Properties and mechanism of d -glucosaminate-6-phosphate ammonia-lyase: An aminotransferase family enzyme with d -amino acid specificity

Phillips, Robert S.; Ting, Samuel C.-K.; Tetsadjio, Ange G.; Anderson, Kaitlin L.; Friez, Kyle M.; Miller, Katherine A.; Hoover, Timothy R.

doi:10.1016/j.bbapap.2017.12.006

Cited by 5 publications

(19 citation statements)

References 28 publications

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“…typhimurium. This exposed pair of vicinal histidines may explain the observed binding of DGL to a Ni 2+ -metal chelate column, even without the addition of a His tag …”

Section: Resultsmentioning

confidence: 98%

“…Salmonella typhimurium DGL was overexpressed and purified on a Ni-metal chelate column as described previously. , It should be noted that native DGL binds selectively to a Ni-metal chelate column without addition of a hexa-His tag. SeMet-substituted DGL was expressed in 0.5 L of Studier PASM-5052 autoinduction medium containing 125 μg/mL l -selenomethionine in a 2 L Erlenmeyer flask at 37 °C and 250 rpm using the previously reported BL21(DE3) cells containing the DGL expression plasmid . The yield of purified selenomethionine-substituted protein was 100 mg.…”

Section: Materials and Methodsmentioning

confidence: 99%

“…The purified protein contained 81% replacement of methionines by selenomethionine as determined by electrospray ionization mass spectrometry (Figure S1). DGL was assayed in 0.1 M triethanolamine-HCl (pH 8.0) containing 4 μL of crude DgaF (2-keto-3-deoxygluconate-6-phosphate aldolase), 10 μg/mL lactate dehydrogenase, and 0.2 mM NADH, at 37 °C, following the decrease in absorbance at 340 nm (Δε = −6220 M –1 cm –1 ) as described previously. , …”

Section: Materials and Methodsmentioning

confidence: 99%

“…1 This is followed by elimination of ammonia by the product of the DgaE gene, D-glucosaminate-6phosphate ammonia-lyase (DGL, EC 4.3.1.29), as shown in eq 1, to produce 2-keto-3-deoxygluconate 6-phosphate (KDG-6-P), a key intermediate in the Entner−Doudoroff pentose phosphate pathway. 1,2 DGL is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that can be assigned to the aminotransferase (AT) superfamily (fold I) on the basis of sequence homology. However, DGL has a homology (<20% identity) quite remote from those of most members of the AT superfamily, and DGL, together with bacterial selenocysteine synthase (SelA), represents a novel subfamily within the AT superfamily.…”

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confidence: 99%

“…The metabolism of d -glucosaminic acid in Salmonella enterica servar typhimurium was shown recently to proceed by phosphorylation at C-6 to give d -glucosaminate 6-phosphate (DGA-6-P) . This is followed by elimination of ammonia by the product of the DgaE gene, d -glucosaminate-6-phosphate ammonia-lyase (DGL, EC 4.3.1.29), as shown in eq , to produce 2-keto-3-deoxygluconate 6-phosphate (KDG-6-P), a key intermediate in the Entner–Doudoroff pentose phosphate pathway. , …”

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confidence: 99%

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Structure and Mechanism of d-Glucosaminate-6-phosphate Ammonia-lyase: A Novel Octameric Assembly for a Pyridoxal 5′-Phosphate-Dependent Enzyme, and Unprecedented Stereochemical Inversion in the Elimination Reaction of a d-Amino Acid

2021

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d-Glucosaminate-6-phosphate ammonia-lyase (DGL) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that produces 2-keto-3-deoxygluconate 6-phosphate (KDG-6-P) in the metabolism of d-glucosaminic acid by Salmonella enterica serovar typhimurium. We have determined the crystal structure of DGL by SAD phasing with selenomethionine to a resolution of 2.58 Å. The sequence has very low identity with most other members of the aminotransferase (AT) superfamily. The structure forms an octameric assembly as a tetramer of dimers that has not been observed previously in the AT superfamily. PLP is covalently bound as a Schiff base to Lys-213 in the catalytic dimer at the interface of two monomers. The structure lacks the conserved arginine that binds the α-carboxylate of the substrate in most members of the AT superfamily. However, there is a cluster of arginines in the small domain that likely serves as a binding site for the phosphate of the substrate. The deamination reaction performed in D2O gives a KDG-6-P product stereospecifically deuterated at C3; thus, the mechanism must involve an enamine intermediate that is protonated by the enzyme before product release. Nuclear magnetic resonance (NMR) analysis demonstrates that the deuterium is located in the pro-R position in the product, showing that the elimination of water takes place with inversion of configuration at C3, which is unprecedented for a PLP-dependent dehydratase/deaminase. On the basis of the crystal structure and the NMR data, a reaction mechanism for DGL is proposed.

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“…typhimurium. This exposed pair of vicinal histidines may explain the observed binding of DGL to a Ni 2+ -metal chelate column, even without the addition of a His tag …”

Section: Resultsmentioning

confidence: 98%

Section: Materials and Methodsmentioning

confidence: 99%

Section: Materials and Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Structure and Mechanism of d-Glucosaminate-6-phosphate Ammonia-lyase: A Novel Octameric Assembly for a Pyridoxal 5′-Phosphate-Dependent Enzyme, and Unprecedented Stereochemical Inversion in the Elimination Reaction of a d-Amino Acid

2021

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His‐163 is a stereospecific proton donor in the mechanism of d‐glucosaminate‐6‐phosphate ammonia‐lyase

2022

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Edited by Christian GriesingerD-Glucosaminate-6-phosphate ammonia-lyase (DGL) catalyzes the conversion of D-glucosaminate-6-phosphate to 2-keto-3-deoxyglutarate-6-phosphate, with stereospecific protonation of C-3 of the product. The crystal structure of DGL showed that His-163 could serve as the proton donor. H163A mutant DGL is fully active in the steady-state reaction, and the pre-steady-state kinetics are very similar to those of wild-type DGL. However, H163A DGL accumulates a transient intermediate with λ max at 293 nm during the reaction that is not seen with wild-type DGL. Furthermore, NMR analysis of the reaction of H163A DGL in D 2 O shows that the product is a mixture of deuterated diastereomers at C-3. These results establish that His-163 is the proton donor in the reaction mechanism of DGL.

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Properties and mechanism of d -glucosaminate-6-phosphate ammonia-lyase: An aminotransferase family enzyme with d -amino acid specificity

Cited by 5 publications

References 28 publications

Structure and Mechanism of d-Glucosaminate-6-phosphate Ammonia-lyase: A Novel Octameric Assembly for a Pyridoxal 5′-Phosphate-Dependent Enzyme, and Unprecedented Stereochemical Inversion in the Elimination Reaction of a d-Amino Acid

Structure and Mechanism of d-Glucosaminate-6-phosphate Ammonia-lyase: A Novel Octameric Assembly for a Pyridoxal 5′-Phosphate-Dependent Enzyme, and Unprecedented Stereochemical Inversion in the Elimination Reaction of a d-Amino Acid

His‐163 is a stereospecific proton donor in the mechanism of d‐glucosaminate‐6‐phosphate ammonia‐lyase

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