing DNA. The Lys-249 acts not only as one of the catalytically important amino acids of the active site of the enzyme, but also plays a significant role in the formation of specific enzyme–substrate complex. The present study significantly complements the molecular-kinetic model of the enzymatic reaction and helps to clarify the origin of the high specificity of hOGG1 to oxidized bases in DNA.     

A thiamin-bound, pre-decarboxylation reaction intermediate analogue in the pyruvate dehydrogenase E1 subunit induces large scale disorder-to-order transformations in the enzyme and reveals novel structural features in the covalently bound adduct

The crystal structure of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined with phosphonolactylthiamin diphosphate (PLThDP) in its active site. PLThDP serves as a structural and electrostatic analogue of the natural intermediate alpha-lactylthiamin diphosphate (LThDP), in which the carboxylate from the natural substrate pyruvate is replaced by a phosphonate group. This represents the first example of an experimentally determined, three-dimensional structure of a thiamin diphosphate (ThDP)-dependent enzyme containing a covalently bound, pre-decarboxylation reaction intermediate analogue and should serve as a model for the corresponding intermediates in other ThDP-dependent decarboxylases. Regarding the PDHc-specific reaction, the presence of PLThDP induces large scale conformational changes in the enzyme. In conjunction with the E1-PLThDP and E1-ThDP structures, analysis of a H407A E1-PLThDP variant structure shows that an interaction between His-407 and PLThDP is essential for stabilization of two loop regions in the active site that are otherwise disordered in the absence of intermediate analogue. This ordering completes formation of the active site and creates a new ordered surface likely involved in interactions with the lipoyl domains of E2s within the PDHc complex. The tetrahedral intermediate analogue is tightly held in the active site through direct hydrogen bonds to residues His-407, Tyr-599, and His-640 and reveals a new, enzyme-induced, strain-related feature that appears to aid in the decarboxylation process. This feature is almost certainly present in all ThDP-dependent decarboxylases; thus its inclusion in our understanding of general thiamin catalysis is important.     

Characterization of a succinyl-CoA radical-cob(II)alamin spin triplet intermediate in the reaction catalyzed by adenosylcobalamin-dependent methylmalonyl-CoA mutase

The electron paramagnetic resonance (EPR) spectrum of an intermediate freeze trapped during the steady state of the reaction catalyzed by the adenosylcobalamin (AdoCbl)-dependent enzyme, methylmalonyl-CoA mutase, has been studied. The EPR spectrum is that of a hybrid triplet spin system created as a result of strong electron-electron spin coupling between an organic radical and the low-spin Co(2+) in cob(II)alamin. The spectrum was analyzed by simulation to obtain the zero-field splitting (ZFS) parameters and Euler angles relating the radical-to-cobalt interspin vector to the g axis system of the low-spin Co(2+). Labeling of the substrate with (13)C and (2)H was used to probe the identity of the organic radical partner in the triplet spin system. The patterns of inhomogeneous broadening in the EPR signals produced by [2'-(13)C]methylmalonyl-CoA and [2-(13)C]methylmalonyl-CoA as well as line narrowing resulting from deuterium substitution in the substrate were consistent with those expected for a succinyl-CoA radical wherein the unpaired electron was centered on the carbon alpha to the free carboxyate group of the rearranged radical. The interspin distance and the Euler angles were used to position this product radical into the active site of the enzyme.     

Involvement of the flavin si-face tyrosine on the structure and function of ferredoxin-NADP+ reductases

In ferredoxin-NADP(+) reductase (FNR), FAD is bound outside of an anti-parallel beta-barrel with the isoalloxazine lying in a two-tyrosine pocket. To elucidate the function of the flavin si-face tyrosine (Tyr-89 in pea FNR) on the enzyme structure and catalysis, we performed ab initio molecular orbital calculations and site-directed mutagenesis. Our results indicate that the position of Tyr-89 in pea FNR is mainly governed by the energetic minimum of the pairwise interaction between the phenol ring and the flavin. Moreover, most of FNR-like proteins displayed geometries for the si-face tyrosine phenol and the flavin, which correspond to the more negative free energy theoretical value. FNR mutants were obtained replacing Tyr-89 by Phe, Trp, Ser, or Gly. Structural and functional features of purified FNR mutants indicate that aromaticity on residue 89 is essential for FAD binding and proper folding of the protein. Moreover, hydrogen bonding through the Tyr-89 hydroxyl group may be responsible of the correct positioning of FAD and the substrate NADP(+)     

Oxygen-dependent coproporphyrinogen III oxidase (HemF) from Escherichia coli is stimulated by manganese

During heme biosynthesis in Escherichia coli two structurally unrelated enzymes, one oxygen-dependent (HemF) and one oxygen-independent (HemN), are able to catalyze the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX. Oxygen-dependent coproporphyrinogen III oxidase was produced by overexpression of the E. coli hemF in E. coli and purified to apparent homogeneity. The dimeric enzyme showed a Km value of 2.6 microm for coproporphyrinogen III with a kcat value of 0.17 min-1 at its optimal pH of 6. HemF does not utilize protoporphyrinogen IX or coproporphyrin III as substrates and is inhibited by protoporphyrin IX. Molecular oxygen is essential for the enzymatic reaction. Single turnover experiments with oxygen-loaded HemF under anaerobic conditions demonstrated electron acceptor function for oxygen during the oxidative decarboxylation reaction with the concomitant formation of H2O2. Metal chelator treatment inactivated E. coli HemF. Only the addition of manganese fully restored coproporphyrinogen III oxidase activity. Evidence for the involvement of four highly conserved histidine residues (His-96, His-106, His-145, and His-175) in manganese coordination was obtained. One catalytically important tryptophan residue was localized in position 274. None of the tested highly conserved cysteine (Cys-167), tyrosine (Tyr-135, Tyr-160, Tyr-170, Tyr-213, Tyr-240, and Tyr-276), and tryptophan residues (Trp-36, Trp-123, Trp-166, and Trp-298) were found important for HemF activity. Moreover, mutation of a potential nucleotide binding motif (GGGXXTP) did not affect HemF activity. Two alternative routes for HemF-mediated catalysis, one metal-dependent, the other metal-independent, are proposed.     

Controlling the reactivity of radical intermediates by coenzyme B(12)-dependent methylmalonyl-CoA mutase

Adenosylcobalamin or coenzyme B(12)-dependent enzymes are members of the still relatively small group of radical enzymes and catalyse 1,2-rearrangement reactions. A member of this family is methylmalonyl-CoA mutase, which catalyses the isomerization of methylmalonyl-CoA to succinyl-CoA and, unlike the others, is present in both bacteria and animals. Enzymes that catalyse some of the most chemically challenging reactions are the ones that tend to deploy radical chemistry. The use of radical intermediates in an active site lined with amino acid side chains that threaten to extinguish the reaction by presenting alternative groups for abstraction poses the conundrum of how the enzymes control their reactivity. In this review, insights into this issue that have emerged from kinetic, mutagenesis and structural studies are described for methylmalonyl-CoA mutase.     

The role of the conserved residues His-246, His-199, and Tyr-255 in the catalysis of catechol 2,3-dioxygenase from Pseudomonas stutzeri OX1

Catechol 2,3-dioxygenase (C2,3O) from Pseudomonas stutzeri OX1, which is able to grow on various aromatic substrates as the sole source of carbon and energy, has been expressed in Escherichia coli, purified, characterized, and found to be very similar to other dioxygenases from Pseudomonas species. Interestingly, the activity of the protein shows a rather unusual pH dependence when assayed on catechol. A model of the catalytic mechanism was developed that is able to reproduce the catalytic behavior of the protein as a function of the pH. The model includes multiple equilibria and four productive intermediates with different ionization states of the enzyme-substrate complex. The fitting of the theoretical curve to the experimental data suggests that a tyrosine and two histidine residues are involved in catalysis. Mutants (H246N)-, (H246A)-, (H199N)- and (Y255F)-C2,3O were produced to investigate the role of highly conserved His-199, His-246, and Tyr-255. The strongly reduced activity of the mutants suggests a primary catalytic role for each of these residues. Moreover, mutants at positions 199 and 246 display pH profiles different from that of the wild-type protein, thus indicating that residues His-246 and His-199 play a role in determining the unusual pH dependence of the enzyme. In addition, electron-withdrawing groups on catechol, which increase the acidity of the phenolic hydroxyl group, are able to counterbalance the effect of the mutation H246N in reducing catalytic activity but cause a further reduction of the activity of (H199N)-C2,3O. This finding suggests that His-246 is involved in the initial catechol deprotonation, whereas His-199 promotes the reaction between oxygen and the aromatic ring.