Exploring the active site of chorismate mutase by combinatorial mutagenesis and selection: the importance of electrostatic catalysis.

Chorismate mutase (EC 5.4.99.5) catalyzes the intramolecular rearrangement of chorismate to prephenate. Arg-90 in the active site of the enzyme from Bacillus subtilis is in close proximity to the substrate's ether oxygen and may contribute to efficient catalysis by stabilizing the presumed dipolar transition state that would result upon scission of the C--O bond. To test this idea, we have developed a novel complementation system for chorismate mutase activity in Escherichia coli by reengineering parts of the aromatic amino acid biosynthetic pathway. The codon for Arg-90 was randomized, alone and in combination with that for Cys-88, and active clones were selected. The results show that a positively charged residue either at position 88 (Lys) or 90 (Arg or Lys) is essential. Our data provide strong support for the hypothesis that the positive charge is required for stabilization of the transition state of the enzymatic chorismate rearrangement. The new selection system, in conjunction with combinatorial mutagenesis, renders the mechanism of the natural enzyme(s) accessible to further exploration and opens avenues for the improvement of first generation catalytic antibodies with chorismate mutase activity.

In Vivo screening and discovery of novel candidate thalidomide analogs in the zebrafish embryo and chicken embryo model systems

This study was supported by a Wellcome Trust-NIH PhD Studentship to SB, WDF and NV. Grant number 098252/Z/12/Z. SB, CHC and WDF are supported by the Intramural Research Program, NCI, NIH. NHG and WL are supported by the Intramural Research Program, NIA, NIH.

Stabilization/solidification of CERCLA and RCRA wastes : physical tests, chemical testing procedures, technology screening, and field activities.

"May 1989."

Combined sewer overflow treatment by screening and terminal ponding : Fort Wayne, Indiana /

Mode of access: Internet.

Assessment of international technologies for Superfund applications : technology identification and selection /

Mode of access: Internet.

Reproduction of farm animals and their improvement through testing and selection,

Mode of access: Internet.

Genetic screening and the handling of high-risk groups in the workplace [microform] : hearings before the Subcommittee on Investigations and Oversight of the Committee on Science and Technology, U.S. House of Representatives, Ninety-seventh Congress, first session, October 14, 15, 1981.

"No. 53."

Grenada documents : an overview and selection.

Item 876

Effects of short term oven aging on volumetrics and selection of N-design /

"Study was conducted in cooperation with the U.S. Department of Transportation, Federal Highway Administration."

Evaluating state-financed, workplace-based retraining programs : a report on the feasibility of a business screening and performance outcome evaluation system /

"May 1990."

Descent of man, and selection in relation to sex.

Mode of access: Internet.

Mentally ill inmates : BOP plans to improve screening and care in federal prisons and jails : report to Congressional requesters /

"B-242958"--P. 1.

The descent of man, and selection in relation to sex.

Mode of access: Internet.

Birth defects and genetic screening and counseling /

Item 985