Structure of crystalline Escherichia coli methionyl-tRNA(f)Met formyltransferase: comparison with glycinamide ribonucleotide formyltransferase.

Abstract

Formylation of the methionyl moiety esterified to the 3′ end of tRNA(f)Met is a key step in the targeting of initiator tRNA towards the translation start machinery in prokaryotes. Accordingly, the presence of methionyl‐tRNA(f)Met formyltransferase (FMT), the enzyme responsible for this formylation, is necessary for the normal growth of Escherichia coli. The present work describes the structure of crystalline E.coli FMT at 2.0 A, resolution. The protein has an N‐terminal domain containing a Rossmann fold. This domain closely resembles that of the glycinamide ribonucleotide formyltransferase (GARF), an enzyme which, like FMT, uses N‐10 formyltetrahydrofolate as formyl donor. However, FMT can be distinguished from GARF by a flexible loop inserted within its Rossmann fold. In addition, FMT possesses a C‐terminal domain with a beta‐barrel reminiscent of an OB fold. This latter domain provides a positively charged side oriented towards the active site. Biochemical evidence is presented for the involvement of these two idiosyncratic regions (the flexible loop in the N‐terminal domain, and the C‐terminal domain) in the binding of the tRNA substrate.

Eukaryotic-type elongator tRNA Met of Trypanosoma brucei becomes formylated after import into mitochondria

“…5. The general structure of MTFs consisting of two domains that are separated by a linker region seems to be retained in trypanosomes (22,23). Interestingly, the linker region (at position 313-368) is longer than in any of the other enzymes.…”

Mammalian Mitochondrial Methionyl-tRNA Transformylase from Bovine Liver

“…The crystal structure of E. coli MTF has recently been determined (35). Analysis of this structure indicates that MTF contains two domains, NH 2 -terminal and COOH-terminal domains.…”

“…Analysis of this structure indicates that MTF contains two domains, NH 2 -terminal and COOH-terminal domains. NH 2 -terminal domain carries the tetrahydrofolate (THF)-binding site in which the THF binding motif (SLLP motif) is contained, as well as a Rossman fold (35). The longest stretch of conserved residues among the MTFs from various sources is located in the THF-binding site proposed for E. coli MTF (35) (Fig.…”

“…The COOH-terminal domain provides a positively charged surface oriented toward the active center of the enzyme and is, presumably, involved in positioning the Met-tRNA substrate for the formylation reaction. The THF binding motif is present as SCLP in bovine MTF mt , and the Phe at position 14, which is assumed to be responsible for the interaction of E. coli MTF with the 3Ј-end of the Met-tRNA (35), is also conserved in the bovine mitochondrial counterpart (Phe-29) (Fig. 3).…”

“…3). The COOH-terminal domain of E. coli MTF is (19,35). The secondary structure of E. coli MTF is designated with red (␣-helix) and blue (␤-strand) lines above the sequence (35).…”