Structure of SAICAR synthetase from Pyrococcus horikoshii OT3: Insights into thermal stability

Manjunath, Kavyashree; Kanaujia, Shankar Prasad; Kanagaraj, S.; Jeyakanthan, Jeyaraman; Sekar, K.

doi:10.1016/j.ijbiomac.2012.10.028

Cited by 6 publications

(15 citation statements)

References 72 publications

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“…A comparison of the structural and sequence parameters for DHDPS from mesophiles and thermophiles is given in Table 2. Also, the percentage composition of amino acid (Manjunath et al 2013) from each of the four different DHDPS was calculated and the mean of the percentages from the mesophiles and thermophiles was plotted on an XY-graph (Supplementary fig. S3) and tabulated (Supplementary Table ST1).…”

Section: Bioinformatic Analysis and Possible Thermostability Of Aqdhdpsmentioning

confidence: 99%

Crystal structure and in silico studies of dihydrodipicolinate synthase (DHDPS) from Aquifex aeolicus

et al. 2014

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Dihydrodipicolinate synthase (DHDPS, E.C.4.2.1.52) catalyzes the first committed step in the lysine biosynthetic pathway: the condensation of (S)-aspartate semialdehyde and pyruvate to form (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. Since (S)-lysine biosynthesis does not occur in animals, DHDPS is an attractive target for rational antibiotic and herbicide design. Here, we report the crystal structure of DHDPS from a hyperthermophilic bacterium Aquifex aeolicus (AqDHDPS). L-Lysine is used as an important animal feed additive where the production is at the level of 1.5 million tons per year. The biotechnological manufacture of lysine has been going for more than 50 years which includes over synthesis and reverse engineering of DHDPS. AqDHDPS revealed a unique disulfide linkage which is not conserved in the homologues of AqDHDPS. In silico mutation of C139A and intermolecular ion-pair residues and the subsequent molecular dynamics simulation of the mutants showed that these residues are critical for the stability of AqDHDPS tetramer. MD simulations of AqDHDPS at three different temperatures (303, 363 and 393 K) revealed that the molecule is stable at 363 K. Thus, this structural and in silico study of AqDHDPS likely provides additional details towards the rational and structure-based design of hyper-L-lysine producing bacterial strains.

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Section: Bioinformatic Analysis and Possible Thermostability Of Aqdhdpsmentioning

confidence: 99%

Crystal structure and in silico studies of dihydrodipicolinate synthase (DHDPS) from Aquifex aeolicus

et al. 2014

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“…We had previously obtained the structure of apo form of P. horikoshii SAICAR synthetase (PhSS) in C222 1 and H3 space groups (Manjunath et al, 2013). Here, we report the ligand bound structures of the same enzyme in C222 1 and H3 space groups.…”

Section: Introductionmentioning

confidence: 94%

“…Protein was extracted and purified as mentioned in our previous work (Manjunath et al, 2013). The purified protein was concentrated to 10-14 mg/ml for crystallization experiments in 20 mM TrisHCl buffer at pH 8.0 with 200 mM NaCl.…”

Section: Purification and Crystallizationmentioning

confidence: 99%

“…All protein-ligand complexes mentioned here were obtained by co-crystallization using microbatch under oil method. The crystallization conditions used for co-crystallization were same as those used for obtaining the apo-structures (in H3 and C222 1 space groups), described previously (Manjunath et al, 2010(Manjunath et al, , 2013, and crystals were obtained within 10-15 days. The dimensions of the crystals were $0.1-0.2 Â 0.5-0.7 Â 0.05 mm and $0.2 Â 0.2 Â 0.1 mm in C222 1 and H3 space groups, respectively.…”

Section: Purification and Crystallizationmentioning

confidence: 99%

“…This structural variability occurs as a result of the underlying sequence differences. Most of the bacterial SAICAR synthetase structures exist as a dimer (Ginder et al, 2006;Manjunath et al, 2013;Wolf et al, 2014;Zhang et al, 2006), the structure from yeast and Mycobacterium abscessus is a monomer (Levdikov et al, 1998), and the bifunctional octameric forms are observed only in higher eukaryotes (Li et al, 2007;Taschner et al, 2013). Thus, the structural variations between microbial and human SAICAR synthetase Abbreviations: SAICAR, phosphoribosylaminoimidazole-succinocarboxamide; CAIR, 4-carboxyaminoimidazole ribonucleotide; N 5 -CAIR, N 5 -carboxyaminoimidazole ribonucleotide; ATP, adenosine triphosphate; AMP, adenosine monophosphate; ASP, aspartate; AMP-PNP, adenosine 5 0 -(b,c-imido)tri phosphate; SS, SAICAR synthetase; Ph, Pyrococcus horikoshii; Sc, Saccharomyces cerevisiae; Ec, Escherichia coli; Sp, Streptococcus pneumoniae.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Catalytic pathway, substrate binding and stability in SAICAR synthetase: A structure and molecular dynamics study

Manjunath

Jeyakanthan

Sekar

2015

Journal of Structural Biology

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Molecular Dynamics Perspective on the Protein Thermal Stability: A Case Study Using SAICAR Synthetase

Manjunath

Sekar

2013

J. Chem. Inf. Model.

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The enzyme SAICAR synthetase ligates aspartate with CAIR (5'-phosphoribosyl-4-carboxy-5-aminoimidazole) forming SAICAR (5-amino-4-imidazole-N-succinocarboxamide ribonucleotide) in the presence of ATP. In continuation with our previous study on the thermostability of this enzyme in hyper-/thermophiles based on the structural aspects, here, we present the dynamic aspects that differentiate the mesophilic (E. coli, E. chaffeensis), thermophilic (G. kaustophilus), and hyperthermophilic (M. jannaschii, P. horikoshii) SAICAR synthetases by carrying out a total of 11 simulations. The five functional dimers from the above organisms were simulated using molecular dynamics for a period of 50 ns each at 300 K, 363 K, and an additional simulation at 333 K for the thermophilic protein. The basic features like root-mean-square deviations, root-mean-square fluctuations, surface accessibility, and radius of gyration revealed the instability of mesophiles at 363 K. Mean square displacements establish the reduced flexibility of hyper-/thermophiles at all temperatures. At the simulations time scale considered here, the long-distance networks are considerably affected in mesophilic structures at 363 K. In mesophiles, a comparatively higher number of short-lived (having less percent existence time) Cα, hydrogen bonds, hydrophobic interactions are formed, and long-lived (with higher percentage existence time) contacts are lost. The number of time-averaged salt-bridges is at least 2-fold higher in hyperthermophiles at 363 K. The change in surface accessibility of salt-bridges at 363 K from 300 K is nearly doubled in mesophilic protein compared to proteins from other temperature classes.

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Structure of SAICAR synthetase from Pyrococcus horikoshii OT3: Insights into thermal stability

Cited by 6 publications

References 72 publications

Crystal structure and in silico studies of dihydrodipicolinate synthase (DHDPS) from Aquifex aeolicus

Crystal structure and in silico studies of dihydrodipicolinate synthase (DHDPS) from Aquifex aeolicus

Catalytic pathway, substrate binding and stability in SAICAR synthetase: A structure and molecular dynamics study

Molecular Dynamics Perspective on the Protein Thermal Stability: A Case Study Using SAICAR Synthetase

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