Crystallographic and kinetic studies of the tryptophan synthase α2β2 complex with a mutation in β subunit lysine-87 that binds pyridoxal phosphate

1994 ◽  
pp. 113-117 ◽  
Author(s):  
E. W. Miles ◽  
U. Banik ◽  
Z. Lu ◽  
S. A. Ahmed ◽  
K. D. Parris ◽  
...  
Keyword(s):  
Pyridoxal Phosphate ◽  
Kinetic Studies ◽  
Tryptophan Synthase ◽  
Β Subunit

scholarly journals Characterization of the putative tryptophan synthase β-subunit from Mycobacterium tuberculosis

2009 ◽  
Vol 41 (5) ◽  
pp. 379-388 ◽  
Author(s):  
Hongbo Shen ◽  
Yanping Yang ◽  
Feifei Wang ◽  
Ying Zhang ◽  
Naihao Ye ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Tryptophan Synthase ◽  
Β Subunit

scholarly journals A Novel Tryptophan Synthase β-Subunit from the HyperthermophileThermotoga maritima

2001 ◽  
Vol 277 (10) ◽  
pp. 8194-8201 ◽  
Author(s):  
Stefan Hettwer ◽  
Reinhard Sterner
Keyword(s):  
Tryptophan Synthase ◽  
Β Subunit

scholarly journals Scalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Gordon Rix ◽  
Ella J. Watkins-Dulaney ◽  
Patrick J. Almhjell ◽  
Christina E. Boville ◽  
Frances H. Arnold ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Thermotoga Maritima ◽  
Enzyme Engineering ◽  
Tryptophan Synthase ◽  
Β Subunit ◽  
Evolutionary Processes ◽  
Separate Species ◽  
Continuous Evolution ◽  
Primary Activity ◽  
Natural Diversity

Abstract Enzyme orthologs sharing identical primary functions can have different promiscuous activities. While it is possible to mine this natural diversity to obtain useful biocatalysts, generating comparably rich ortholog diversity is difficult, as it is the product of deep evolutionary processes occurring in a multitude of separate species and populations. Here, we take a first step in recapitulating the depth and scale of natural ortholog evolution on laboratory timescales. Using a continuous directed evolution platform called OrthoRep, we rapidly evolve the Thermotoga maritima tryptophan synthase β-subunit (TmTrpB) through multi-mutation pathways in many independent replicates, selecting only on TmTrpB’s primary activity of synthesizing l-tryptophan from indole and l-serine. We find that the resulting sequence-diverse TmTrpB variants span a range of substrate profiles useful in industrial biocatalysis and suggest that the depth and scale of evolution that OrthoRep affords will be generally valuable in enzyme engineering and the evolution of biomolecular functions.

scholarly journals Directed evolution of the tryptophan synthase β-subunit for stand-alone function recapitulates allosteric activation

2015 ◽  
Vol 112 (47) ◽  
pp. 14599-14604 ◽  
Author(s):  
Andrew R. Buller ◽  
Sabine Brinkmann-Chen ◽  
David K. Romney ◽  
Michael Herger ◽  
Javier Murciano-Calles ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Pyrococcus Furiosus ◽  
Tryptophan Synthase ◽  
Β Subunit ◽  
Allosteric Activation ◽  
Α Subunit ◽  
X Ray ◽  
Catalytic Activities ◽  
Catalytic Potential ◽  
Protein Partners

Enzymes in heteromeric, allosterically regulated complexes catalyze a rich array of chemical reactions. Separating the subunits of such complexes, however, often severely attenuates their catalytic activities, because they can no longer be activated by their protein partners. We used directed evolution to explore allosteric regulation as a source of latent catalytic potential using the β-subunit of tryptophan synthase from Pyrococcus furiosus (PfTrpB). As part of its native αββα complex, TrpB efficiently produces tryptophan and tryptophan analogs; activity drops considerably when it is used as a stand-alone catalyst without the α-subunit. Kinetic, spectroscopic, and X-ray crystallographic data show that this lost activity can be recovered by mutations that reproduce the effects of complexation with the α-subunit. The engineered PfTrpB is a powerful platform for production of Trp analogs and for further directed evolution to expand substrate and reaction scope.

GLUTAMIC CARBOXYLASE OF THE MATURE WHEAT LEAF

1952 ◽  
Vol 30 (6) ◽  
pp. 755-763 ◽  
Author(s):  
P. Weinberger ◽  
K. A. Clendenning
Keyword(s):  
Pyridoxal Phosphate ◽  
Kinetic Studies ◽  
Wheat Plant ◽  
Leaf Extracts ◽  
Ph Optimum ◽  
Carboxylase Activity ◽  
Mature Leaves ◽  
Heat Stable ◽  
Wheat Leaf ◽  
Weak Activity

A study of the distribution of glutamic carboxylase within the developing wheat plant revealed that it was absent in young plants, and was present only in traces in mature roots, and that it accumulated in mature leaves. Glutamic carboxylase was particularly abundant in the mature and senescent third leaf. Extracts of leaves of other cereals showed only weak activity, while extracts of roots, other than barley, were inactive. The high enzyme activity of the barley root extracts was exceeded only by that of mature wheat leaf extracts. A convenient method is described for enzyme storage at −40 °C. and a purification procedure was developed which effected a 500-fold concentration (nitrogen basis). The glutamic carboxylase activity of crude extracts was enhanced by preparatory exposure to phosphate buffer; after selective salt precipitation and lengthy dialysis, activity was reduced, but could be restored by the addition of pyridoxal phosphate. A heat-stable inhibitor of glutamic carboxylase at its pH optimum was found in the ether-soluble organic acid fraction of the cell sap of Kalanchoe leaves. Similar inhibiting effects were shown by malate, tartrate, and citrate, but not by succinate, fumarate, aspartate, and alanine. Kinetic studies indicated that the inhibition of plant glutamic carboxylase by cyanide is noncompetitive.

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