Biomedical subjects

Michael L Neidig

Publications and source records attributed to Michael L Neidig.

7 recordsLinked to original sources

PubMed2006

Spectroscopic and electronic structure studies of the role of active site interactions in the decarboxylation reaction of alpha-keto acid-dependent dioxygenases.

The alpha-ketoglutate (alpha-KG)-dependent dioxygenases are a large class of mononuclear non-heme iron enzymes that require Fe(II), alpha-KG and dioxygen for catalysis, with the alpha-KG cosubstrate supplying the two additional electrons required for dioxygen activation. A sub-class of these enzymes exists in which the alpha-keto acid is covalently attached to the substrate, including (4-hydroxy)mandelate synthase (HmaS) and (4-hydroxyphenyl)pyruvate dioxygenase (HPPD) which utilize the same substrate but exhibit two different general reactivities (H-atom abstraction and electrophilic attack). Previous kinetic studies of Streptomyces avermitilis HPPD have shown that the substrate analog phenylpyruvate (PPA), which only differs from the normal substrate (4-hydroxyphenyl)pyruvate (HPP) by the absence of a para-hydroxyl group on the aromatic ring, does not induce a reaction with dioxygen. While an Fe(IV)O intermediate is proposed to be the reactive species in converting substrate to product, the key step utilizing O(2) to generate this species is the decarboxylation of the alpha-keto acid. It has been generally proposed that the two requirements for decarboxylation are bidentate coordination of the alpha-keto acid to Fe(II) and the presence of a 5C Fe(II) site for the O(2) reaction. Circular dichroism and magnetic circular dichroism studies have been performed and indicate that both enzyme complexes with PPA are similar with bidentate alpha-KG coordination and a 5C Fe(II) site. However, kinetic studies indicate that while HmaS reacts with PPA in a coupled reaction similar to the reaction with HPP, HPPD reacts with PPA in an uncoupled reaction at an approximately 10(5)-fold decreased rate compared to the reaction with HPP. A key difference is spectroscopically observed in the n-->pi( *) transition of the HPPD/Fe(II)/PPA complex which, based upon correlation to density functional theory calculations, is suggested to result from H-bonding between a nearby residue and the carboxylate group of the alpha-keto acid. Such an interaction would disfavor the decarboxylation reaction by stabilizing electron density on the carboxylate group such that the oxidative cleavage to yield CO(2) is disfavored.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed2006

Kinetic, spectroscopic, and structural investigations of the soybean lipoxygenase-1 first-coordination sphere mutant, Asn694Gly.

In wild-type soybean LO-1 (WT sLO-1), Asn694 is a weak sixth ligand that is thought to be critical for enzymatic catalysis. In this investigation, N694G sLO-1 was studied to probe its contribution at this sixth ligand position to the kinetic and spectroscopic properties. The k(cat) value of N694G is approximately 230 times lower than that of WT sLO-1 at 25 degrees C, which can be partially explained by a lowered reduction potential of the iron as seen as a shift in the visible ligand-to-metal charge-transfer band (lambda(max) = 410 nm for N694G and lambda(max) = 425 nm for WT sLO-1). This conclusion was supported by a faster rate of oxidation of N694G by the product than that of WT sLO-1 (k(2) = 606 s(-)(1) for N694G and k(2) = 349 s(-)(1) for WT sLO-1). These results suggest a stronger ligand at the active site iron than the native Asn694, which is confirmed to be a water bound to the Fe(II) in the crystal structure. This produces a six-coordinate circular dichroism/magnetic circular dichroism (CD/MCD) spectra for ferrous N694G and an intermediate rhombic electron paramagnetic resonance (EPR) signal for ferric N694G. The EPR spectrum and its pH dependence suggest that the coordination environment of ferric N694G contains one hydroxide and one water. On the basis of both kinetic and structural factors, we propose that the Asn694 water-derived ligand would likely be a hydroxide and the active site, water-derived ligand a water in the ferric state, hence lowering the reaction rate of N694G more than would be expected from the lowered reduction potential alone.

Amino Acid Substitution↗

PubMed2006

Spectroscopic and electronic structure studies of aromatic electrophilic attack and hydrogen-atom abstraction by non-heme iron enzymes.

(4-Hydroxy)mandelate synthase (HmaS) and (4-hydroxyphenyl)pyruvate dioxygenase (HPPD) are two alpha-keto acid dependent mononuclear non-heme iron enzymes that use the same substrate, (4-hydroxyphenyl)pyruvate, but exhibit two different general reactivities. HmaS performs hydrogen-atom abstraction to yield benzylic hydroxylated product (S)-(4-hydroxy)mandelate, whereas HPPD utilizes an electrophilic attack mechanism that results in aromatic hydroxylated product homogentisate. These enzymes provide a unique opportunity to directly evaluate the similarities and differences in the reaction pathways used for these two reactivities. An Fe(II) methodology using CD, magnetic CD, and variable-temperature, variable-field magnetic CD spectroscopies was applied to HmaS and compared with that for HPPD to evaluate the factors that affect substrate interactions at the active site and to correlate these to the different reactivities exhibited by HmaS and HPPD to the same substrate. Combined with density functional theory calculations, we found that HmaS and HPPD have similar substrate-bound complexes and that the role of the protein pocket in determining the different reactivities exhibited by these enzymes (hydrogen-atom abstraction vs. aromatic electrophilic attack) is to properly orient the substrate, allowing for ligand field geometric changes along the reaction coordinate. Elongation of the Fe(IV) O bond in the transition state leads to dominant Fe(III) O(*-) character, which significantly contributes to the reactivity with either the aromatic pi-system or the C H sigma-bond.

4-Hydroxyphenylpyruvate Dioxygenase↗

PubMed2005

Structure-function correlations in oxygen activating non-heme iron enzymes.

A large group of mononuclear non-heme iron enzymes exist which activate dioxygen to catalyze key biochemical transformations, including many of medical, pharmaceutical and environmental significance. These enzymes utilize high-spin Fe(II) active sites and additional reducing equivalents from cofactors or substrates to react with O2 to yield iron-oxygen intermediates competent to transform substrate to product. While Fe(II) sites have been difficult to study due to the lack of dominant spectroscopic features, a spectroscopic methodology has been developed which allows the elucidation of the geometric and electronic structures of these active sites and provides molecular level insight into the mechanisms of catalysis. This review provides a summary of this methodology with emphasis on its application to the determination of important active site structure-function correlations in mononuclear non-heme iron enzymes. These studies provide key insight into the mechanisms of oxygen activation, active site features that contribute to differences in reactivity and, combined with theoretical calculations and model studies, the nature of oxygen intermediates active in catalysis.

Enzyme Activation↗

PubMed2005

Spectroscopic and computational studies of NTBC bound to the non-heme iron enzyme (4-hydroxyphenyl)pyruvate dioxygenase: active site contributions to drug inhibition.

(4-Hydroxyphenyl)pyruvate dioxygenase (HPPD) is an alpha-keto-acid-dependent dioxygenase which catalyzes the conversion of (4-hydroxyphenyl)pyruvate (HPP) to homogentisate as part of tyrosine catabolism. While several di- and tri-ketone alkaloids are known as inhibitors of HPPD and used commercially as herbicides, one such inhibitor, [2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC), has also been used therapeutically to treat type I tyrosinemia and alkaptonuria in humans. To gain further insight into the mechanism of inhibition by NTBC, a combination of CD/MCD spectroscopy and DFT calculations of HPPD/Fe(II)/NTBC has been performed to evaluate the contribution of the Fe(II)-NTBC bonding interaction to the high affinity of this drug for the enzyme. The results indicate that the bonding of NTBC to Fe(II) is very similar to that for HPP, both involving similar pi-backbonding interactions between NTBC/HPP and Fe(II). Combined with the result that the calculated binding energy of NTBC is, in fact, approximately 3 kcal/mol less than that for HPP, the bidentate coordination of NTBC to Fe(II) is not solely responsible for its extremely high affinity for the enzyme. Thus, the pi-stacking interactions between the aromatic rings of NTBC and two phenyalanine residues, as observed in the crystallography of the HPPD/Fe(II)/NTBC complex, appear to be responsible for the observed high affinity of drug binding.

4-Hydroxyphenylpyruvate Dioxygenase↗

PubMed2004

CD and MCD studies of the non-heme ferrous active site in (4-hydroxyphenyl)pyruvate dioxygenase: correlation between oxygen activation in the extradiol and alpha-KG-dependent dioxygenases.

(4-Hydroxyphenyl)pyruvate dioxygenase (HPPD) is an unusual alpha-keto acid-dependent non-heme iron dioxygenase as it incorporates both atoms of dioxygen into a single substrate, paralleling the extradiol dioxygenases. CD/MCD studies of the catalytically active ferrous site and its interaction with substrate reveal a geometic and electronic structure and mechanistic approach to oxygen activation which bridges those of the alpha-KG-dependent and the extradiol dioxygenases.

4-Hydroxyphenylpyruvate Dioxygenase↗

PubMed2003

Spectroscopic characterization of soybean lipoxygenase-1 mutants: the role of second coordination sphere residues in the regulation of enzyme activity.

Lipoxygenases are non-heme iron enzymes, which catalyze the stereo- and regiospecific hydroperoxidation of unsaturated fatty acids. Spectroscopic studies on soybean lipoxygenase have shown that the ferrous form of the enzyme is a mixture of five- and six-coordinate species (40 and 60%, respectively). Addition of substrate leads to a purely six-coordinate form. A series of mutations in the second coordination sphere (Q697E, Q697N, Q495A, and Q495E) were generated, and the structures of the mutants were solved by crystallography [Tomchick et al. (2001) Biochemistry 40, 7509-7517]. While this study clearly showed the contribution of H-bond interactions between the first and the second coordination spheres in catalysis, no correlation with the coordination environment of the Fe(II) was observed. A recent study using density-functional theory [Lehnert and Solomon (2002) J. Biol. Inorg. Chem. 8, 294-305] indicated that coordination flexibility, involving the Asn694 ligand, is regulated via H-bond interactions. In this paper, we investigate the solution structures of the second coordination sphere mutants using CD and MCD spectroscopy since these techniques are more sensitive indicators of the first coordination sphere ligation of Fe(II) systems. Our data demonstrate that the iron coordination environment directly relates to activity, with the mutations that have the ability to form a five-coordinate/six-coordinate mixture being more active. We propose that the H-bond between the weak Asn694 ligand and the Gln697 plays a key role in the modulation of the coordination flexibility of Asn694, and thus, is crucial for the regulation of enzyme reactivity.

Amino Acid Substitution↗