Crystal Structure of Glyceraldehyde-3-Phosphate Dehydrogenase 1 from Methicillin-Resistant Staphylococcus aureus MRSA252 Provides Novel Insights into Substrate Binding and Catalytic Mechanism

Mukherjee, Somnath; Dutta, Debajyoti; Saha, Barun Kumar; Das, Amit Kumar

doi:10.1016/j.jmb.2010.07.002

Cited by 44 publications

(53 citation statements)

References 49 publications

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“…MBP (Uniprot: P0AEX9), SETD7 (Uniprot: Q8WTS6), GAP1 (Uniprot: Q6GIL8), PGK (Uniprot: Q6GB57) and bromo domain (Uniprot: B4NS38) were amplified from pHFT2/MBP 33, 34 , pNICBio2/SETD7, pQE30/GAP1 35 , pQE30/PGK 36 and pET21/bromo respectively. ESRRA (Uniprot: P11474), NEDD9 (Uniprot: Q14511), ZSCAN29 (Uniprot: Q8IWY8), TSC22D4 (Uniprot: Q9Y3Q8), VENTX (Uniprot: O95231), MAFF (Uniprot: Q9ULX9), LIN28A (Uniprot: Q9H9Z2) and JARID2 (Uniprot: Q92833) were amplified from the plasmids HR7097C, HR5554A, HR8429A, HR7683A, HR7703A, HR8265A, HR7525A and HR8400C respectively.…”

Section: Methodsmentioning

confidence: 99%

A New Versatile Immobilization Tag Based on the Ultra High Affinity and Reversibility of the Calmodulin–Calmodulin Binding Peptide Interaction

Mukherjee

Ura

Hoey

et al. 2015

Journal of Molecular Biology

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Reversible, high affinity immobilization tags are critical tools for myriad biological applications. However, inherent issues are associated with a number of the current methods of immobilization. Particularly, a critical element in phage display sorting is functional immobilization of target proteins. To circumvent these problems, we have used a mutant (N5A) of calmodulin binding peptide (CBP) as an immobilization tag in phage display sorting. The immobilization relies on the ultra high affinity of calmodulin to N5A mutant CBP (RWKKNFIAVSAANRFKKIS) in presence of calcium (KD ~ 2pM), which can be reversed by EDTA allowing controlled “capture and release” of the specific binders. To evaluate the capabilities of this system, we chose eight targets, some of which were difficult to overexpress and purify with other tags and some had failed in sorting experiments. In all cases, specific binders were generated using a Fab phage display library with CBP fused constructs. KD of the Fabs were in sub to low nanomolar (nM) ranges and were successfully used to selectively recognize antigens in cell-based experiments. Some of these targets were problematic even without any tag, so the fact that all led to successful selection endpoints means that borderline cases can be worked on with a high probability of positive outcome. Taken together with examples of successful case specific high level applications like generation of conformation, epitope and domain specific Fabs, we feel that the CBP tag embodies all the attributes of covalent immobilization tags, but does not suffer from some of their well documented drawbacks.

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Section: Methodsmentioning

confidence: 99%

A New Versatile Immobilization Tag Based on the Ultra High Affinity and Reversibility of the Calmodulin–Calmodulin Binding Peptide Interaction

Mukherjee

Ura

Hoey

et al. 2015

Journal of Molecular Biology

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“…A DALI search indicated that TtLysY had higher structural homology to N-acetyl-␥-glutamyl-phosphate reductase (ArgC) from Mycobacterium tuberculosis (14) (PDB code 2I3G) with a Z-score of 44.8. Despite its low sequence identity (Ͻ21%), TtLysY also showed structural homology to GAPDH family members such as archaeal malonyl-CoA reductase from Sulfolobus tokodaii (PDB code 4DPK) (25), aspartate-␤-semialdehyde dehydrogenases from Streptococcus pneumoniae (PDB code 4R3N) (26), and D-glyceraldehyde-3-phosphate dehydrogenases from Staphylococcus aureus (PDB code 3LC7) (27) with Z-scores of 30.8, 29.9, and 25.6, respectively. The monomer of TtLysY was composed of the N-terminal nucleotide-binding domain (Met-1-Lys-138 and Thr-317-Pro-349) and C-terminal dimerization domain (Gly-137-Gly-316), which was similar to the structural homologs.…”

Section: Ttlysy Catalyzes the Nadph-dependent Reduction Ofmentioning

confidence: 99%

Crystal Structure of the LysY·LysW Complex from Thermus thermophilus

Shimizu

Tanaka

Kuzuyama

et al. 2016

Journal of Biological Chemistry

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Several bacteria and archaea utilize the amino group-carrier protein, LysW, for lysine biosynthesis, in which an isopeptide bond is formed between the C-terminal Glu of LysW and an amino group of ␣-aminoadipate (AAA). The resulting LysW-␥-AAA is phosphorylated by LysZ to form LysW-␥-AAA phosphate, which is subsequently reduced to LysW-␥-aminoadipic semialdehyde (LysW-␥-AASA) through a reaction catalyzed by LysY. In this study, we determined the crystal structures of LysY from Thermus thermophilus HB27 (

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“…Moreover, among the 104 entries for GAPDH crystal structures that have been deposited in the Protein Data Bank, substrate/product- or substrate-analog-bound structures are available only for three enzymes. Structures of ternary complexes are available only for GAPDHs from Bacillus stearothermophilus ( Bs GAPDH) [11–12], Staphylococcus aureus ( Sa GAPDH) [13] and Cryptosporidium parvum ( Cp GAPDH) [14]. To capture the enzyme in the substrate-bound state in most cases the active site cysteine was mutated to serine, alanine or glycine.…”

Section: Introductionmentioning

confidence: 99%

Crystal Structures of Group B Streptococcus Glyceraldehyde-3-Phosphate Dehydrogenase: Apo-Form, Binary and Ternary Complexes

et al. 2016

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Glyceraldehyde 3-phosphate dehydrogenase or GAPDH is an evolutionarily conserved glycolytic enzyme. It catalyzes the two step oxidative phosphorylation of D-glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate using inorganic phosphate and NAD+ as cofactor. GAPDH of Group B Streptococcus is a major virulence factor and a potential vaccine candidate. Moreover, since GAPDH activity is essential for bacterial growth it may serve as a possible drug target. Crystal structures of Group B Streptococcus GAPDH in the apo-form, two different binary complexes and the ternary complex are described here. The two binary complexes contained NAD+ bound to 2 (mixed-holo) or 4 (holo) subunits of the tetrameric protein. The structure of the mixed-holo complex reveals the effects of NAD+ binding on the conformation of the protein. In the ternary complex, the phosphate group of the substrate was bound to the new Pi site in all four subunits. Comparison with the structure of human GAPDH showed several differences near the adenosyl binding pocket in Group B Streptococcus GAPDH. The structures also reveal at least three surface-exposed areas that differ in amino acid sequence compared to the corresponding areas of human GAPDH.

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Crystal Structure of Glyceraldehyde-3-Phosphate Dehydrogenase 1 from Methicillin-Resistant Staphylococcus aureus MRSA252 Provides Novel Insights into Substrate Binding and Catalytic Mechanism

Cited by 44 publications

References 49 publications

A New Versatile Immobilization Tag Based on the Ultra High Affinity and Reversibility of the Calmodulin–Calmodulin Binding Peptide Interaction

A New Versatile Immobilization Tag Based on the Ultra High Affinity and Reversibility of the Calmodulin–Calmodulin Binding Peptide Interaction

Crystal Structure of the LysY·LysW Complex from Thermus thermophilus

Crystal Structures of Group B Streptococcus Glyceraldehyde-3-Phosphate Dehydrogenase: Apo-Form, Binary and Ternary Complexes

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