Identification And Enzymatic Properties Of Serine Hydroxymethyltransferase From The Silkworm,Bombyx Mori

Serine hydroxymethyltransferases （SHMTs, EC:2.1.2.1） are a family of5-pyridoxal phosphate （PLP）-dependent enzyme of aspartate transaminase enzyme superfamily member class I folding,which are widely distributed in eukaryotes, prokaryotes and archaea. The homologous in the SHMTs family share a highly conserved amino acid sequence, and show diversity of substrate specificity SHMTs catalyze the reversible transformation of serine and tetrahydrofolate to glycine and N5, N10-methylenetetrahydrofolate. The reaction is the main source of one-carbon units,and involved in synthesis of neurotransmitter, thymidylate, purine derivatives and methionine. Moreover, SHMTs have an important role in the photorespiration, regulation of cancer cells, insect resistance and adversity resistance. The SHMTs already become a model enzyme that study the catalytic mechanism of enzymes and the allosteric regulation of protein structure.Bombyx mori silk is mainly composed of fibroin and sericin. The higher content of amino acids in fibroin are glycine, alanine, serine and tyrosine.The enzyme activity of glycine synthesis is higher than that of alanine, which is consistent with the prime characteristic that the highest content of silk is glycine. The physiological substrates of serine hydroxymethyl transferase are glycine and serine. The study of silkworm serine hydroxymethyl transferase gene（BmSHMT） can help us understand the mechanism of amino acid supply when silkworm silk protein synthesis.The main results are as follows:1.Bioinformatics analysis and expression pattern analysis of BmSHMT geneUse HcSHMT amino acid sequence downloaded from the NCBI to retrieval silkworm genome database, get a candidate gene BGIBMGA007079-TA, named BmSHMT.Use BmSHMT amino acid sequence re-BLASTP in NCBI, we found only one gene sequence report about BmSHMT, and the sequence number is NP₀01040279.1. Predicting the ORF of BmSHMT gene by BioEdit software, we get the full sequence in length of1398bp. BmSHMT gene locates on chromosome17in nscaf2865, with seven exons and six introns. The gene encodes465amino acids, with molecular weight is about53kDa, and pⅠ is7.56. Domain prediction for protein sequence shows that BmSHMT containing the SHMT family domain without signal peptide and transmembrane region.Use ClastalX software to align SHMT amino acid sequences of other species downloaded from the NCBI with BmSHMT, and then use the MEGA5software build phylogenetic tree, derive the amino acid sequences using GENEDOC software. The results showed that the phylogenetic tree constructed roughly divides into four major clusters.Insects, mammals, plants, and prokaryotes are respectively clustered into a cluster. BmSHMT gene and Monarch butterflies SHMT gene homology. The amino acid sequence alignment showed a higher conserved sequence of SHMTs in the selected species.The Expression pattern of BmSHMT gene in different tissues and different periods are analyzed by qRT-PCR. The results showed that BmSHMT gene is in high amount of expression in the epidermis, fat body and silk glands, especially the posterior silk gland. SHMT gene is higher expressed in the posterior silk gland than that in anterior/middle silk gland of872breed. This difference is even more obvious when compared with Dz breed. Yet the expression level of SHMT gene in Nd breed’s silk gland is close to that in anterior/middle silk gland of872or Dz. The expression of BmSHMT gene is higher in mulberry period than that of dormancy period, and the expression in the early fifth instar is higher than in the late fifth instar. BmSHMT gene has an increased expression quantity in the pre-pupal stage again.The5upstream in2kb length of BmSHMT gene was analyzed. Promoter sequence analysis and prediction of transcription factor binding sites showed that BmSHMT gene has no typical TATA box and CAAT box, no CpGF Island. Transcription factor binding sites can roughly divide into three categories:cell growth and differentiation factors, stress response and immune-related factors and hormones regulating factor. HSF transcription factor repeated many times and BR-C Z transcription factors have also been concerns.2.Full-length clone, prokaryotic expression, purification and antibody preparation of BmSHMT geneTotal RNA extracted from fat body in3days of the fifth instar of silkworm in reverse transcription of cDNA as a template, clone the full-length sequence in length of1398bp.We construct the protein expression vector, BmSHMT-pET28a, and transformed into E. coli BL21（DE3） expression strain. Cells were induced in the condition of16℃, for20h. The soluble recombinant protein was purified by nickel column, and the target protein was eluted at with200mM imidazole. After further purification and concentration using30K ultrafiltration, we obtain a large number of protein at the purity of90%. The commercial rabbit polyclonal antibody titer of BmSHMT prepared is 1:100,000.3.Western blot analysis and subcellular localization of BmSHMTDetection of the BmSHMT organizational differences at the protein level by western blot showed that BmSHMT expressed in the epidermis, fat body, silk glands at a higher level, consistent with the qRT-PCR results.The immunofluorescence localization of middle silk gland and posterior silk gland showed that the fluorescence signal of BmSHMT in the posterior silk gland was stronger than that in the middle silk gland.4.Enzyme activity detection of BmSHMTThe basic function of SHMT is to catalytic the reversible transformation from serine and tetrahydrofolate to glycine and N5, N10-methylenetetrahydrofolate. We select spectrophotometry to detect the activity of BmSHMT. All assay was performed at under laboratory conditions. Detection method is as follows:The reaction conditions is30℃, in a standard assay mixture, pH8.5. BmSHMT couples with methylene tetrahydrofolate dehydrogenase2（BmMTHFD2）, the detection of NADPH absorbance at340nm （extinction coefficient of6.22mM·L-1·cm-1） within1min of the change, and then calculated indirectly activity of BmSHMT. Ser and THF maximum concentration of5mM, and400μM, respectively, and to establish a series of substrate concentration gradient measured Km value for L-Ser and THF. Activity measurement results show that when the enzyme concentration is BmSHMT0.002mg/mL MTHFD-2protein concentration of4mg/ml, BmSHMT enzyme activity was1.32±0.26) μM·min-1·mg-1. According to Michaelis-Menten equation using software Origin deduced Km for L-serine was0.52±0.23mM, Km for THF was0.07±0.04mM. The activity measurement results BmSHMT SHMT enzymatic activity and other species have been reported in the comparison, the type and activity of human cytoplasmic SHMT considerable.5.Site-directed mutagenesis and activity determination of silkworm BmSHMTComparison of silkworm and human cytosolic SHMT （HcSHMT） amino acid sequence, and review of existing research literature related SHMT mutations, a series of amino acids were selected for mutagenesis. By-step method of site-directed mutagenesis and overlapping PCR method for specific sites BmSHMT amino acid sequence mutagenesis, get BmSHMT （H132D） and BmSHMT （P246C） and other mutants. Its determination of enzyme activity found that their activity were0.32±0.08μM`min-1· mg-1, significantly lower than the wild-type BmSHMT of normal activity. Compare to the wild-type, the color of BmSHMT （H132D） protein in7mg/mL showing is white, suggesting the mutant of H132D loss the ability of binding the cofactor of PLP.