Prokaryotic translation produces proteins that all begin with N-formyl methionine. This residue iscommonly removed in a multi-step process beginning with deformylation. Later steps do not proceed in the presence of the formyl group and often the resulting protein is not biologically active with the N-terminal Met in place. Thus inactivation of deformylase (DEF) activity is lethal and is being developed as an antibiotic strategy. Chloroplasts have recently been shown to contain two homologous DEF enzymes (DEF1 and DEF2). Our collaborators in the Houtz and Williams groups have cloned the Arabidopsis thaliana DEF genes and shown that DEF2 is responsible for most of the DEF activity in At chloroplasts. Thus, inhibitors of AtDEF are being pursued as potential herbicides. However, DEF employs a metal ion as a Lewis acid catalyst of hydrolysis, and it has recently been shown that although over-expressed E. coli DEF is commonly found to contain predominantly Zn2+, the Fe2+-containing form is 1000 x more active. Upon exposure to air, FeDEF auto-inactivates, so other metal-containing versions have been characterized. For the E. coli DEF, the Fe- and Nicontaining versions retain full activity, Co substitution produces 10% activity and Zn substitution leaves only residual activity (possibly representing contaminating transition metal ions. Thus the identity of the metal ion is crucial to catalytic activity, and Lewis acidity per-se does not suffice for activity. (Note however, that Meinnelís group claims that although DEF1 is most active with Fe bound, DEF2 is most active with Zn. Thus the identity of the protein also appears to dictate which metal performs best.)
The Miller group is studying the interplay between protein and metal ion by which proteins modulate the subtly different reactive tendencies of different metal ions, to produce (and enforce) specific chemistry, often activating the metal ion, compensating for tendencies it does not have and suppressing undesirable tendencies it does have. Understanding of these interactions is fundamental to understanding how enzymes work (40% contain metal ions), and how life evolved (based on inorganic substrates and catalysis). We are able to justify our studies of DEF on at least two bases. In order to produce inhibitors specific of DEF that do not inhibit related metalloproteases (such as thermolysin), or in order to target chloroplast DEFs without inhibiting bacterial DEFs or DEFs that may exist in human mitochondria, one should understand how chloroplast DEFís active site differs from those of other DEFs, and know what are the determinants of chloroplast DEF activity. In addition, the Miller lab would like to advance the possibility of mechanism-based inhibitors (since these can be much more specific and can be used in much smaller quantities since their action is irreversible). Therefore, we seek to define the mechanism of DEF activity.
This material is based upon
work supported by the National Science Foundation under Grant No. 0240165.
"Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation."
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