Recombinant production of serine hydroxymethyl transferase from Streptococcus thermophilus and its preliminary evaluation as a biocatalyst

Abstract

The glyA gene encoding a serine hydroxymethyl transferase (SHMT) with threonine aldolase activity was isolated from Streptococcus thermophilus YKA-184 chromosomal DNA. This aldolase is a pyridoxal 5'-phosphate-dependent enzyme that stereospecifically catalyzes the interconversion of L-threonine to glycine and acetaldehyde. The enzyme was overexpressed in Escherichia coli M15 as a recombinant protein of 45 kDa with a His6-tag at its N-terminus. The recombinant enzyme was purified to homogeneity by a single chromatographic step using Ni-nitrilotriacetic acid affinity, obtaining a high activity-recovery yield (83%). Lyophilized and precipitated enzymes were stable at least for 10 weeks when stored at -20 degrees C and 4 degrees C. It was observed that the Km for L-allo-threonine was 38-fold higher than that for L-threonine, suggesting this enzyme can be classified as a specific L-allo-threonine aldolase. The optimum pH range of threonine aldolase activity for the recombinant SHMT was pH 6-7. When tested for aldol addition reactions with non-natural aldehydes, such as benzyloxyacetaldehyde and (R)-N-Cbz-alaninal, two possible beta-hydroxy-alpha-amino acid diastereoisomers were produced, but with moderate stereospecificity. The enzyme showed potential as a biocatalyst for the stereoselective synthesis of beta-hydroxy-alpha-amino acids.

Characterization of Central Carbon Metabolism of Streptococcus pneumoniae by Isotopologue Profiling

“…The de novo synthesis of Ser in bacteria typically involves dehydrogenation of 3-phosphoglycerate, which is followed by reductive transamination and dephosphorylation. This pathway has been reported for Streptococcus thermophilus (65,66) and also L. monocytogenes (12,14). In L. pneumophila, Ser supports the growth and serves as a major carbon substrate (13).…”

The Presequence of Arabidopsis Serine Hydroxymethyltransferase SHM2 Selectively Prevents Import into Mesophyll Mitochondria

“…This in vitro experiment showed distinct SHM activity for both recombinant enzymes (Fig. 2), and the measured activities were in the range of reported values for recombinant and native SHM from other organisms (Bourguignon et al, 1988;JagathReddy et al, 1995;diSalvo et al, 1998;Vidal et al, 2005). The somewhat higher specific activity of recombinant SHM2 was likely because of a lower level of contaminating protein.…”

Amino Acid Catabolic Pathways of Lactic Acid Bacteria

“…It has been demonstrated that SHMT and Thr aldolase activities are due to the same enzyme (encoded by glyA) in S. thermophilus since the inactivation of glyA led to an almost complete elimination of Thr aldolase activity (Chaves et al 2002). Further biochemical characterization of this enzyme confirmed its Thr aldolase activity (Vidal et al 2005).…”