Structure and Mechanism of HpcH: A Metal Ion Dependent Class II Aldolase from the Homoprotocatechuate Degradation Pathway of Escherichia coli

Rea, Dean; Fülöp, Vilmos; Bugg, Timothy D. H.; Roper, David I.

doi:10.1016/j.jmb.2007.06.048

Cited by 28 publications

(39 citation statements)

References 41 publications

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“…Its precise role in the enzyme is not understood. Here we show, contrary to previous report [7], that His45 mutants of HpcH/HpaI are still active, although activities are less than 1% of the wild-type. Using various biochemical approaches we demonstrate that this residue is important for optimal metal cofactor binding and for base catalysis in the enzyme.…”

Section: Introductioncontrasting

confidence: 99%

“…Catalytic efficiencies (k cat /K m ) of the mutant enzymes are therefore decreased by 3-to 4-orders of magnitude. The high K m values for HOPA in the mutants may account for the previous report that H45A mutant of HpcH is inactive when tested with low concentrations (1.5 mM) of substrate [7].…”

Section: Steady-state Kinetic Analysis and Pyruvate Proton Exchangementioning

confidence: 54%

“…It has been proposed that histidine 45 also forms a proton relay to the water ligand of the metal cofactor, which in turn acts as the catalytic acid in donating a proton to the C3 of pyruvate enolate [7]. However, due to the perturbed metal binding, we cannot unequivocally determine if the reduced pyruvate proton exchange observed in the His 45 mutants is due to the disruption of this proton relay.…”

Section: Ph-activity Profilementioning

confidence: 79%

“…Crystal structures of DDG aldolase [6] and HpcH/HpaI [7] show common (ab) 8 TIM barrel folds with similar active site architectures. Pyruvate or the pyruvate analogue, oxamate, binds in a bidentate fashion, via the C1 carboxylate and C2 carbonyl, to the divalent metal ion cofactor.…”

Section: Introductionmentioning

confidence: 99%

“…An enzyme bound phosphate was previously postulated to be the catalytic acid [6], but this was subsequently disputed since in phosphate-free buffer, pyruvate proton exchange catalyzed by HpaI is not impaired [2]. A histidine residue (His 45) within the active site of HpcH has been replaced with alanine and the resulting enzyme was reported to be inactive [7]. This histidine resides on a flexible loop, and it is distal to N of oxamate or C3 of pyruvate and 3.4 Å from a water ligand to the metal cofactor in the structures of DDG aldolase and HpcH.…”

Section: Introductionmentioning

confidence: 99%

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The role of a conserved histidine residue in a pyruvate‐specific Class II aldolase

Wang

Seah

2008

FEBS Letters

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Histidine 45 in HpaI was replaced with alanine (H45A) and glutamine (H45Q). In the aldol cleavage reaction, k cat values were lowered by 78-and 2059-fold while K m values were increased by 100-and 42-fold in H45A and H45Q, respectively, compared to the wild-type enzyme. Both mutants displayed higher dissociation constants towards the metal cofactor, pyruvate and the transition state analogue, oxalate. Pyruvate proton exchange rates are consequently reduced in H45A and H45Q. pK a for a catalytic base (6.5) is lost in the mutant enzymes and catalysis is dependent on hydroxide ions. The results show that histidine 45 is important for metal cofactor binding and for facilitating C4-OH proton abstraction of the substrate in the reaction mechanism. Crown

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Section: Introductioncontrasting

confidence: 99%

Section: Steady-state Kinetic Analysis and Pyruvate Proton Exchangementioning

confidence: 54%

Section: Ph-activity Profilementioning

confidence: 79%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The role of a conserved histidine residue in a pyruvate‐specific Class II aldolase

Wang

Seah

2008

FEBS Letters

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CH‐π Interactions Promote the Conversion of Hydroxypyruvate in a Class II Pyruvate Aldolase

et al. 2019

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The class II hydroxy ketoacid aldolase A5VH82 from Sphingomonas wittichii RW1 (SwHKA) accepts hydroxypyruvate as nucleophilic donor substrate, giving access to synthetically challenging 3,4dihydroxy-α-ketoacids. The crystal structure of holo-SwHKA in complex with hydroxypyruvate revealed CH-π interactions between the CÀ H bonds at C3 of hydroxypyruvate and a phenylalanine residue at position 210, which in this case occupies the position of a conserved leucine residue. Mutagenesis to tyrosine further increased the electron density of the interacting aromatic system and effected a rate enhancement by twofold. While the leucine variant efficiently catalyses the enolisation of hydroxypyruvate as the first step in the aldol reaction, the enol intermediate then becomes trapped in a disfavoured configuration that considerably hinders subsequent CÀ C bond formation. In SwHKA, micromolar concentrations of inorganic phosphate increase the catalytic rate constant of enolisation by two orders of magnitude. This rate enhancement was now shown to be functionally conserved across the structurally distinct (α/β) 8 barrel and αββα sandwich folds of two pyruvate aldolases. Characterisation of the manganese (II) cofactor by electron paramagnetic resonance excluded ionic interactions between the metal centre and phosphate. Instead, histidine 44 was shown to be primarily responsible for the binding of phosphate in the micromolar range and the observed rate enhancement in SwHKA.

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Chemoenzymatic Platform for Synthesis of Chiral Organofluorines Based on Type II Aldolases

et al. 2019

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Aldolases are C À Cbond forming enzymes that have become prominent tools for sustainable synthesis of complex synthons.H owever,e nzymatic methods of fluorine incorporation into such compounds are lacking due to the rarity of fluorine in nature.Recently,the use of fluoropyruvate as anonnative aldolase substrate has arisen as as olution. Here,w e report that the type II HpcH aldolases efficiently catalyze fluoropyruvate addition to diverse aldehydes,w ith exclusive (3S)-selectivity at fluorine that is rationalized by DFT calculations on am echanistic model. We also measure the kinetic parameters of aldol addition and demonstrate engineering of the hydroxylgroup stereoselectivity.Our aldolase collection is then employed in the chemoenzymatic synthesis of novel fluoroacids and ester derivatives in high stereopurity (d.r.8 0-98 %). The compounds made available by this method serve as precursors to fluorinated analogs of sugars,a mino acids,a nd other valuable chiral building blocks.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

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Structure and Mechanism of HpcH: A Metal Ion Dependent Class II Aldolase from the Homoprotocatechuate Degradation Pathway of Escherichia coli

Cited by 28 publications

References 41 publications

The role of a conserved histidine residue in a pyruvate‐specific Class II aldolase

The role of a conserved histidine residue in a pyruvate‐specific Class II aldolase

CH‐π Interactions Promote the Conversion of Hydroxypyruvate in a Class II Pyruvate Aldolase

Chemoenzymatic Platform for Synthesis of Chiral Organofluorines Based on Type II Aldolases

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