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Biomedical subjects

Marvin L Hackert

Publications and source records attributed to Marvin L Hackert.

6 recordsLinked to original sources
PubMed2006

Evolution of enzymatic activity in the tautomerase superfamily: mechanistic and structural consequences of the L8R mutation in 4-oxalocrotonate tautomerase.

4-Oxalocrotonate tautomerase (4-OT) and trans-3-chloroacrylic acid dehalogenase (CaaD) are members of the tautomerase superfamily, a group of structurally homologous proteins that share a beta-alpha-beta fold and a catalytic amino-terminal proline. 4-OT, from Pseudomonas putida mt-2, catalyzes the conversion of 2-oxo-4-hexenedioate to 2-oxo-3-hexenedioate through the dienol intermediate 2-hydroxymuconate in a catabolic pathway for aromatic hydrocarbons. CaaD, from Pseudomonas pavonaceae 170, catalyzes the hydrolytic dehalogenation of trans-3-chloroacrylate in the trans-1,3-dichloropropene degradation pathway. Both reactions may involve an arginine-stabilized enediolate intermediate, a capability that may partially account for the low-level CaaD activity of 4-OT. Two active-site residues in 4-OT, Leu-8 and Ile-52, have now been mutated to the positionally conserved and catalytic ones in CaaD, alphaArg-8, and alphaGlu-52. The L8R and L8R/I52E mutants show improved CaaD activity (50- and 32-fold increases in k(cat)/K(m), respectively) and diminished 4-OT activity (5- and 1700-fold decreases in k(cat)/K(m), respectively). The increased efficiency of L8R-4-OT for the CaaD reaction stems primarily from an 8.8-fold increase in k(cat), whereas that of the L8R/I52E mutant is due largely to a 23-fold decrease in K(m). The presence of the additional arginine residue in the active site of L8R-4-OT does not alter the pK(a) of the Pro-1 amino group from that measured for the wild type (6.5 +/- 0.1 versus 6.4 +/- 0.2). Moreover, the crystal structure of L8R-4-OT is comparable to that of the wild type. Hence, the enhanced CaaD activity of L8R-4-OT is likely due to the additional arginine residue that can participate in substrate binding and/or stabilization of the putative enediolate intermediate. The results also suggest that the evolution of new functions within the tautomerase superfamily could be quite facile, requiring only a few strategically placed active-site mutations.

Binding Sites↗
PubMed2006

Inactivation of the phenylpyruvate tautomerase activity of macrophage migration inhibitory factor by 2-oxo-4-phenyl-3-butynoate.

Macrophage migration inhibitory factor (MIF) is an important immunoregulatory protein that has been implicated in several inflammatory diseases. MIF also has a phenylpyruvate tautomerase (PPT) activity, the role of which remains elusive in these biological activities. The acetylene compound, 2-oxo-4-phenyl-3-butynoate (2-OPB), has been synthesized and tested as a potential irreversible inhibitor of its enzymatic activity. Incubation of the compound with MIF results in the rapid and irreversible loss of the PPT activity. Mass spectral analysis established that the amino-terminal proline, previously implicated as a catalytic base in the PPT-catalyzed reaction, is the site of covalent modification. Inactivation of the PPT activity likely occurs by a Michael addition of Pro-1 to C-4 of the inhibitor. Attempts to crystallize the inactivated complex to confirm the structure of the adduct on the covalently modified Pro-1 by X-ray crystallography were not successful. Nor was it possible to unambiguously interpret electron density observed in the active sites of the native crystals soaked with the inhibitor. This may be due to crystal packing in that the side chain of Glu-16 from an adjacent trimer occupies one active site. However, this crystal contact may be partially responsible for the high-resolution quality of these MIF crystals. Nonetheless, because MIF is a member of the tautomerase superfamily, a group of structurally homologous proteins that share a beta-alpha-beta structural motif and a catalytic Pro-1, 2-OPB may find general use as a probe of tautomerase superfamily members that function as PPTs.

Alkynes↗
PubMed2005

Crystal structures of the wild-type, P1A mutant, and inactivated malonate semialdehyde decarboxylase: a structural basis for the decarboxylase and hydratase activities.

Malonate semialdehyde decarboxylase (MSAD) from Pseudomonas pavonaceae 170 is a tautomerase superfamily member that converts malonate semialdehyde to acetaldehyde by a mechanism utilizing Pro-1 and Arg-75. Pro-1 and Arg-75 have also been implicated in the hydratase activity of MSAD in which 2-oxo-3-pentynoate is processed to acetopyruvate. Crystal structures of MSAD (1.8 A resolution), the P1A mutant of MSAD (2.7 A resolution), and MSAD inactivated by 3-chloropropiolate (1.6 A resolution), a mechanism-based inhibitor activated by the hydratase activity of MSAD, have been determined. A comparison of the P1A-MSAD and MSAD structures reveals little geometric alteration, indicating that Pro-1 plays an important catalytic role but not a critical structural role. The structures of wild-type MSAD and MSAD covalently modified at Pro-1 by 3-oxopropanoate, the adduct resulting from the incubation of MSAD and 3-chloropropiolate, implicate Asp-37 as the residue that activates a water molecule for attack at C-3 of 3-chloropropiolate to initiate a Michael addition of water. The interactions of Arg-73 and Arg-75 with the C-1 carboxylate group of the adduct suggest these residues polarize the alpha,beta-unsaturated acid and facilitate the addition of water. On the basis of these structures, a mechanism for the inactivation of MSAD by 3-chloropropiolate can be formulated along with mechanisms for the decarboxylase and hydratase activities. The results also provide additional evidence supporting the hypothesis that MSAD and trans-3-chloroacrylic acid dehalogenase, a tautomerase superfamily member preceding MSAD in the trans-1,3-dichloropropene degradation pathway, diverged from a common ancestor but retained the key elements for the conjugate addition of water.

Arginine↗
PubMed2005

Solution structure of a conserved domain of antizyme: a protein regulator of polyamines.

Antizyme and its isoforms are members of an unusual yet broadly conserved family of proteins, with roles in regulating polyamine levels within cells. Antizyme has the ability to bind and inhibit the enzyme ornithine decarboxylase (ODC), targeting it for degradation at the proteasome; antizyme is also known to affect the transport of polyamines and interact with the antizyme inhibitor protein (AZI), as well as the cell-cycle protein cyclin D1. In the present work, NMR methods were used to determine the solution structure of a stable, folded domain of mammalian antizyme isoform-1 (AZ-1), consisting of amino acid residues 87-227. The protein was found to contain eight beta strands and two alpha helices, with the strands forming a mixed parallel and antiparallel beta sheet. At the level of primary sequence, antizyme is not similar to any protein of known structure, and results show that antizyme exhibits a novel arrangement of its strands and helices. Interestingly, however, the fold of antizyme is similar to that found in a family of acetyl transferases, as well as translation initiation factor IF3, despite a lack of functional relatedness between these proteins. Structural results, combined with amino acid sequence comparisons, were used to identify conserved features among the various homologues of antizyme and their isoforms. Conserved surface residues, including a cluster of acidic amino acids, were found to be located on a single face of antizyme, suggesting this surface is a possible site of interaction with target proteins such as ODC. This structural model provides an essential framework for an improved future understanding of how the different parts of antizyme play their roles in polyamine regulation.

Amino Acid Sequence↗
PubMed2002

The crystal structure of YdcE, a 4-oxalocrotonate tautomerase homologue from Escherichia coli, confirms the structural basis for oligomer diversity.

The tautomerase superfamily consists of three major families represented by 4-oxalocrotonate tautomerase (4-OT), 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), and macrophage migration inhibitory factor (MIF). The members of this superfamily are structurally homologous proteins constructed from a simple beta-alpha-beta fold that share a key mechanistic feature; they use an amino-terminal proline, which has an unusually low pK(a), as the general base in a keto-enol tautomerization. Several new members of the 4-OT family have now been identified using PSI-BLAST and categorized into five subfamilies on the basis of multiple-sequence alignments and the conservation of key catalytic and structural residues. The members of subfamily 5, which includes a hypothetical protein designated YdcE from Escherichia coli, are predicted not to form hexamers. The crystal structure of YdcE has been determined to 1.35 A resolution and confirms that it is a dimer. In addition, YdcE complexed with (E)-2-fluoro-p-hydroxycinnamate, identified as a potent competitive inhibitor of this enzyme, as well as N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) and benzoate are also presented. These latter crystal structures reveal the location of the active site and suggest a mechanism for the observed YdcE-catalyzed tautomerization reaction. The dimeric arrangement of YdcE represents a new structure in the 4-OT family and demonstrates structural diversity within the 4-OT family not previously reported.

Amino Acid Sequence↗