Crystal Structures of Protein Glutaminase and Its Pro Forms Converted into Enzyme-Substrate Complex

Hashizume, R.; Maki, Yukiko; Mizutani, Kimihiko; Takahashi, Nobuyuki; Matsubara, Hiroyuki; Sugita, Akiko; Sato, Kimihiko; Yamaguchi, Shotaro; Mikami, Bunzo

doi:10.1074/jbc.m111.255133

Cited by 36 publications

(30 citation statements)

References 41 publications

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“…One example is in the NADH-forming step during glyceraldehyde-3-P dehydrogenase (GAPDH) action when a 3-P-glyceryl- S -Cys enzyme thioester is generated during NADH formation. A second set of widely distributed examples are the γ-glutamyl- S -enzyme thioester intermediates generated during glutaminase actions 70 : nascent NH 3 is released to be used as nucleophilic amination cosubstrate in many contexts of nucleotide and amino acid metabolism. 71 …”

Section: Acetyl-coamentioning

confidence: 99%

Eight Kinetically Stable but Thermodynamically Activated Molecules that Power Cell Metabolism

Walsh

Tang³

2017

Chem. Rev.

238

203

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Contemporary analyses of cell metabolism have called out three metabolites: ATP, NADH, and acetyl-CoA, as sentinel molecules whose accumulation represent much of the purpose of the catabolic arms of metabolism and then drive many anabolic pathways. Such analyses largely leave out how and why ATP, NADH, and acetyl-CoA (Figure 1) at the molecular level play such central roles. Yet, without those insights into why cells accumulate them and how the enabling properties of these key metabolites power much of cell metabolism, the underlying molecular logic remains mysterious. Four other metabolites, S-adenosylmethionine, carbamoyl phosphate, UDP-glucose, and Δ2-isopentenyl-PP play similar roles in using group transfer chemistry to drive otherwise unfavorable biosynthetic equilibria. This review provides the underlying chemical logic to remind how these seven key molecules function as mobile packets of cellular currencies for phosphoryl transfers (ATP), acyl transfers (acetyl-CoA, carbamoyl-P), methyl transfers (SAM), prenyl transfers (IPP), glucosyl transfers (UDP-glucose), and electron and ADP-ribosyl transfers (NAD(P)H/NAD(P)+) to drive metabolic transformations in and across most primary pathways. The eighth key metabolite is molecular oxygen (O2), thermodynamically activated for reduction by one electron paths, leaving it kinetically stable to the vast majority of organic cellular metabolites.

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Section: Acetyl-coamentioning

confidence: 99%

Eight Kinetically Stable but Thermodynamically Activated Molecules that Power Cell Metabolism

Walsh

Tang³

2017

Chem. Rev.

238

203

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“…The result shows that protein-glutaminase from Chryseobacterium proteolyticum (PDB ID: 3A54) is the closest structural homolog of hNtaq1 (Z score 7.0 and RMSD distance 3.6 Å for 123 equivalent C α positions out of 280 residues) and shares only 15% sequence identity. The protein-glutaminase from Chryseobacterium proteolyticum converts a glutamine residue to a glutamate and has catalytic triad comprising Cys156, His197, and Asp217 [27] ( Figure 1D ). The secreted effector protein SseI from Sallonella typhimurium (PDB ID: 4G2B) is the second best structural homolog of hNtaq1 (Z score 6.3 and RMSD distance 3.7 Å for 116 equivalent C α positions out of 169 residues) and shares only 9% sequence identity.…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of Human Protein N-Terminal Glutamine Amidohydrolase, an Initial Component of the N-End Rule Pathway

et al. 2014

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The N-end rule states that half-life of protein is determined by their N-terminal amino acid residue. N-terminal glutamine amidohydrolase (Ntaq) converts N-terminal glutamine to glutamate by eliminating the amine group and plays an essential role in the N-end rule pathway for protein degradation. Here, we report the crystal structure of human Ntaq1 bound with the N-terminus of a symmetry-related Ntaq1 molecule at 1.5 Å resolution. The structure reveals a monomeric globular protein with alpha-beta-alpha three-layer sandwich architecture. The catalytic triad located in the active site, Cys-His-Asp, is highly conserved among Ntaq family and transglutaminases from diverse organisms. The N-terminus of a symmetry-related Ntaq1 molecule bound in the substrate binding cleft and the active site suggest possible substrate binding mode of hNtaq1. Based on our crystal structure of hNtaq1 and docking study with all the tripeptides with N-terminal glutamine, we propose how the peptide backbone recognition patch of hNtaq1 forms nonspecific interactions with N-terminal peptides of substrate proteins. Upon binding of a substrate with N-terminal glutamine, active site catalytic triad mediates the deamination of the N-terminal residue to glutamate by a mechanism analogous to that of cysteine proteases.

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“…1 PG is expressed in C. proteolyticum in a pre-pro-protein form that comprises a putative signal peptide (21 amino acids), proregion sequence (114 amino acids) and mature domain (185 amino acids). 13 PG is only enzymatically active after the pro-region is cleaved off by an extracellular protease from C. proteolyticum. 14 As an environment-friendly enzyme requiring mild reaction conditions with high reaction specificity, PG has received attention in the food industry.…”

Section: Introductionmentioning

confidence: 99%

“…In 2000, Yamaguchi et al reported the discovery of PG from a culture medium of C. proteolyticum that was isolated from rice field soil in Japan 1 . PG is expressed in C. proteolyticum in a pre‐pro‐protein form that comprises a putative signal peptide (21 amino acids), pro‐region sequence (114 amino acids) and mature domain (185 amino acids) 13 . PG is only enzymatically active after the pro‐region is cleaved off by an extracellular protease from C. proteolyticum 14 .…”

Section: Introductionmentioning

confidence: 99%

Mass production of active recombinant Chryseobacterium proteolyticum protein glutaminase in Escherichia coli using a sequential dual expression system and one‐step purification

Poon

Zhang

2020

IUBMB Life

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Protein glutaminase (PG) is an enzyme that specifically catalyzes the deamidation of glutamine residues on proteins or peptides, remarkably improving the solubility, emulsification and foaming properties of food proteins and, thereby, conferring great potential in food industry applications. PG is primarily produced from wild strains of Chryseobacterium proteolyticum and the low enzyme production yield restricts large-scale industrial applications. In this context, by evaluating different cleavage site insertions between the pro-region and mature domain of PG as well as different linkers flanking the cleavage site, an E. coli expression and purification protocol has been developed to produce active recombinant PG. To simplify the production workflow, we developed a sequential dual expression system. More than 15 mg of pure and active PG was obtained from 1 L of shaking-flask bacteria culture by one-step nickel affinity chromatography purification. The enzymatic characteristics of the recombinant PG protein were similar to those of native PG. For the deamidation effect of recombinant PG, the deamidation degree (DD) of gliadin reached up to 67% and the solubility increased 84-fold. Thus, this study provides a practical approach to mass producing active PG proteins and investigates its potential applications on food proteins.

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Crystal Structures of Protein Glutaminase and Its Pro Forms Converted into Enzyme-Substrate Complex

Cited by 36 publications

References 41 publications

Eight Kinetically Stable but Thermodynamically Activated Molecules that Power Cell Metabolism

Eight Kinetically Stable but Thermodynamically Activated Molecules that Power Cell Metabolism

Crystal Structure of Human Protein N-Terminal Glutamine Amidohydrolase, an Initial Component of the N-End Rule Pathway

Mass production of active recombinant Chryseobacterium proteolyticum protein glutaminase in Escherichia coli using a sequential dual expression system and one‐step purification

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