The unexpected catalytic properties of a heterodimer of GAR transformylase

Canhui Liu, Jae Hoon Shim, Stephen J. Benkovic

Research output: Contribution to journalArticlepeer-review

Abstract

We have developed an efficient expression and purification protocol for a heterodimer of glycinamide ribonucleotide transformylase that was identified in incremental truncation libraries, a general combinatorial method for protein fragment complementation (M. Ostermeier, A. E. Nixon, J. H. Shim, and S. J. Benkovic, [1999], Proc. Natl. Acad. Sci. USA 96, 3562-3567). This heterodimer (B 13) containing both a bisection point and a deletion in conserved residues close to the active site was expressed and purified in high yield using Intein methodology. The N-terminus fragment (1-111) and C-terminus fragment (M114-212) were also expressed separately as stable proteins. When these two fragments were mixed together, they associate at a highly specific 1:1 ratio to give only the active heterodimer, B 13. The activity of B 13 is comparable to that of the wild type and the pH-dependent kinetics of B 13 turned out to be nearly identical to those of the wild type, indicating that B 13 operates in the same mechanism as the wild type. This result demonstrated that cutting within conserved regions is a viable domain separation and confirmed the generality of using incremental truncation for protein fragment complementation.

Original languageEnglish (US)
Pages (from-to)316-323
Number of pages8
JournalBioorganic Chemistry
Volume28
Issue number6
DOIs
StatePublished - Jan 1 2000

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Drug Discovery
  • Organic Chemistry

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