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Blickling, Stefan; Knäblein, Jörg

doi:10.1515/bchm.1997.378.3-4.207

Cited by 7 publications

References 14 publications

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Mimicking natural evolution in vitro : An N -acetylneuraminate lyase mutant with an increased dihydrodipicolinate synthase activity

Joerger

Mayer

Fersht

2003

Proc. Natl. Acad. Sci. U.S.A.

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N-acetylneuraminate lyase (NAL) and dihydrodipicolinate synthase (DHDPS) belong to the NAL subfamily of (␤͞␣) 8-barrels. They share a common catalytic step but catalyze reactions in different biological pathways. By rational design, we have introduced various mutations into the NAL scaffold from Escherichia coli to switch the activity toward DHDPS. These mutants were tested with respect to their catalytic properties in vivo and in vitro as well as their stability. One point mutation (L142R) was sufficient to create an enzyme that could complement a bacterial auxotroph lacking the gene for DHDPS as efficiently as DHDPS itself. In vitro, this mutant had an increased DHDPS activity of up to 19-fold as defined by the specificity constant k cat͞KM for the new substrate L-aspartate-␤-semialdehyde when compared with the residual activity of NAL wild-type, mainly because of an increased turnover rate. At the same time, mutant L142R maintained much of its original NAL activity. We have solved the crystal structure of mutant L142R at 1.8 Å resolution in complex with the inhibitor ␤-hydroxypyruvate. This structure reveals that the conformations of neighboring active site residues are left virtually unchanged by the mutation. The high flexibility of R142 may favor its role in assisting in catalysis. Perhaps, nature has exploited the catalytic promiscuity of many enzymes to evolve novel enzymes or biological pathways during the course of evolution.protein ͉ engineering ͉ aldolase

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Mimicking natural evolution in vitro : An N -acetylneuraminate lyase mutant with an increased dihydrodipicolinate synthase activity

Joerger

Mayer

Fersht

2003

Proc. Natl. Acad. Sci. U.S.A.

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Cloning, expression, purification and crystallization of dihydrodipicolinate synthase from the psychrophileShewanella benthica

Wubben¹,

Dogovski²,

Dobson³

et al. 2010

Acta Cryst Sect F

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Dihydrodipicolinate synthase (DHDPS) is an oligomeric enzyme that catalyzes the first committed step of the lysine-biosynthesis pathway in plants and bacteria, which yields essential building blocks for cell-wall and protein synthesis. DHDPS is therefore of interest to drug-discovery research as well as to studies that probe the importance of quaternary structure to protein function, stability and dynamics. Accordingly, DHDPS from the psychrophilic (colddwelling) organism Shewanella benthica (Sb-DHDPS) was cloned, expressed, purified and crystallized. The best crystals of Sb-DHDPS were grown in 200 mM ammonium sulfate, 100 mM bis-tris pH 5.0-6.0, 23-26%(w/v) PEG 3350, 0.02%(w/v) sodium azide and diffracted to beyond 2.5 Å resolution. Processing of diffraction data to 2.5 Å resolution resulted in a unit cell with space group P2 1 2 1 2 1 and dimensions a = 73.1, b = 84.0, c = 143.7 Å . These studies of the first DHDPS enzyme to be characterized from a bacterial psychrophile will provide insight into the molecular evolution of enzyme structure and dynamics.

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Cloning, expression, purification, crystallization and X-ray diffraction analysis of dihydrodipicolinate synthase from the human pathogenic bacteriumBartonella henselaestrain Houston-1 at 2.1 Å resolution

et al. 2016

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The enzyme dihydrodipicolinate synthase catalyzes the committed step in the synthesis of diaminopimelate and lysine to facilitate peptidoglycan and protein synthesis. Dihydrodipicolinate synthase catalyzes the condensation of L-aspartate 4-semialdehyde and pyruvate to synthesize L-2,3-dihydrodipicolinate. Here, the cloning, expression, purification, crystallization and X-ray diffraction analysis of dihydrodipicolinate synthase from the pathogenic bacterium Bartonella henselae, the causative bacterium of cat-scratch disease, are presented. Protein crystals were grown in conditions consisting of 20%(w/v) PEG 4000, 100 mM sodium citrate tribasic pH 5.5 and were shown to diffract to ∼2.10 Å resolution. They belonged to space group P212121, with unit-cell parameters a = 79.96, b = 106.33, c = 136.25 Å. The final R values were Rr.i.m. = 0.098, Rwork = 0.183, Rfree = 0.233.

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Cited by 7 publications

References 14 publications

Mimicking natural evolution in vitro : An N -acetylneuraminate lyase mutant with an increased dihydrodipicolinate synthase activity

Mimicking natural evolution in vitro : An N -acetylneuraminate lyase mutant with an increased dihydrodipicolinate synthase activity

Cloning, expression, purification and crystallization of dihydrodipicolinate synthase from the psychrophileShewanella benthica

Cloning, expression, purification, crystallization and X-ray diffraction analysis of dihydrodipicolinate synthase from the human pathogenic bacteriumBartonella henselaestrain Houston-1 at 2.1 Å resolution

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