Reactivation of methionine synthase from Thermotoga maritima (TM0268) requires the downstream gene product TM0269

Abstract

The crystal structure of the Thermotoga maritima gene product TM0269, determined as part of genome-wide structural coverage of T. maritima by the Joint Center for Structural Genomics, revealed structural homology with the fourth module of the cobalamin-dependent methionine synthase (MetH) from Escherichia coli, despite the lack of significant sequence homology. The gene specifying TM0269 lies in close proximity to another gene, TM0268, which shows sequence homology with the first three modules of E. coli MetH. The fourth module of E. coli MetH is required for reductive remethylation of the cob(II)alamin form of the cofactor and binds the methyl donor for this reactivation, S-adenosylmethionine (AdoMet). Measurements of the rates of methionine formation in the presence and absence of TM0269 and AdoMet demonstrate that both TM0269 and AdoMet are required for reactivation of the inactive cob(II)alamin form of TM0268. These activity measurements confirm the structure-based assignment of the function of the TM0269 gene product. In the presence of TM0269, AdoMet, and reductants, the measured activity of T. maritima MetH is maximal near 80 degrees C, where the specific activity of the purified protein is approximately 15% of that of E. coli methionine synthase (MetH) at 37 degrees C. Comparisons of the structures and sequences of TM0269 and the reactivation domain of E. coli MetH suggest that AdoMet may be bound somewhat differently by the homologous proteins. However, the conformation of a hairpin that is critical for cobalamin binding in E. coli MetH, which constitutes an essential structural element, is retained in the T. maritima reactivation protein despite striking divergence of the sequences.

Scheme 1.

The catalytic cycle of MetH (red) and the reactions that deactivate and reactivate the enzyme from E. coli (green). In methionine synthesis, cobalamin serves as an intermediate methyl carrier, receiving a methyl group from CH₃-H₄folate and, in turn, donating the group to Hcy. Inactivation by reaction with oxygen generates the cob(II)alamin form, which must be reduced and then remethylated by AdoMet in order to return to the catalytic cycle.

Figure 1.

(Solid line) The visible absorbance spectrum of TM0268 reconstituted with methylcobalamin. (Dashed line) For comparison, the spectrum of E. coli MetH in the methylcobalamin form.

Figure 2.

Specific activity of T. maritima methionine synthase as a function of temperature. Assay mixtures included Hcy (saturating), AdoMet, the chemical reducing system, and 1:1 mixtures of TM0268 and TM0269. After temperature equilibration for 2 min, turnover was initiated by addition of CH₃-H₄folate. The amount of product formed was measured in samples quenched at 2 min. (See Materials and Methods for additional details.)

Figure 3.

Reactivation of activity of TM0268 requires TM0269 and AdoMet. TM0268 in the cob(II)alamin form was generated by reaction of the methylcobalamin form with homocysteine under aerobic conditions at 64°C. Assays were performed with 9.2 nM TM0268 in the presence of (•) equimolar TM0269 and 19 μM AdoMet; (□) AdoMet only; (▴) TM0269 only; or (△) neither.

Figure 4.

Electrochemical methylation of TM0268 in the cob(II)alamin form requires TM0269. TM0268 in the cob(III)alamin form was generated by aerobic photolysis (Jarrett et al. 1996) and then incubated under argon in the presence of 1 mM AdoMet and 500 μM methylviologen at −0.450 V versus standard hydrogen electrode for 45 min at 25°C in the presence or absence of TM0269. Following incubation, the proteins were subjected to gel filtration to remove AdoMet and methylviologen, and the spectra were recorded. (Blue line) The absorbance of the hydroxycob(III)alamin form of TM0268 before electrochemical methylation; (black line) the TM0268 absorbance in the cob(II)alamin form after incubation in the absence of TM0269; (red line) the absorbance observed after incubation in the presence of equimolar TM0269. The black line indicates that incubation in the absence of TM0269 results in the formation of cob(II)alamin, which is characterized by an absorbance peak at 477 nm. The red line indicates that TM0268 is converted to methylcobalamin in the presence of TM0269 and AdoMet.

Figure 5.

(A) The structure of TM0269 (1J6R, yellow) is superimposed on the structure of the C-terminal domain of MetH from E. coli (1MSK, blue) (Dixon et al. 1996), with bound AdoMet displayed in ball-and-stick mode (1MSK). Residues used to match the structures are from a structurally conserved core that includes the E. coli helices α5 and α6 and strands β2, β5, and β8 shown in the sequence alignment C. The hairpin that positions cobalamin in the structure of the reactivation complex from E. coli (1163–1173) is above and to the right of the adenine ring in this view. (B) Close-up view of the binding site for AdoMet in TM0269. (C) Structure-based sequence alignment of TM0269 and the activation domain of E. coli MetH. Similar structural elements that overlay each other or show small displacements in A are blue, and identities are highlighted. Italicized sections of the T. maritima sequence are structurally dissimilar to the E. coli structure and demonstrate the shortened loops of the thermophile.