A pH-dependent stabilization of an active site loop observed from low and high ph crystal structures of mutant monomeric glycinamide ribonucleotide transformylase at 1.8 to 1.9 Å

Su, Ying; Yamashita, M M; Greasley, S.E.; Mullen, Christine A; Shim, Jae Hoon; Jennings, Patricia A.; Benkovic, S.J.; Wilson, Ian A.

doi:10.1006/jmbi.1998.1931

Cited by 36 publications

(63 citation statements)

References 39 publications

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“…The position of the Mg 2+ in the current M. tuberculosis structure approximates the expected position of the GAR α-amino group, as deduced from substrate and substrate analogue binding studies on EcPurN and HsPurN. 9,[13][14][15]20 The interactions made by this Mg 2+ , if it is regarded as a surrogate for the GAR α-amino group, would be consistent with the involvement of Asn116, His118, and Asp154 in activation of this amino group to facilitate nucleophilic attack. At least two of the three amino acids Asn106, His108 and Asp144 in EcPurN are absolutely required for in vivo activity, and it has been suggested that any two of these three polar residues could activate the amino group of GAR.…”

Section: Ion Binding and Loop Variability In The Substrate-binding Sitementioning

confidence: 59%

Structures of Glycinamide Ribonucleotide Transformylase (PurN) from Mycobacterium tuberculosis Reveal a Novel Dimer with Relevance to Drug Discovery

Zhang¹,

Caradoc-Davies²,

Dickson³

et al. 2009

Journal of Molecular Biology

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Section: Ion Binding and Loop Variability In The Substrate-binding Sitementioning

confidence: 59%

Structures of Glycinamide Ribonucleotide Transformylase (PurN) from Mycobacterium tuberculosis Reveal a Novel Dimer with Relevance to Drug Discovery

Zhang¹,

Caradoc-Davies²,

Dickson³

et al. 2009

Journal of Molecular Biology

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“…This implies that N-terminal IMPCH domain somewhat stabilizes on interaction with Cterminal domain as it attains a more stable structural fold in full-length SlugATIC as compared with a molten globule structure in free-form. This could be due to the unavailability of ATIC crystal structure with naturally occurring 10-f-THF (due to its unstability) with a reduced pterin flexible ring which could have more favorable interactions in the folate binding site/pocket in comparison to the available crystal structures of ATIC with the folate analogs which have rigid aromatic deazafolate ring [6,23]. Interestingly, a reduction in T m (38.9, 43.4, and 46.1°C) of melting transitions were observed on DSC analysis of an equimolar mixture of two domains with none of peaks as prominent as was case in full-length SlugATIC (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Also, the lower T m of the major transition peak in case of SlugATIC as compared to individual domains could be attributed to stabilization of N-terminal domain and could have functional implications (Table 3). Interestingly, GAR TFase another enzyme utilizing 10f-THF in the de novo pathway had been reported to show large conformational change in the folate binding loop (open and closed conformation) which sequesters the folate from bulk solvent [23,24]. 7D).…”

Section: Discussionmentioning

confidence: 99%

Characterization of AICAR transformylase/IMP cyclohydrolase (ATIC) from Staphylococcus lugdunensis

et al. 2017

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The 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) catalyzes final two steps of purine nucleotide de novo biosynthetic pathway. This study reports the characterization of ATIC from Staphylococcus lugdunensis (SlugATIC). Apart from kinetic analysis and a detailed biophysical characterization of SlugATIC, the role of ATIC in cell proliferation has been demonstrated for the first time. The purified recombinant SlugATIC and its truncated domains exist mainly in dimeric form was revealed in gel-filtration and glutaraldehyde cross-linking studies. The two activities reside on separate domains was demonstrated in kinetic analysis of SlugATIC and reconstituted truncated N-terminal IMP cyclohydrolase (IMPCHase) and C-terminal AICAR transformylase (AICAR TFase) domains. Site-directed mutagenesis showed that Lys255 and His256 are the key catalytic residues, while Asn415 substantially contributes to AICAR TFase activity in SlugATIC. The differential scanning calorimetry (DSC) analysis revealed a molten globule-like structure for independent N-terminal domain as compared with a relatively stable conformational state in full-length SlugATIC signifying the importance of covalently linked domains. Unlike reported crystal structures, the DSC studies revealed significant conformational changes on binding of leading ligand to AICAR TFase domain in SlugATIC. The cell proliferation activity of SlugATIC was observed where it promoted proliferation and viability of NIH 3T3 and RIN-5F cells, exhibited in vitro wound healing in NIH 3T3 fibroblast cells, and rescued RIN-5F cells from the cytotoxic effects of palmitic acid and high glucose. The results suggest that ATIC, an important drug target, can also be exploited for its cell proliferative properties.

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“…The enzyme glycinamide ribonucleotide transformylase (GART) participates in the de novo biosynthetic pathway of purines (Garrett and Grishnan 1995). GART undergoes two major conformational changes as a function of pH, a coil–helix transition (Almassy et al 1992; Su et al 1998) and a dimer–monomer transition (Almassy et al 1992; Mullen and Jennings 1996; Su et al 1998), both with pK a ∼ 7. Specifically, an 8‐residue segment (residues 120–127) of a 21‐residue‐long loop (residues 111–131), called hereafter the activation loop, becomes an α‐helix at high pH, called hereafter the activation helix (Fig.…”

mentioning

confidence: 99%

“…At high pH the activation loop–helix caps and shields the active site from solvent, to facilitate catalysis (Almassy et al 1992; Greasley et al 1999). In addition to the coil–helix transition, GART converts from a dimeric form at low pH to a monomeric form at high pH (Almassy et al 1992; Mullen and Jennings 1996; Su et al 1998). The two pH‐dependent conformational changes appear to be independent because they occur at different surfaces of the enzyme; however, cooperativity cannot be excluded at present.…”

mentioning

confidence: 99%

Native‐state conformational dynamics of GART: A regulatory pH‐dependent coil–helix transition examined by electrostatic calculations

et al. 2001

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Glycinamide ribonucleotide transformylase (GART) undergoes a pH-dependent coil-helix transition with pK a ∼ 7. An ␣-helix is formed at high pH spanning 8 residues of a 21-residue-long loop, comprising the segment Thr120-His121-Arg122-Gln123-Ala124-Leu125-Glu126-Asn127. To understand the electrostatic nature of this loop-helix, called the activation loop-helix, which leads to the formation and stability of the ␣-helix, pK a values of all ionizable residues of GART have been calculated, using Poisson-Boltzmann electrostatic calculations and crystallographic data. Crystallographic structures of high and low pH E70A GART have been used in our analysis. Low pK a values of 5.3, 5.3, 3.9, 1.7, and 4.7 have been calculated for five functionally important histidines, His108, His119, His121, His132, and His137, respectively, using the high pH E70A GART structure. Ten theoretical single and double mutants of the high pH E70A structure have been constructed to identify pairwise interactions of ionizable residues, which have aided in elucidating the multiplicity of electrostatic interactions of the activation loop-helix, and the impact of the activation helix on the catalytic site. Based on our pK a calculations and structural data, we propose that: (1) His121 forms a molecular switch for the coil-helix transition of the activation helix, depending on its protonation state; (2) a strong electrostatic interaction between His132 and His121 is observed, which can be of stabilizing or destabilizing nature for the activation helix, depending on the relative orientation and protonation states of the rings of His121 and His132; (3) electrostatic interactions involving His119 and Arg122 play a role in the stability of the activation helix; and (4) the activation helix contains the helixpromoting sequence Arg122-Gln123-Ala124-Leu125-Glu126, but its alignment relative to the N and C termini of the helix is not optimal, and is possibly of a destabilizing nature. Finally, we provide electrostatic evidence that the formation and closure of the activation helix create a hydrophobic environment for catalytic-site residue His108, to facilitate catalysis.

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A pH-dependent stabilization of an active site loop observed from low and high ph crystal structures of mutant monomeric glycinamide ribonucleotide transformylase at 1.8 to 1.9 Å

Cited by 36 publications

References 39 publications

Structures of Glycinamide Ribonucleotide Transformylase (PurN) from Mycobacterium tuberculosis Reveal a Novel Dimer with Relevance to Drug Discovery

Structures of Glycinamide Ribonucleotide Transformylase (PurN) from Mycobacterium tuberculosis Reveal a Novel Dimer with Relevance to Drug Discovery

Characterization of AICAR transformylase/IMP cyclohydrolase (ATIC) from Staphylococcus lugdunensis

Native‐state conformational dynamics of GART: A regulatory pH‐dependent coil–helix transition examined by electrostatic calculations

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