PLP undergoes conformational changes during the course of an enzymatic reaction

Ngo, H.P.T.; Cerqueira, Nuno M. F. S. A.; Kim, Jin-Kwang; Hong, Myoung-Ki; Fernandes, Pedro A.; Ramos, María J.; Kang, Lin‐Woo

doi:10.1107/s1399004713031283

Cited by 27 publications

(38 citation statements)

References 40 publications

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“…44 Extensive interactions that stabilize this reaction intermediate are formed. The carboxyl group of substrate L-Met forms a salt bridge with R375 (Figure 5D), which is further supported by a hydrogen bond from the side chain of N161 (3 Å) and the main chain amide group of S340.…”

Section: Resultsmentioning

confidence: 99%

Structural Snapshots of an Engineered Cystathionine-γ-lyase Reveal the Critical Role of Electrostatic Interactions in the Active Site

2017

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Enzyme therapeutics that can degrade L-methionine (L-Met) are of great interest as numerous malignancies are exquisitely sensitive to L-Met depletion. To exhaust the pool of methionine in human serum, we previously engineered an L-Met-degrading enzyme based on the human cystathionine-γ-lyase scaffold (hCGL-NLV) to circumvent immunogenicity and stability issues observed in the preclinical application of bacterially derived methionine-γ-lyases. To gain further insights into the structure−activity relationships governing the chemistry of the hCGL-NLV lead molecule, we undertook a biophysical characterization campaign that captured crystal structures (2.2 Å) of hCGL-NLV with distinct reaction intermediates, including internal aldimine, substrate-bound, gem-diamine, and external aldimine forms. Curiously, an alternate form of hCGL-NLV that crystallized under higher-salt conditions revealed a locally unfolded active site, correlating with inhibition of activity as a function of ionic strength. Subsequent mutational and kinetic experiments pinpointed that a salt bridge between the phosphate of the essential cofactor pyridoxal 5′-phosphate (PLP) and residue R62 plays an important role in catalyzing β- and γ-eliminations. Our study suggests that solvent ions such as NaCl disrupt electrostatic interactions between R62 and PLP, decreasing catalytic efficiency.

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Section: Resultsmentioning

confidence: 99%

Structural Snapshots of an Engineered Cystathionine-γ-lyase Reveal the Critical Role of Electrostatic Interactions in the Active Site

2017

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“…When we superimposed the ToCDS GD structure into the ToCDS NAT structure, the conformations of the PLP pyridine ring and His114 of both structures were well conserved (Figure 5(a)). In the catalytic intermediate structures of XometC [18], a PLP-dependent enzyme of L-serine hydratase, both the PLP pyridine ring and Tyr112 (His114 in ToCDS) showed the tilting conformational changes (Figure 5(b)). However, no tilting of the PLP pyridine ring and His114 was observed in ToCDS.…”

Section: Resultsmentioning

confidence: 99%

“…Two amino groups of Lys216 and product alanine had the similar distances of approximate 2.9 Å with the hydroxyl group in an equilateral triangle form. The hydroxyl group of PLP was proposed as catalytic base to deprotonate the incoming substrate amino group in XometC [18]. In Escherichia coli , NAT (PDB ID: 1jf9) and GD (in complex with selenocysteine; PDB ID: 1kmk) structures of CsdB (EcNifS_CsdB), which is a NifS-like CDS [32], were determined [33].…”

Section: Resultsmentioning

confidence: 99%

“…Transaldimine reaction is the conserved critical step of all PLP-dependent enzymes. Recently, the dihedral angle change between the PLP pyridine ring and Schiff-base linkage was proposed to play an essential role in the transaldimination reaction of XometC based on the catalytic intermediate crystal structures and high-level computational calculations [18] (Figure S3). The conformational change of the dihedral angle had two main roles.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, the conformational change of PLP, especially the dihedral angle between the PLP pyridine ring and the Schiff-base linkage, was proposed to play an essential role in the transaldimination reaction in a bacterial L-serine hydratase (XometC) [18]. In this study, we cloned the ToCDS gene from Thermococcus onnurineus NA1 and determined the crystal structures of ToCDS alone and in complex with catalytic intermediate ligands from four different crystals, which were then compared with Escherichia coli CDS structures.…”

Section: Introductionmentioning

confidence: 99%

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Catalytic Intermediate Crystal Structures of Cysteine Desulfurase from the Archaeon Thermococcus onnurineus NA1

Huynh

Nguyen

et al. 2017

Archaea

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Thermococcus onnurineus NA1 is an anaerobic archaeon usually found in a deep-sea hydrothermal vent area, which can use elemental sulfur (S0) as a terminal electron acceptor for energy. Sulfur, essential to many biomolecules such as sulfur-containing amino acids and cofactors including iron-sulfur cluster, is usually mobilized from cysteine by the pyridoxal 5′-phosphate- (PLP-) dependent enzyme of cysteine desulfurase (CDS). We determined the crystal structures of CDS from Thermococcus onnurineus NA1 (ToCDS), which include native internal aldimine (NAT), gem-diamine (GD) with alanine, internal aldimine structure with existing alanine (IAA), and internal aldimine with persulfide-bound Cys356 (PSF) structures. The catalytic intermediate structures showed the dihedral angle rotation of Schiff-base linkage relative to the PLP pyridine ring. The ToCDS structures were compared with bacterial CDS structures, which will help us to understand the role and catalytic mechanism of ToCDS in the archaeon Thermococcus onnurineus NA1.

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The Catalytic Mechanism of Pdx2 Glutaminase Driven by a Cys‐His‐Glu Triad: A Computational Study

2021

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The catalytic mechanism of Pdx2 was studied with atomic detail employing the computational ONIOM hybrid QM/MM methodology. Pdx2 employs a Cys‐His‐Glu catalytic triad to deaminate glutamine to glutamate and ammonia – the source of the nitrogen of pyridoxal 5’‐phosphate (PLP). This enzyme is, therefore, a rate‐limiting step in the PLP biosynthetic pathway of Malaria and Tuberculosis pathogens that rely on this mechanism to obtain PLP. For this reason, Pdx2 is considered a novel and promising drug target to treat these diseases. The results obtained show that the catalytic mechanism of Pdx2 occurs in six steps that can be divided into four stages: (i) activation of Cys87, (ii) deamination of glutamine with the formation of the glutamyl‐thioester intermediate, (iii) hydrolysis of the formed intermediate, and (iv) enzymatic turnover. The kinetic data available in the literature (19.1–19.5 kcal mol−1) agree very well with the calculated free energy barrier of the hydrolytic step (18.2 kcal.mol−11), which is the rate‐limiting step of the catalytic process when substrate is readily available in the active site. This catalytic mechanism differs from other known amidases in three main points: i) it requires the activation of the nucleophile Cys87 to a thiolate; ii) the hydrolysis occurs in a single step and therefore does not require the formation of a second tetrahedral reaction intermediate, as it is proposed, and iii) Glu198 does not have a direct role in the catalytic process. Together, these results can be used for the synthesis of new transition state analogue inhibitors capable of inhibiting Pdx2 and impair diseases like Malaria and Tuberculosis.

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PLP undergoes conformational changes during the course of an enzymatic reaction

Cited by 27 publications

References 40 publications

Structural Snapshots of an Engineered Cystathionine-γ-lyase Reveal the Critical Role of Electrostatic Interactions in the Active Site

Structural Snapshots of an Engineered Cystathionine-γ-lyase Reveal the Critical Role of Electrostatic Interactions in the Active Site

Catalytic Intermediate Crystal Structures of Cysteine Desulfurase from the Archaeon Thermococcus onnurineus NA1

The Catalytic Mechanism of Pdx2 Glutaminase Driven by a Cys‐His‐Glu Triad: A Computational Study

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