Browsing by Subject "Tautomerase superfamily"

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Characterization of the activities of trans-3-chloroacrylic acid dehalogenase and cis-3-chloroacrylic acid dehalogenase and malonate semialdehyde decarboxylase homologues : mechanism and evolutionary implications
(2009-12) Serrano, Hector, doctor of pharmacy; Whitman, Christian P.
Members of the tautomerase superfamily are characterized by a [beta-alpha-beta] structural fold motif as well as a catalytic N-terminal proline (Pro-1). Three members of the superfamily are involved in the degradation of the nematocide 1,3-dichloropopene; trans-3-chloroacrylic acid dehalogenase (CaaD), cis-3-chloroacrylic acid dehalogenase (cis-CaaD) and malonate semialdehyde decarboxylase (MSAD). CaaD and cis-CaaD are involved in the hydration of their respective 3-chloroacrylic acid isomers to generate malonate semialdehyde. Subsequently, MSAD is responsible for catalyzing the decarboxylation of malonate semialdehyde to generate acetaldehyde. All three of these enzymes contain an N-terminal proline (Pro-1) that functions as a general acid, in contrast to other tautomerase superfamily members, such as 4-oxalocrotonate tautomerase (4-OT) and macrophage migration inhibitory factor (MIF), where Pro-1 acts as a catalytic base. Two new members of the tautomerase superfamily have been cloned and characterized; FG41 MSAD, a homologue of MSAD from Coryneform Bacterium strain FG41, and Cg10062, a homologue of cis-CaaD from Corynebacterium glutamicum, with low-level cis-CaaD and CaaD activities. As part of an effort to delineate the mechanisms of CaaD, cis-CaaD and Cg10062, secondary activities for all three enzymes were characterized. The three enzymes function as efficient phenylpyruvate tautomerases (PPT), converting phenylenolpyruvate to phenylpyruvate. The activity also indicates that the active site of these three enzymes can ketonize enol compounds, thereby providing evidence for the presence of an enediolate intermediate. The characterization of FG41 MSAD uncovered an activity it shares with MSAD. FG41 MSAD catalyzes the hydration of 2-oxo-3-pentynoate, but at a rate that is 50-fold less efficient than that of MSAD (as assessed by kcat/Km values). Mutagenesis studies of FG41 MSAD revealed that a single mutation resulted in a 8-fold increase in the activity. The characterization of Cg10062 and attempts to enhance the low-level cis-CaaD activity demonstrated the need for a bacterial screen that could screen a library of mutants. The resulting bacterial screen could be used to screen other members of the superfamily for dehalogenase activity. An in-depth exploration of the Cg10062 and FG41 MSAD activities may lead to a better understanding of the mechanism of cis-CaaD and MSAD and further delineate the evolutionary pathway for the tautomerase superfamily.
Investigations of halogenated intermediates in the meta-fission pathway and of the tautomerase superfamily in biosynthetic pathways
(2017-01-09) Stack, Tyler Mason Maxwell; Whitman, Christian P.; Fast, Walter L; Liu, Hung-wen (Ben); Hoffman, David; Zhang, Yan (Jessie)
The tautomerase superfamily (TSF) is a group of relatively short monomers (60-150 amino acid residues) that is characterized by a core β-α-β fold, along with an N-terminal proline. In the characterized TSF members, Pro-1 is a critical catalytic group. 4-oxalocrotonate tautomerase (4-OT) is the first characterized member of this superfamily, and is part of the bacterial meta-fission pathway in Pseudomonas putida mt-2. 5-Halo-2-hydroxy-2,4-pentadienoates are intermediates in a similar pathway in Comamonas testosteroni CNB-1, yet the intermediates contain a halogen. The enzymes in the Leptothrix cholodnii SP-6 meta-fission pathway are used as representative C. testosteroni enzymes. The presence of a halogen on these intermediates yielded two insights into how Nature processes halogenated species. First, 5-halo-2-hydroxy-2,4-pentadienoates are mechanism-based inactivators of 4-OT. Mass spectral and crystallographic analysis of the covalently inactivated enzymes showed distinct mechanisms for the fluoro- and bromo-/chloro-substituted species. A kinetic analysis suggests that the L. cholodnii 4-OT is more resistant to inactivation. Second, a stereochemical analysis on two vinylpyruvate hydratases (VPHs), which convert 2-hydroxy-2,4-pentadienoate to 2-keto-4S-hydroxypentanoate, showed identical mechanisms. A stereochemical analysis in D₂O using the 5-methyl and 5-chloro derivatives of 2-hydroxy-2,4-pentadienoate showed that deuterons are incorporated stereospecifically and identically at the C-3 and C-5 positions of the products for both enzymes. These initial observations could suggest that the 4-OT-catalyzed reaction may have evolved in L. cholodnii (and C. testosteroni) to prevent inactivation, yet the VPH enzymes have similar mechanisms that could possibly be common to this group of enzymes. The common starting unit of streptogramin B and pyridomycin is 3-hydroxypicolinate (3-HP), which is likely derived from lysine. The biosynthetic steps required for the conversion of lysine to 3-HP have not been elucidated, but the genes for Pyr5 and SnbT have been identified. These two TSF members (72% identical, 87% similar) are implicated in the transformation. Preliminary work shows that Pyr5 and SnbT might function as tautomerases, due to the 1,3-keto-enol tautomerase activity with 2-hydroxypentadienoate and the inactivation by 2-oxo-3-pentynoate. The latter observation suggests a low pK [subscript a] for Pro-1, like that of 4-OT. A biosynthetic pathway consisting of four enzymes to convert lysine to 3HP is proposed.
Random and rational evolution of tautomerase superfamily members : analysis and implications
(2008-12) Darty, Joseph Edward; Whitman, Christian P.
P[Kappa]a is not responsible for the improved activity. Hence, stabilization of an enediolate intermediate may be important for catalysis. In the second part of this work, the Chloroflexus aurantiacus J-10-fl heterohexameric 4-OT tautomerase was employed in random and rational directed evolution studies to introduce a CaaD activity. Genetic selection and a high throughput screening assay were used to identify mutants. Genetic selection was unsuccessful due to plasmid instability in the host strain. A small mutant library in the screening assay precluded the identification of any mutants with CaaD activity. Finally, rational design using structure-function relationships was investigated and a single mutant was discovered for hh4-OT that incorporated CaaD activity into the enzyme, the [alpha]L9R hh4-OT, this mutant has been characterized kinetically and the evolutionary implications for the tautomerase superfamily are described.
Sequence, structure, and function relationships in the aldolase and tautomerase superfamilies
(2022-05-06) Lancaster, Emily Beatrice; Whitman, Christian P.; Fast, Walter; Dalby, Kevin; Hoffman, David
Studying the structure of an enzyme and how it relates to its function has been a goal of enzymologists for decades. Although informative and useful, classical techniques, such as BLAST searches or manual analysis of multisequence alignments may be too focused. These techniques tend to rely on comparing a manageable quantity of sequences. As a result, patterns in conservation of certain residues across a limited number of sequences carry more weight in the study. In the first section, we present a study that used these classical approaches to compare only 3 sequences of Aldolase Superfamily members as described below. NahE is a hydratase-aldolase that converts o-substituted trans-benzylidenepyruvates (where the ortho-substituent is H, OH, or CO₂⁻) to benzaldehyde, salicylaldehyde, or 2-carboxybenzaldehyde, respectively, and pyruvate. The enzyme is part of a bacterial pathway for the degradation of naphthalene, which is a toxic and persistent environmental contaminant. Sequence, crystallographic, and mutagenic analysis identified the enzyme as a member of the N-acetylneuraminate lyase (NAL) subgroup in the aldolase superfamily. As such, it has a conserved lysine (Lys183) and tyrosine (Tyr155), for Schiff base formation, as well as a GXXGE motif for binding of the pyruvoyl carboxylate group. NahE crystal structures show these core active site elements along with other nearby residues that might be involved in the mechanism and/or specificity. Mutations of five active site amino acids (Thr65, Trp128, Tyr155, Asn157, and Asn281) were constructed and kinetic parameters measured in order to assess the effect(s) on binding, catalysis, and/or the reaction step (hydration vs aldol cleavage). The results show that the two Trp128 mutants (Phe and Tyr) have the least effect on catalysis, whereas amino acids with bulky side chains at Thr65 (Val) and Asn281 (Leu) have the greatest effect. The Y155F mutation also significantly hinders catalysis and falls in between these extremes. These observations are put into a structural context. Finally, trapping experiments were carried out with substrate, NaCNBH₃, and wild type and selected mutations. The mass spectral analysis is consistent with the observed activities and suggests that pyruvate is released quickly from the active site, but salicylaldehyde is not. In the second section, we utilize a more modern technique of sequence analysis. We present studies done to analyze trends observed within the Tautomerase Superfamily (TSF) that have been identified by a sequence similarity network (SSN). Hidden trends in sequences that appear insignificant at the small scale may be revealed on a larger scale, such as in the second section. The amino-terminal proline (Pro1) has long been thought to be a mechanistic imperative for tautomerase superfamily (TSF) enzymes, functioning as a general base or acid in all characterized reactions. However, a global examination of more than 11,000 nonredundant sequences of the TSF uncovered 346 sequences that lack Pro1. The majority (~85%) are found in the malonate semialdehyde decarboxylase (MSAD) subgroup where most of the 294 sequences form a separate cluster. Four sequences within this cluster retain Pro1. Because these four sequences might provide clues to assist in the identification and characterization of activities of nearby sequences without Pro1, they were examined by kinetic, inhibition, and crystallographic studies. The most promising of the four (from Calothrix sp. PCC 6303 designated 437) exhibited decarboxylase and tautomerase activities and was covalently modified at Pro1 by 3-bromopropiolate. A crystal structure was obtained for the apo enzyme (2.35 Å resolution). The formation of a 3-oxopropanoate adduct with Pro1 provides clues to build a molecular model for the bound ligand. The modeled ligand extends into a region that allows interactions with three residues (Lys37, Arg56, Glu98), suggesting that these residues could play roles in the observed decarboxylation and tautomerization activities. Moreover, these same residues are conserved in 16 nearby, non-Pro1 sequences in a sequence similarity network. Thus far, these residues have not been implicated in the mechanisms of any other TSF members. The collected observations provide starting points for the characterization of the non-Pro1 sequences. Five non-Pro1 sequences were studied in this section, as well, containing either glycine, alanine, valine, threonine, or serine at the N-terminus. The most promising was NJ7 (from Nostoc sp. strain PCC 7120/SAG 25.82/UTEX 2576 derived from the UniProt Accession code Q8YNJ7). Kinetic analysis showed that this enzyme with Val1 has tautomerization and decarboxylation activity. The introduction of Pro1 enhanced NJ7’s performance as a tautomerase and decarboxylase.
Structure-based mechanism of hydratase-aldolases in the Type I aldolase superfamily and structures of tautomerase superfamily members
(2017-05-05) LeVieux, Jake Allen; Whitman, Christian P.; Fast, Walter L; Hoffman, David W; Kerwin, Sean M; Zhang, Yan J
Polycyclic Aromatic Hydrocarbons (PAHs) are composed of multiple benzene-like rings and their bacterial catabolism has potential utility for bioremediation. In the breakdown of each ring, oxygenation is followed by ring cleavage and side chain removal, the latter of which is catalyzed by hydratase-aldolases. These enzymes belong to the N-acetylneuraminate lyase (NAL) subgroup of the Type I aldolase superfamily. NahE and PhdJ are hydratase-aldolases that process benzylidenepyruvate compounds in PAH degradative pathways from Pseudomonas putida G7 and Mycobacterium vanbaalenii PYR-1, respectively. Crystal structures of these enzymes in liganded and unliganded states are reported here and reveal new details about their catalytic mechanisms. Stabilization of the developing hydroxide anion during hydration could involve the amide nitrogen of Asn-157 (NahE numbering), conserved in hydratase-aldolases, but not other NAL subgroup members. For NahE, Tyr-155 might act as the general base for addition, but the identity of the base is less certain for PhdJ. The crystal structure of PhdG reveals a carbinolamine intermediate hydrogen bonding to Tyr-152, consistent with the participation of the residue in Schiff base formation. In addition, this dissertation describes structures within the tautomerase superfamily (TSF). The enzymes reported are from 4-oxalocrotonate tautomerase (4-OT), cis-3-chloroacrylic acid dehalogenase (cis-CaaD), and malonate semialdehyde decarboxylase (MSAD) subgroups. 4-OT catalyzes the ketonization of 2-hydroxymuconate (2-HM) in the meta-fission pathway. cis-CaaD and MSAD respectively catalyze dehalogenation and decarboxylation reactions in the catabolic 1,3-dichloropropene pathway, respectively. TSF members have a catalytic Pro-1 and share a common β-α-β fold. TSF members designated Ps01740, Pt0534, and Fused 4-OT have sequence similarity with the cis-CaaD and 4-OT subgroups and may provide insight into gene duplication and fusion events in the TSF. These enzymes have tautomerase activity and low or absent cis-CaaD activity. Liganded and unliganded structures suggest a structural basis for their activation. The trimeric structure of Fused 4-OT is unusual in that one monomer is inverted relative to the others. Mechanisms for 2-HM tautomerization are proposed based on a 2-HM-bound structure and kinetic parameters of mutants. Crystal structures for the MSAD homologues BP4401 and YusQ were obtained, and provide clues about their activities