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J Biol Chem, Vol. 274, Issue 27, 19041-19047, July 2, 1999

Circular Permutation Analysis as a Method for Distinction of Functional Elements in the M20 Loop of Escherichia coli Dihydrofolate Reductase

Tsutomu NakamuraDagger § and Masahiro IwakuraDagger

From the Dagger  National Institute of Bioscience and Human-Technology, Higashi 1-1, Tsukuba, Ibaraki 305-8566, Japan and the § Osaka National Research Institute, Midorigaoka 1-8-31, Ikeda, Osaka 563-8577, Japan

A functional element of an enzyme can be defined as the smallest unit of the local peptide backbone of which the connectivity is crucial for the catalytic activity. In order to elucidate the distribution of functional elements in an active site flexible loop (the M20 loop) of Escherichia coli dihydrofolate reductase, systematic cleavage of main chain connectivity was performed using circular permutation. Our analysis is based on the assumption that a permutation within a functional element would significantly reduce enzyme function, whereas ones outside or at the boundaries of the elements would affect the function only slightly. Thus, a functional element would be assigned as the minimum peptide chain between the identified boundaries. Comparison of the activities of the circularly permuted variants revealed that the peptide chain around the M20 loop could be divided into four regions (regions 1-4). Region 1 was found to play an important role in overall tertiary fold because most variants permuted at region 1 did not accumulate in E. coli cells stably. A distinction between region 2 and region 3 was in agreement with the extent of movements calculated from the coordinates of alpha  carbons, supporting the idea that the movement of peptide backbone is a key feature of enzyme function. The boundary between region 3 and region 4 coincided with that between the M20 loop and the following alpha  helix. From equilibrium binding studies, region 2 was found to be involved in the binding of nicotinamide substrates, whereas region 4 appeared to be very important for the binding of pterin substrates.

Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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