Molecular cloning and expression of the gene for serine hydroxymethyltransferase from an obligate methylotroph Hyphomicrobium methylovorum GM2

Abstract

The gene encoding serine hydroxymethyltransferase (SHMT), one of the key enzymes of the one-carbon-compound assimilation of a methylotroph, Hyphomicrobium methylovorum GM2, and its flanking regions were isolated using a DNA fragment encoding Escherichia coli SHMT as a probe. Nucleotide sequencing of the recombinant plasmids revealed the SHMT gene codes for the 434-amino-acid protein with a calculated molecular mass of 46 068 Da. The amino-acid sequence of the enzyme showed identity to the sequences of the enzymes from E. coli (55%) and rabbit liver (44%). The recombinant plasmid, which was constructed by ligation of the cloned gene and an expression vector pKK223-3, was introduced to an SHMT-deficient E. coli mutant ME5427 (glyA-). The transformed E. coli cells expressed SHMT, which was immunologically and enzymologically indistinguishable from the enzyme isolated from H. methylovorum GM2.Serine hydroxymethyltransferase (SHMT) catalyzes the reversible interconversion of serine and glycine, with tetrahydrofolate as the one-carbon ( C , ) acceptor. This enzyme activity is widely distributed in nature, being found in prokaryote and eukaryote. Its major functions are to provide glycine for protein and purine synthesis, and 5,lO-methylenetetrahydrofolate for the C , pool. Recently, primary structures of the enzymes from rabbit liver, Salmonella typhimurium, Bacillus stearothermophilus, Escherichia coli, Bradyrhizobium japonicum and Campilobacter jejuni have been determined [l -71. A number of methylotrophic bacteria possess the serine pathway for the assimilation of C , compounds, which provides precursors for the biosyntheses of cell components from C , substrates. In such bacteria, SHMT plays an important role as the first enzyme in the assimilation of C , compounds through the transfer of formaldehyde (via 5,lO-Correspondence to

The Heterodisulfide Reductase from Methanobacterium Thermoautotrophicum Contains Sequence Motifs Characteristic of pyridine‐Nucleotide‐Dependent Thioredoxin Reductases

“…Further downstream, 196 nucleotides from the stop codon, an open reading frame was detected (Fig. 1A) which by sequence comparison appears to encode a hydroxymethyltransferase (Myata et al, 1993), probably the serine hydroxymethyltransferase described by Hoyt et al (1986).…”

Genetics of the serine cycle in Methylobacterium extorquens AM1: identification of sgaA and mtdA and sequences of sgaA, hprA, and mtdA

“…Neither of these fragments overlaps the hprA-ppcmclA-containing region (10). The genes responsible for the synthesis of glycerate kinase and the enzymes participating in oxidation of acetyl-CoA to glyoxylate were not precisely mapped, and the number of genes involved in the latter * (36), but its location relative to other methylotrophy genes is unknown.…”

Genetics of the serine cycle in Methylobacterium extorquens AM1: cloning, sequence, mutation, and physiological effect of glyA, the gene for serine hydroxymethyltransferase

“…Neither of these fragments overlaps the region containing sgaA, hprA, mtdA, mtkAB, ppcA, and mcL4 (7). The gene (glyA) for another serine cycle enzyme, serine hydroxymethyltransferase (SHMT), has been recently cloned and sequenced from an obligate methylotroph, Hyphomicrobium methylovorum GM2 (24); however, its location relative to other methylotrophy genes in that bacterium is unknown.…”

“…To isolate glyA from M. extorquens AM1, an oligonucleotide probe was developed on the basis of an amino acid sequence (GGHLTHG; amino acid residues 124 to 130 in E. coli SHMT [27]) which is known to be conserved in SHMTs (24). The known preference of M extorquens AM1 for G or C in the third triplet position was taken into consideration, resulting in the oligonucleotide GGCGGCCACCT(C/G)AC (C/G)CACGGC, with only a fourfold degeneracy.…”