Catalytic mechanism, molecular engineering and applications of threonine aldolases / 生物工程学报

Threonine aldolases catalyze the aldol condensation of aldehydes with glycine to furnish β-hydroxy-α-amino acid with two stereogenic centers in a single reaction. This is one of the most promising green methods for the synthesis of optically pure β-hydroxy-α-amino acid with high atomic economy and less negative environmental impact. Several threonine aldolases from different origins have been identified and characterized. The insufficient -carbon stereoselectivity and the challenges of balancing kinetic versus thermodynamic control to achieve the optimal optical purity and yield hampered the application of threonine aldolases. This review summarizes the recent advances in discovery, catalytic mechanism, high-throughput screening, molecular engineering and applications of threonine aldolases, with the aim to provide some insights for further research in this field.

Evaluating the clinical significance of SHMT2 and its co-expressed gene in human kidney cancer

BACKGROUND: Kidney cancer is one of the most common cancers in the world. It is necessary to clarify its underlying mechanism and find its prognostic biomarkers. Current studies showed that SHMT2 may be participated in several kinds of cancer. METHODS: Our studies investigated the expression of SHMT2 in kidney cancer by Oncomine, Human Protein Atlas database and ULCAN database. Meanwhile, we found its co-expression gene by cBioPortal online tool and validated their relationship in A498 and ACHN cells by cell transfection, western blot and qRT-PCR. Besides these, we also explored their prognostic values via the Kaplan-Meier plotter database in different types of kidney cancer patients. RESULTS: SHMT2 was found to be increased in 7 kidney cancer datasets, compared to normal renal tissues. For the cancer stages, ages and races, there existed significant difference in the expression of SHMT2 among different groups by mining of the UALCAN database. High SHMT2 expression is associated with poor overall survival in patients with kidney cancer. Among all co-expressed genes, NDUFA4L2 and SHMT2 had a high co-expression efficient. SHMT2 overexpression led to the increased expression of NDUFA4L2 at both mRNA and protein levels. Like SHMT2, overexpressed NDUFA4L2 also was associated with worse overall survival in patients with kidney cancer. CONCLUSION: Based on above results, overexpressed SHMT2 and its co-expressed gene NDUFA4L2 were all correlated with the prognosis in kidney cancer. The present study might be benefit for better understanding the clinical significance of SHMT2 and provided a potential therapeutic target for kidney cancer in future.

SHMT1 C1420T polymorphism contributes to the risk of non-Hodgkin lymphoma: evidence from 7309 patients / 癌症

<p><b>BACKGROUND</b>Serine hydroxymethyltransferase 1 (SHMT1) is a key enzyme in the folate metabolic pathway that plays an important role in biosynthesis by providing one carbon unit. SHMT1 C1420T may lead to the abnormal biosynthesis involved in DNA synthesis and methylation, and it may eventually increase cancer susceptibility. Many epidemiologic studies have explored the association between C1420T polymorphism and the risk of non-Hodgkin lymphoma (NHL), but the results have been contradictory. Therefore, we performed this meta-analysis to evaluate the relationship.</p><p><b>METHODS</b>The meta-analyses were conducted to evaluate the effect of SHMT1 C1420T polymorphism on NHL risk. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to measure the strength of the association.</p><p><b>RESULTS</b>Eight studies encompassing 3232 cases and 4077 controls were included. A statistically significant association was found between SHMT1 C1420T polymorphism and NHL risk under the allelic comparison (T vs. C: OR = 1.09, 95% CI 1.01-1.17); a borderline association was found between SHMT1 C1420T polymorphism and NHL risk under the homozygote model (TT vs. CC: OR = 1.18, 95% CI 1.00-1.39) and the dominant model (CT+TT vs. CC: OR = 1.10, 95% CI 1.00-1.21).</p><p><b>CONCLUSION</b>SHMT1 C1420T polymorphism may be associated with NHL risk, which needs to be validated in large, prospective studies.</p>

Genetic variation in genes involved in folate and drug metabolism in a south Indian population.

BACKGROUND: Genetic variations represented as single nucleotide polymorphisms (SNPs) vary across the world population. This genetic polymorphism (such as SNPs) plays an important role in pharmacogenomics. SNPs that affects cellular metabolism, by altering the enzyme activity, have an important role in therapeutic outcome. Allele frequencies in number of clinically relevant SNPs within south Indian populations are not yet known. Hence, we genotyped randomly selected unrelated south Indian subjects from different locations of south India representing the heterogeneous ethnic background of the population. MATERIALS AND METHODS: Common variants of MTHFD1, TYMS, SHMT1, MTR, MTRR, CBS and SULT1A1 gene polymorphisms were screened from healthy unrelated south Indian volunteers. Genotypes were determined using RFLP analysis of polymerase chain reaction-amplified products and confirmed by DNA sequencing. Chi-square test was performed to test for deviation from the Hardy-Weinberg equilibrium for each locus. RESULTS: Gene allele frequency for several polymorphisms in our study differed significantly between the populations of other nations reported for several of the SNPs. These results demonstrate that the populations in different geographic regions may have widely varying genetic allele frequencies for clinically relevant SNPs. CONCLUSION: The present study reports, for the first time, the frequency distribution of MTHFD1, TYMS, SHMT1, MTR, MTRR, CBS and SULTIA1 gene polymorphisms in a south Indian population. Population-specific genetic polymorphism studies will help in practicing pharmacogenomic principles in the clinics.

Construction of co-expression SHMT and TPase recombinant vector and dual-enzymatic synthesis of L-tryptophan / 生物工程学报

Hydroxymethyltransferase (SHMT) and tryptophanase (TPase) are key enzymes in biosynthesis of L-tryptophan. We constructed three recombinant plasmids, including pET-SHMT, pET-TPase, and pET-ST for over-expression or co-expression of SHMT and TPase in Escherichia coli BL21 (DE3). The SDS-PAGE analysis showed that the recombinant proteins of 47 kDa and 50 kDa were expressed of pET-SHMT and pET-TPase, respectively. As compared to the host stain, the enzyme activity of SHMT and TPase was increased by 6.4 and 8.4 folds, respectively. Co-expression of both recombinant proteins, 47 kDa and 50 kDa, was also successful by using pET-ST and the enzyme activities were enhanced by 6.1 and 6.9 folds. We designed two pathways of dual-enzymatic synthesis of L-tryptophan by using these recombinant strains as source of SHMT and TPase. In the first pathway, the pET-SHMT carrying strain was used to catalyze synthesis of L-serine, which was further transformed into L-tryptophan by the pET-TPase expressing strain. These two steps sequentially took place in different bioreactors. In the second pathway, the pET-ST carrying strain, in which two enzymes were co-expressed, was used to catalyze simultaneously two steps in a single bioreactor. HPLC analysis indicated a high yield of 41.5 g/L of L-tryptophan was achieved in the first pathway, while a lower yield of 28.9 g/L was observed in the second pathway. In the first pathway, the calculated conversion rates for L-glycine and indole were 83.3% and 92.5%, respectively. In the second pathway, a comparable conversion rate, 82.7%, was achieved for L-glycine, while conversion of indole was much lower, only 82.9%.

Overexpression and characterization of dimeric and tetrameric forms of recombinant serine hydroxymethyltransferase from Bacillus stearothermophilus.

Serine hydroxymethyltransferase (SHMT), a pyridoxal-5' -phosphate (PLP) dependent enzyme catalyzes the interconversion of L-Ser and Gly using tetrahydrofolate as a substrate. The gene encoding for SHMT was amplified by PCR from genomic DNA of Bacillus stearothermophilus and the PCR product was cloned and overexpressed in Escherichia coli. The purified recombinant enzyme was isolated as a mixture of dimer (90%) and tetramer (10%). This is the first report demonstrating the existence of SHMT as a dimer and tetramer in the same organism. The specific activities at 37 C of the dimeric and tetrameric forms were 6 7 U/mg and 4 1 U/mg, respectively. The purified dimer was extremely thermostable with a T(m) of 85 degrees C in the presence of PLP and L-Ser. The temperature optimum of the dimer was 80 degrees C with a specific activity of 32 4 U/mg at this temperature. The enzyme catalyzed tetrahydrofolate-independent reactions at a slower rate compared to the tetrahydrofolate-dependent retro-aldol cleavage of L-Ser. The interaction with substrates and their analogues indicated that the orientation of PLP ring of B. stearothermophilus SHMT was probably different from sheep liver cytosolic recombinant SHMT (scSHMT).

Affinity properties of phosvitin: interaction of phosvitin with serine hydroxymethyl transferase.

The affinity of phosvitin with serine hydroxymethyl transferase (SHMT), an acidic multi-subunit protein, was evaluated by measurements of enzyme activity, sedimentation velocity, steady-state fluorescence, circular dichroism and kinetic thermal stability. While the presence of phosvitin had no effect on the SHMT activity, the sedimentation coefficient of SHMT increased from 8.7 S to 12.5 S suggesting the formation of a complex at a SHMT:phosvitin molar ratio of 2:1. Based on steady-state fluorescence quenching measurements an association constant of 2.4 +/- 0.2 x 10(5) M-1 at 25 degrees C was obtained for the interaction of phosvitin with SHMT. The temperature dependency of the association constant in the range 15-35 degrees C suggests the involvement of ionic forces in the interaction. The thermal inactivation of SHMT followed first order kinetics. In the presence of phosvitin the rate constant decreased and half time increased. The circular dichroism measurements suggest that phosvitin interaction does not involve pyridoxal phosphate binding domain of the enzyme. Although minor changes in the secondary structure of the enzyme were observed, the environment around aromatic amino acids did not change significantly.

Partial amino acid sequence of sheep liver serine hydroxymethyltransferase and comparison of peptide maps of the enzyme from human, ox livers and Escherichia coli.

A comparison of the tryptic peptide maps of serine hydroxymethyltransferase from sheep, human, ox livers and Escherichia coli revealed that the mammalian enzymes were similar, while the bacterial enzyme exhibited differences in the primary structure. N-terminus of the reduced carboxymethylated sheep liver enzyme was acetylated. Serine hydroxymethyltransferase was hydrolyzed with trypsin and fragments of peptides were separated by high performance liquid chromatography using a combination of gel permeation, reverse phase and ion-pair chromatography. The peptides were sequenced manually using the 4-N,N'-dimethyl aminoazobenzene-4'-isothiocyanate/phenyl isothiocyanate double coupling method. The tryptic peptides with 80% homology or above were aligned on the rabbit liver enzyme sequence.

Interactions of methoxyamine with pyridoxal-5'-phosphate-Schiff's base at the active site of sheep liver serine hydroxymethyltransferase.

The mechanism of interaction of methoxyamine with sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) (SHMT) was established by measuring changes in enzyme activity, visible absorption spectra, circular dichroism and fluorescence, and by evaluating the rate constant by stopped-flow spectrophotometry. Methoxyamine can be considered as the smallest substituted aminooxy derivative of hydroxylamine. It was a reversible noncompetitive inhibitor (Ki = 25 microM) of SHMT similar to O-amino-D-serine. Like in the interaction of O-amino-D-serine and aminooxyacetic acid, the first step in the reaction was very fast. This was evident by the rapid disappearance of the enzyme-Schiff base absorbance at 425 nm with a rate constant of 1.3 x 10(3) M-1 sec-1 and CD intensity at 430 nm. Concomitantly, there was an increase in absorbance at 388 nm (intermediate I). The next step in the reaction was the unimolecular conversion (1.1 x 10(-3) sec-1) of this intermediate to the final oxime absorbing at 325 nm. The identity of the oxime was established by its characteristic fluorescence emission at 460 nm when excited at 360 nm and by high performance liquid chromatography. These results highlight the specificity in interactions of aminooxy compounds with sheep liver serine hydroxymethyltransferase and that the carboxyl group of the inhibitors enhances the rate of the initial interaction with the enzyme.

Isolation and characterization of a naturally occurring inhibitor from mung bean (Vigna radiata) seedlings for serine hydroxymethyltransferase.

A naturally occurring inhibitor of serine hydroxymethyltransferase (EC 2.1.2.1) in mung bean seedlings extracts was purified by ammonium sulphate precipitation, phenyl-Sepharose chromatography followed by heating to release the inhibitor bound to the protein. The inhibitor had an absorption maximum at 200 nm, was not precipitated by trichloroacetic acid, was dialysable and resistant to inactivation by heating at 98 degrees C for 4 hr, protease and ribonuclease digestion; but was acid labile. The chromatographically pure preparation inhibited both mung bean and sheep liver SHMT. Qualitative and quantitative analyses indicated that it contained a carbohydrate moiety, an O-amino and vicinal diol groups. Paper electrophoresis at pH 4.3 suggested that the inhibitor was positively charged.