The glyA was cloned into the vector pGEX-6p-1 and expressed in DE3. Expression, purification and activity of the AnSHMT and the mutant Site-directed mutagenesisĪ leucine residue was introduced into the 249 position using site-directed mutagenesis and conformed, followed by the test of its effects on enzymatic activity using purification I249L-SHMT. The glycine rich region GQQGGP (268-273) (Figure 2c), was highly conserved sequence, significant homologous sequence, and proposed to be essential for PLP binding, the region of which was indicated on the three-dimensional structure of AnSHMT (Figure 3). nicotianae) using degenerate primers, (ii) study characteristics of recombinant SHMT by isolating the enzyme with high activity and (iii) improve its catalytic efficiency by site-directed mutagenesis.īased on multiple sequence comparisons, two well-known conserved sequences LTNKYAEGYPGRRYYGG (61-77) and GGHLTHG (134-140) (Figure 2a and 2b) were also detected in AnSHMT.
![pbp3 catalytic serine pbp3 catalytic serine](https://els-jbs-prod-cdn.jbs.elsevierhealth.com/cms/attachment/413bac8c-02cd-4b9f-b1d6-bec0e48ae88b/gr1_lrg.jpg)
The objectives of the current study were to (i) clone the glyA gene from Arthrobacter nicotianae ( A. Structure-based site-directed mutagenesis is usually applied to produce variants with dramatically improved specificities and the residues in or near the active site tend to be chosen as their special roles in the activity of the enzymes. And the exact active-site residues have never been confirmed using kinetic analyses of the wild-type and variant enzymes. coli) are reported, little is known at present about the structure-function relationship among these enzymes. Although the crystal structures of SHMT from the human, rabbit and Escherichia coli ( E. Protein engineers redesign proteins in order to improve their biomedical or industrial utility. Based on unknown genome information circumstances, using DOP-PCR to obtain glyA (encoding SHMT) can overcome the shortcomings of existing methods, such as cDNA library, southern-blot hybridization, direct PCR amplification according to the known genome information, and shotgun technology, for their waste time and material under the same conditions. The degenerate oligonucleotide-primed PCR (DOP-PCR) has been applied to the characterization of abnormal chromosomes and also in the cloning of new markers for specific chromosome regions. SHMT is widely used in the synthesis of the serine using glycine and formaldehyde, while the activity of the wild-type is not sufficient for industrial production. SHMT is ubiquitous, highly conserved PLP-dependent enzyme with tetrahydrofolate (THFA) as the C 1 acceptor, purified from the animals, plants and bacteria. The crystal structures of SHMT from the human and rabbit confirm that SHMT belongs to the α-family of enzymes and shares a similar tertiary fold and mechanism. SHMT (EC 2.1.2.1), a member of the α-class of pyridoxal phosphate enzymes, catalyzes the reversible interconversion of serine and glycine, changes the chemical bonding at the C α-C β bond of the serine side-chain mediated by the pyridoxal phosphate cofactor.
![pbp3 catalytic serine pbp3 catalytic serine](https://journals.asm.org/cms/10.1128/JB.185.13.3726-3734.2003/asset/b70c3072-a6bf-4d33-9197-64ce6f28d3d1/assets/graphic/jb1330223003.jpeg)
Therefore, it is necessary to obtain a SHMT with high activity of some new microorganisms and improve the catalytic efficiency through in vitro directed evolution. The SHMT is coded by the glyA genes and act as the first enzyme in the assimilation of C 1 compounds through the addition of formaldehyde to glycine, producing the principal intermediate in the pathway, serine.
![pbp3 catalytic serine pbp3 catalytic serine](https://files.rcsb.org/pub/pdb/validation_reports/un/6un1/6un1_multipercentile_validation.png)
PBP3 CATALYTIC SERINE PLUS
Currently, L-serine production mainly relies on enzymatic conversion from glycine precursor plus a C 1 compound, and the key enzyme in L-serine enzymatic conversion is SHMT.
![pbp3 catalytic serine pbp3 catalytic serine](https://journals.asm.org/cms/10.1128/mBio.03058-20/asset/217e1af2-b195-42ad-9c1c-34c96ded432c/assets/images/medium/mbio.03058-20-f0009.gif)
There have been only a few reports about the enzymatic characterization and the change of the enzymatic properties of SHMT by protein engineering. This research provides useful information about the interesting site, and the application of DOP-PCR in cloning a novel glyA gene. The obtained protein AnSHMT showed the optimal activity at 40☌ and pH 7.5, and was more stable in weakly alkali conditions (pH 6.5-8.5) than Hyphomicrobium methylovorum’s SHMT (pH 6.0-7.5), In order to improve the catalytic efficiency of the wild type, the site-directed mutagenesis based on sequences alignment and bioinformatics prediction, was used and the catalytic efficiency of the mutant I249L was found to be 2.78-fold higher than that of the wild-type, with the replacement of isoleucine by leucine at the 249 position. Here, a novel SHMT gene, glyA, was obtained through degenerate oligonucleotide-primed PCR and encoded a novel SHMT with 54.3% similarity to the known SHMT from Escherichia coli. Serine hydroxymethyltransferase (SHMT) is the key enzyme in L-serine enzymatic production, suggesting the importance of obtaining a SHMT with high activity.