Stereoselectivity of Enoyl-CoA Hydratase Results from Preferential Activation of One of Two Bound Substrate Conformers

Bell, Alasdair F.; Feng, Yixiang; Hofstein, Hilary A.; Parikh, Sapan; Wu, Joy T.; Rudolph, M.; Kisker, Caroline; Whitty, Adrian; Tonge, Peter J.

doi:10.1016/s1074-5521(02)00263-6

Cited by 34 publications

(45 citation statements)

References 55 publications

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“…The key active site geometry, shaped by the two catalytic glutamates, the catalytic water and the oxyanion hole, is preserved. The proposed protonation state (Glu123 protonated, Glu103 deprotonated) for the MFE1 hydratase reaction is also consistent with reaction mechanistic studies of the monofunctional enoyl‐CoA hydratase and the enzyme kinetic studies of the α‐chain of the TFE‐enzyme , suggesting that at least one of the catalytic glutamates should be deprotonated in the active site competent for hydratase catalysis. As in the monofunctional enoyl‐CoA hydratase, the catalytic water is tightly anchored between the OE1‐carboxylate atoms of Glu103 and Glu123, which act jointly to achieve catalysis.…”

Section: Discussionsupporting

confidence: 76%

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The isomerase and hydratase reaction mechanism of the crotonase active site of the multifunctional enzyme (type‐1), as deduced from structures of complexes with 3S‐hydroxy‐acyl‐CoA

et al. 2013

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The multifunctional enzyme, type-1 (MFE1) is involved in several lipid metabolizing pathways. It catalyses: (a) enoyl-CoA isomerase and (b) enoyl-CoA hydratase (EC 4.2.1.17) reactions in its N-terminal crotonase part, as well as (3) a 3S-hydroxy-acyl-CoA dehydrogenase (HAD; EC 1.1.1.35) reaction in its C-terminal 3S-hydroxy-acyl-CoA dehydrogenase part. Crystallographic binding studies with rat peroxisomal MFE1, using unbranched and branched 2E-enoyl-CoA substrate molecules, show that the substrate has been hydrated by the enzyme in the crystal and that the product, 3S-hydroxy-acyl-CoA, remains bound in the crotonase active site. The fatty acid tail points into an exit tunnel shaped by loop-2. The thioester oxygen is bound in the classical oxyanion hole of the crotonase fold, stabilizing the enolate reaction intermediate. The structural data of these enzyme product complexes suggest that the catalytic base, Glu123, initiates the isomerase reaction by abstracting the C2-proton from the substrate molecule. Subsequently, in the hydratase reaction, Glu123 completes the catalytic cycle by reprotonating the C2 atom. A catalytic water, bound between the OE1-atoms of the two catalytic glutamates, Glu103 and Glu123, plays an important role in the enoyl-CoA isomerase and the enoyl-CoA hydratase reaction mechanism of MFE1. The structural variability of loop-2 between MFE1 and its monofunctional homologues correlates with differences in the respective substrate preferences and catalytic rates. DatabaseThe structures have been deposited in the Protein Data Bank under accession numbers: 3ZW8 (MFE1 apo), 3ZW9 (MFE1 2S-methyl-3S-hydroxy-butanoyl-CoA complex), 3ZWA (MFE1 3S-hydroxy-hexanoyl-CoA complex), 3ZWB (MFE1-E123A 2E-hexenoyl-CoA complex) and 3ZWC (MFE1 3S-hydroxy-decanoyl-CoA complex). Structured digital abstract

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Section: Discussionsupporting

confidence: 76%

“…) correlates with different substrate specificities . The two catalytic glutamates in MFE1 are conserved in the hydratases , as well as in the TFE α‐chain (Fig. ).…”

Section: Introductionmentioning

confidence: 99%

The isomerase and hydratase reaction mechanism of the crotonase active site of the multifunctional enzyme (type‐1), as deduced from structures of complexes with 3S‐hydroxy‐acyl‐CoA

et al. 2013

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“…Again, the rows represent individual protein structures and the columns represent spatial positions in the alignment. The known catalytic residues, A98, G141, E144, and E164 [40,41], are shown in boldface . Residues predicted by POOL are shown in uppercase and residues not predicted are shown in lowercase.…”

Section: Resultsmentioning

confidence: 99%

Protein function annotation with Structurally Aligned Local Sites of Activity (SALSAs)

et al. 2013

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BackgroundThe prediction of biochemical function from the 3D structure of a protein has proved to be much more difficult than was originally foreseen. A reliable method to test the likelihood of putative annotations and to predict function from structure would add tremendous value to structural genomics data. We report on a new method, Structurally Aligned Local Sites of Activity (SALSA), for the prediction of biochemical function based on a local structural match at the predicted catalytic or binding site.ResultsImplementation of the SALSA method is described. For the structural genomics protein PY01515 (PDB ID 2aqw) from Plasmodium yoelii, it is shown that the putative annotation, Orotidine 5'-monophosphate decarboxylase (OMPDC), is most likely correct. SALSA analysis of YP_001304206.1 (PDB ID 3h3l), a putative sugar hydrolase from Parabacteroides distasonis, shows that its active site does not bear close resemblance to any previously characterized member of its superfamily, the Concanavalin A-like lectins/glucanases. It is noted that three residues in the active site of the thermophilic beta-1,4-xylanase from Nonomuraea flexuosa (PDB ID 1m4w), Y78, E87, and E176, overlap with POOL-predicted residues of similar type, Y168, D153, and E232, in YP_001304206.1. The substrate recognition regions of the two proteins are rather different, suggesting that YP_001304206.1 is a new functional type within the superfamily. A structural genomics protein from Mycobacterium avium (PDB ID 3q1t) has been reported to be an enoyl-CoA hydratase (ECH), but SALSA analysis shows a poor match between the predicted residues for the SG protein and those of known ECHs. A better local structural match is obtained with Anabaena beta-diketone hydrolase (ABDH), a known β-diketone hydrolase from Cyanobacterium anabaena (PDB ID 2j5s). This suggests that the reported ECH function of the SG protein is incorrect and that it is more likely a β-diketone hydrolase.ConclusionsA local site match provides a more compelling function prediction than that obtainable from a simple 3D structure match. The present method can confirm putative annotations, identify misannotation, and in some cases suggest a more probable annotation.

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“…The disorder of the A-loop is a common observation among crotonase superfamily members (enoyl-CoA hydratase (ECH) (46, 57–58), methylmalonyl CoA decarboxylase (MMCD) (45), Δ 3 - Δ 2 -enoyl-CoA isomerase (ECI) (59), and hydroxycinnamoyl-CoA hydratase-lyase (HCHL) (48)), while in MenB the flexibility of the B-loop and C-loop has also been observed (8, 22). These loops together form the interface between the two trimers in the hexamer that buries more than 3450 Å, or 24% of the total surface area, and which is likely the reason for the stability of the mtMenB structure (22).…”

Section: Resultsmentioning

confidence: 99%

Mechanism of the Intramolecular Claisen Condensation Reaction Catalyzed by MenB, a Crotonase Superfamily Member

Li¹,

Li²,

Liu³

et al. 2011

Biochemistry

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MenB, the 1,4-dihydroxy-2-naphthoyl-CoA synthase from the bacterial menaquinone biosynthesis pathway, catalyzes an intramolecular Claisen condensation (Dieckmann reaction) in which the electrophile is an unactivated carboxylic acid. Mechanistic studies on this crotonase family member have been hindered by partial active site disorder in existing MenB X-ray structures. In the current work the 2.0 Å structure of O-succinylbenzoyl-aminoCoA (OSB-NCoA) bound to the MenB from Escherichia coli provides important insight into the catalytic mechanism by revealing the position of all active site residues. This has been accomplished by the use of a stable analogue of the O-succinylbenzoyl-CoA (OSB-CoA) substrate in which the CoA thiol has been replaced by an amine. The resulting OSB-NCoA is stable and the X-ray structure of this molecule bound to MenB reveals the structure of the enzyme-substrate complex poised for carbon-carbon bond formation. The structural data support a mechanism in which two conserved active site Tyr residues, Y97 and Y258, participate directly in the intramolecular transfer of the substrate α-proton to the benzylic carboxylate of the substrate, leading to protonation of the electrophile and formation of the required carbanion. Y97 and Y258 are also ideally positioned to function as the second oxyanion hole required for stabilization of the tetrahedral intermediate formed during carbon-carbon bond formation. In contrast, D163, which is structurally homologous to the acid-base catalyst E144 in crotonase, is not directly involved in carbanion formation and may instead play a structural role by stabilizing the loop that carries Y97. When similar studies were performed on the MenB from Mycobacterium tuberculosis, a twisted hexamer was unexpectedly observed, demonstrating the flexibility of the interfacial loops that are involved in the generation of the novel tertiary and quaternary structures found in the crotonase superfamily. This work reinforces the utility of using a stable substrate analogue as a mechanistic probe in which only one atom has been altered leading to a decrease in α-proton acidity.

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Stereoselectivity of Enoyl-CoA Hydratase Results from Preferential Activation of One of Two Bound Substrate Conformers

Cited by 34 publications

References 55 publications

The isomerase and hydratase reaction mechanism of the crotonase active site of the multifunctional enzyme (type‐1), as deduced from structures of complexes with 3S‐hydroxy‐acyl‐CoA

The isomerase and hydratase reaction mechanism of the crotonase active site of the multifunctional enzyme (type‐1), as deduced from structures of complexes with 3S‐hydroxy‐acyl‐CoA

Protein function annotation with Structurally Aligned Local Sites of Activity (SALSAs)

Mechanism of the Intramolecular Claisen Condensation Reaction Catalyzed by MenB, a Crotonase Superfamily Member

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