Comparison of cobalamin-independent and cobalamin-dependent methionine synthases from Escherichia coli: two solutions to the same chemical problem.
In Escherichia coli, two enzymes catalyze the synthesis of methionine from homocysteine using methyltetrahydrofolate as the donor of the required methyl group: cobalamin-dependent and cobalamin-independent methionine synthases. Comparison of the mechanisms of these two enzymes offers the opportunity to examine two different solutions to the same chemical problem. We initiated the research described here to determine whether the two enzymes were evolutionarily related by comparing the deduced amino acid sequences of the two proteins. We have determined the nucleotide sequence for the metE gene, encoding the cobalamin-independent methionine synthase. Our results reveal an absence of similarity between the deduced amino acid sequences of the cobalamin-dependent and cobalamin-independent proteins and suggest that the two have arisen by convergent evolution. We have developed a rapid one-step purification of the recombinant cobalamin-independent methionine synthase (MetE) that yields homogeneous protein in high yield for mechanistic and structural studies. In the course of these studies, we identified a highly reactive thiol in MetE that is alkylated by chloromethyl ketones and by iodoacetamide. We demonstrated that alkylation of this residue, shown to be cysteine 726, results in complete loss of activity. While we are unable to deduce the role of cysteine 726 in catalysis at this time, the identification of this reactive residue suggests the possibility that this thiol functions as an intermediate methyl acceptor in catalysis, analogous to the role of cobalamin in the reaction catalyzed by the cobalamin-dependent enzyme.
González
J C
JC
Biophysics Research Division, University of Michigan, Ann Arbor 48109.
Banerjee
R V
RV
Huang
S
S
Sumner
J S
JS
Matthews
R G
RG
D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000478:Alkylation; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006710:Homocysteine / Q000378:metabolism; D007460:Iodoacetamide / Q000378:metabolism; D008715:Methionine / Q000096:biosynthesis; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D013763:Tetrahydrofolates / Q000378:metabolism; D014107:Tosyllysine Chloromethyl Ketone / Q000378:metabolism; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014805:Vitamin B 12 / Q000494:pharmacology*
D011994:Recombinant Proteins; D013763:Tetrahydrofolates; D006710:Homocysteine; D014107:Tosyllysine Chloromethyl Ketone; D014108:Tosylphenylalanyl Chloromethyl Ketone; D008715:Methionine; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D014357:Trypsin; D003545:Cysteine; D014805:Vitamin B 12; C005984:5-methyltetrahydrofolate; D007460:Iodoacetamide