CN101781637B - Human 5,10-methenyltetrahydrofolate and its compound crystallization methods, crystals and application thereof - Google Patents
Human 5,10-methenyltetrahydrofolate and its compound crystallization methods, crystals and application thereof Download PDFInfo
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- CN101781637B CN101781637B CN2009101344744A CN200910134474A CN101781637B CN 101781637 B CN101781637 B CN 101781637B CN 2009101344744 A CN2009101344744 A CN 2009101344744A CN 200910134474 A CN200910134474 A CN 200910134474A CN 101781637 B CN101781637 B CN 101781637B
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- anhydroleucovorin
- synthetic enzyme
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- acid
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Abstract
The invention relates to a crystal of humaniz 5,10-methenyltetrahydrofolate (MTHFS) and a crystallization method thereof, also relates to a crystal of a composite of the human 5,10-methenyltetrahydrofolate and ADP and a crystallization method thereof, a crystal of a composite of the human 5,10-methenyltetrahydrofolate and glutamic acid and a crystallization method thereof, a crystal of a composite of the human 5,10-methenyltetrahydrofolate and N5-imide phosphoric acid and a crystallization method thereof, and a crystal of a composite of the human 5,10-methenyltetrahydrofolate and a product, namely 10- methenyltetrahydrofolate and a crystallization method thereof, and also relates to important structure domains or peptide fragments in the crystals, and a method for designing and screening medicaments by using the structure domains or the peptide fragments.
Description
Technical field
The present invention relates to a kind of people source 5; The crystal of 10-anhydroleucovorin synthetic enzyme (MTHFS) and crystalline method thereof; Also relate to simultaneously people source 5; The crystal of the mixture of 10-anhydroleucovorin synthetic enzyme and ADP and crystalline method thereof, people source 5; The crystal of the mixture of 10-anhydroleucovorin synthetic enzyme and L-glutamic acid and crystalline method thereof, people source 5, the crystal of the mixture of 10-anhydroleucovorin synthetic enzyme and N5-imines phosphoric acid transition state and crystalline method thereof, people source 5, the crystal and the crystalline method thereof of 10-anhydroleucovorin synthetic enzyme and product 10-LEUCOVORIN ACETATE mixture; Also relate in these crystal for people source 5 structural domain or peptide fragment that the enzymic activity of 10-anhydroleucovorin synthetic enzyme plays a crucial role; And utilize these important structure territories or peptide fragment to carry out medicinal design and method for screening.
Background technology
Folic acid, this water miscible vitamin B group comes to light as far back as the forties in 19th century.Folate-mediated single carbon pathways metabolism is present in from the mikrobe to the higher plant, in the vital movement of animal; The growth of this pathways metabolism pair cell and breed extremely important; And in a lot of physiological activities, play important effect, so reduced form folic acid is wherein important cofactor.Folic acid can only synthesize in prokaryotic organism, yeast, plant, but but is necessary to human body, if folate-mediated single carbon pathways metabolism is blocked, in human body with cause cancer, cardiovascular disorder and heteroplasia etc.The enzyme that relates in this pathways metabolism just is used as treatment cancer (Appling, 1991 for a long time; Fox and Strover, 2008; Chattopadhyay et.al., 2007; Farber et.al., 1948).At present, the antifolic thing not only is used as chemotherapeutics, also in diseases such as treatment rheumatic arthritis and psoriatic, plays a role.
Folic acid exists with multiple oxidation state in vivo, as: folic acid, dihydrofolic acid, THFA etc.Single carbosilane unit that the folic acid of these different states carries also exists different oxidation state such as methyl alcohol or formic acid, and they carry out mutual conversion through the zymetology reaction.The THFA of different states can accept or provide single carbosilane unit to multiple vital movement.Comprise: the de novo synthesis of (1) purine and pyrimidine, di-phosphate uridylic methylate and form the di-phosphate thymus pyrimidine; (2) the homocysteine formation methionine(Met) that methylates, methionine(Met) can form S-adenosylmethionine, and the methylation reaction in latter's pair cell is most important.Synthesizing of the de novo synthesis of purine, pyrimidine and homocysteine is all very sensitive to the synthetic blocking-up at the pathways metabolism upper reaches; Can cause the di-phosphate uridylic to mix in the middle of the DNA by error; And can promote intracellular homocysteine concentration, destroy S-adenosylmethionine dependent form methylation reaction.The disorder of folic acid metabolism approach will cause multiple serious disease, so a lot of enzymes of this approach all are used as the important target spot of medicinal design.Single glutamic acid deriv of folic acid passes cytolemma and gets into cell; In cell by the glutamic acid deriv of the synthetic different lengths of polyglutamic acid synthetic enzyme catalysis; The polyglutamic acid side chain can make folic acid be present in the cell, also can increase itself and the interior binding ability that combines enzyme of born of the same parents simultaneously.
5,10-anhydroleucovorin synthetic enzyme (Homo sapiens5,10-methenyltetrahydrofolate synthetase are called for short MTHFS) is also referred to as 5-LEUCOVORIN ACETATE cyclase (5-formyltetrahydrofolatecyclo-ligase).Zymetology classification number: (EC) 6.3.3.2.
The catalytic internal metabolism reaction of MTHFS is:
5-LEUCOVORIN ACETATE 5, the 10-anhydroleucovorin
The substrate of reaction is 5-LEUCOVORIN ACETATE (5-formyltetrahydrofolate is called for short 5-FTHF), and the product of reaction is 5, and (5,10-methenyltetrahydrofolate is called for short 5,10-MTHF) to the 10-anhydroleucovorin.This catalyzed reaction at room temperature can be carried out, and divalence mg ion and ATP (Triphosaden) are 5, the cofactor of 10-anhydroleucovorin synthetic enzyme.
MTHFS is present in a lot of biologies, comprising: bacterium, plant and animal show that it has very important effect in folic acid metabolism.At present multiple important proteic zymetology The Characteristics result in the folic acid metabolism approach is shown that the 5-LEUCOVORIN ACETATE is the present stable reduced form folic acid derivatives of known unique thermokinetics, and is not used directly as single carbon support.In prokaryotic organism, the 5-LEUCOVORIN ACETATE is considered to a kind of storing state of single carbon support, mainly is present in seed and the spore, and its state is with 5, the expression of 10-anhydroleucovorin synthetic enzyme and changing.In Mammals; The 5-LEUCOVORIN ACETATE accounts for the 10%-15% of total folate content in the born of the same parents; The 5-LEUCOVORIN ACETATE has restraining effect to the enzyme in some single carbon pathways metabolisms, as: serine hydroxymethylase (serine hydroxymethyltransferase) and 5-aminoimidazole-4-carbozamide yl nucleosides acid transformylase (AICAR Tfase).5,10-anhydroleucovorin synthetic enzyme is unique enzyme that can the 5-LEUCOVORIN ACETATE be converted into other type reduced form folic acid.Through the concentration of 5-LEUCOVORIN ACETATE in the control agent, 5,10-anhydroleucovorin synthetic enzyme can be controlled a lot of folic acid dependent form pathways metabolisms, for example: metabolism approach such as purine, pyrimidine, amino acid.Above-mentioned pathways metabolism and life are grown, and cancer prevention closely links to each other with committed step in treating.
There are some researches show: use 5-Calcium Folinate-SF homofolic acid, can suppress 5, the activity of 10-anhydroleucovorin synthetic enzyme, and then suppress human breast carcinoma.From Fig. 1, can see, 5, the effect of 10-anhydroleucovorin synthetic enzyme in single carbon pathways metabolism, it simultaneously can forward and reverse this pathways metabolism of regulating.
5,10-anhydroleucovorin synthetic enzyme is the key enzyme at single carbon cycle upper reaches, and through the analysis of pathways metabolism, it is more remarkable than Tetrahydrofolate dehydrogenase to the metabolic influence in downstream.Unusual and the integrity of DNA and RNA of the genomic methylation that causes for cancer, the aspects such as repair ability of DNA, 5,10-anhydroleucovorin synthetic enzyme all is a very crucial regulation and control target protein.
5, aminoacid sequence and the people intravital amino acid sequence homology of 10-anhydroleucovorin synthetic enzyme in different biologies relatively listed in the table 1: (homology is relatively used the BLAST software of NCBI)
Table 1
Though 5 in the prokaryotic organism, 10-anhydroleucovorin synthetic enzyme three-dimensional structure is resolved, itself and people source 5, and the homology of 10-anhydroleucovorin synthetic enzyme aminoacid sequence is merely 28%.And Mammals and people 5, the three-dimensional structure of 10-anhydroleucovorin synthetic enzyme is resolved and is not but appeared in the newspapers.
Because the verivate of folic acid is unstable in physiological environment, most folic acid derivatives are to exist with the form that is combined on the albumen in the body.Exist the bottoms stream transmission in the enzyme reaction chain in the single carbon metabolism of the folic acid dependent form network, promptly substrate with react after enzyme combines, the product that reacts after the completion does not separate with enzyme, but is delivered in the next enzyme reaction with this bonding state.Research also shows, in this pathways metabolism: come regulatory enzyme active through forming intermediate state, and then regulate the trend of pathways metabolism and keep running balance.As: the 10-formyltetrahydrofolate dehydrogenase that from pork liver, extracts is exactly the THFA that has been combined with 6 L-glutamic acid.So just cause: if an enzyme morphs in the reaction chain, wherein between product accumulate, then the influence to whole pathways metabolism all is far-reaching.
MTHFS catalysis 5-LEUCOVORIN ACETATE is irreversible to turn to 5; 10-anhydroleucovorin (Jolivet, 1997), this catalyzed reaction depends on Triphosaden (adenosine-triphosphate; Be called for short ATP) and can be by product feedback inhibition (Huennekens et.al., 1984; Jolivet et.al., 1996; Maras et.al., 1994).Nucleus magnetic resonance (NMR) wave spectrum and the research of isotopic exchange method show: there are two intermediate states (Huang and Schirch, 1995) in the catalytic reaction of MTHFS.(1) phosphate group of the formyl group nucleophillic attack ATP of 5-LEUCOVORIN ACETATE produces first N5-imines phosphoric acid intermediate state, and this part reaction is a reversible.(2) second nucleophillic attacks occur in intramolecularly, and N10 is connected with the N5 of N5-imines phosphoric acid, form a tetrahedron intermediate state phosphoimidazole quinoline.Phosphoimidazole quinoline intermediate state removes phosphate group, forms catalysate 5 at last, the 10-anhydroleucovorin.5, the 10-anhydroleucovorin is the intermediate product of folic acid metabolism, can with enzyme bonded state under be converted into the 10-LEUCOVORIN ACETATE.The final step of reaction makes irreversible by the catalytic reaction of MTHFS.At present, the information of the three-dimensional structure aspect of this reaction mechanism is known little.That report is arranged is the MTHFS (mpMTHFS) that comes from mycoplasma pneumoniae (Mycoplasmapnuemoniae); And in conjunction with ADP (adenonisine disphosphate; Be called for short ADP) and the ternary complex structure (Chen et.al., 2005) of 5-LEUCOVORIN ACETATE, another one is to come from Bacillus anthracis (Bacillus anthracis) MTHFS (bMTHFS); And be combined with ADP (Meieret.al., 2007).The aminoacid sequence of people source MTHFS (hMTHFS) homology of comparing with the primary structure in the Protein Data Bank (http://www.rcsb.org/pdb/) is less than 28% (Berman et.al., 2000).Find out from known structure: the one-piece construction of hMTHFS and mpMTHFS, bMTHFS is quite similar, is indicating that MTHFS trends towards a kind of conservative folding mode.In muroid research, utilize enzyme stability kinetics and Molecular Simulation Technique to show, the methylated folic acid derivatives of N5 and N10 is used as the feasibility less (Field et.al., 2006,2007) of MTHFS inhibition.
Conventional 5 FU 5 fluorouracil (the 5-fluorouracil that uses in the rectum cancer and pancreas cancer chemotherapy; Be called for short 5-FU) be through suppressing thymidylate synthetase (thymidylate synthase; Abbreviation TS) (the Longley et.al. that plays a role; 2003), thymidylate synthetase (TS) obtains a single carbon unit and comes catalytic deoxidation uridylic acid (deoxyuridylate) to be converted into deoxythymidylic acid (deoxythymidylate) from 5 on the 10-methylene tetrahydrofolate; This reaction is for DNA synthetic most important (Carreras and Santi, 1995).Clinically, in order to improve result of treatment, 5-LEUCOVORIN ACETATE (5-formylTHF) usually and 5-FU use together.The benefit of using like this is: MTHFS can synthesize 5 by catalysis 5-LEUCOVORIN ACETATE, the 10-anhydroleucovorin, and 5, the 10-anhydroleucovorin can be converted into 5, the 10-methylene tetrahydrofolate.5 of low levels, 10-methylene tetrahydrofolate cause 5-FU to disintegrate down from the TS tetramer, thereby reduce inhibition effect (Bertino, 1977 of thymidine analogue; Sirotnak et.al., 2000).5, the 10-methylene tetrahydrofolate also can be synthetic in transforming the process that Serine is a glycocoll by serine hydroxymethylase (serinehydroxymethyltransferase is called for short SHMT).The enzymic activity of SHMT can directly be regulated by MTHFS, because the substrate of MTHFS: the 5-LEUCOVORIN ACETATE can suppress SHMT (Renwick et.al., 1998; Schirch and Szebenyi, 2005; Stover and Schirch, 1990).5-aminoimidazole-4-carbozamide yl nucleosides acid transformylase (phosphoribosylaminoimidazolecarboxamide transformylase; Be called for short AICARFT) and glycinamide ribonucleotide transformylase (Glycinamideribonucleotide transformylase; Be called for short GARFT) be two enzymes of participating in purine metabolism in single carbon pathways metabolism; Can obtain single carbon unit from the 10-LEUCOVORIN ACETATE; Also be used as target spot (Allegra et.al., 1985 of cancer therapy drug; Piper et.al., 1988).Lometrexol (Lometrexol) is the representative of s-generation antifol, is used for suppressing the GARFT activity, and then it is synthetic to suppress purine, reaches the order ground of treatment cancer.
Use methotrexate (Methotrexate; Be called for short MTX) and aminopterin (aminopterin) inhibition Tetrahydrofolate dehydrogenase (dihydrofolatereductase; Be called for short DHFR) cause the polyglutamic acid verivate of dihydrofolic acid to accumulate; And then enzymic activity (Quinlivan et.al., 2000 of inhibition AICARFT; Shih et.al., 1997).Equally; Analogue through the 5-LEUCOVORIN ACETATE suppresses 5; 10-anhydroleucovorin synthetic enzyme (5,10-methenyltetrahydrofolate synthetase is called for short MTHFS) causes the accumulation of 5-LEUCOVORIN ACETATE; And then the enzymic activity of inhibition serine hydroxymethylase (serine hydroxymethyltransferase is called for short SHMT) and AICARFT.As stated, the transfer in pathways metabolism has negative effect to carbon unit in the inhibition of enzymic activity, causes the synthetic blocking-up of some important component in the cell, and this also is to use folacin to carry out the mechanism of cancer chemotherapeutic at present.Use folic acid derivatives to carry out cancer therapy, have two problems, (one) these medicines in a lot of cases, found the resistance phenomenon, and concerning some patients, these two kinds of effect of drugs are obvious inadequately through after the use of decades; (2) these medicines have more intense cytotoxicity more.Therefore, to be used for cancer chemotherapeutic be present research focus to development of new folic acid derivatives medicine.
Summary of the invention
The present invention provides a kind of people source 5; The crystal of 10-anhydroleucovorin synthetic enzyme; It is characterized in that said crystalline spacer is: C2221; Unit cell parameters is
α=90.00 °; β=90.00 °, γ=90.00 °.
The present invention provides a kind of people source 5; The crystal of the mixture of 10-anhydroleucovorin synthetic enzyme and ADP; It is characterized in that said crystalline spacer is: C2221; Unit cell parameters is
α=90.00 °; β=90.00 °, γ=90.00 °.
The present invention provides a kind of people source 5; The crystal of the mixture of 10-anhydroleucovorin synthetic enzyme and ADP; It is characterized in that said crystalline spacer is: P21212; Unit cell parameters is
α=90.00 °; β=90.00 °, γ=90.00 °.
The present invention provides a kind of people source 5; The crystal of the mixture of 10-anhydroleucovorin synthetic enzyme and L-glutamic acid; It is characterized in that said crystalline spacer is: C2221; Unit cell parameters is
α=90.00 °; β=90.00 °, γ=90.00 °.
The present invention provides a kind of people source 5; The crystal of the mixture of 10-anhydroleucovorin synthetic enzyme and N5-imines phosphoric acid transition state; It is characterized in that said crystalline spacer is: C2221; Unit cell parameters is
α=90.00 °; β=90.00 °, γ=90.00 °.
The present invention provides a kind of people source 5; The crystal of the mixture of 10-anhydroleucovorin synthetic enzyme and 10-LEUCOVORIN ACETATE; It is characterized in that said crystalline spacer is: C2221; Unit cell parameters is
α=90.00 °; β=90.00 °, γ=90.00 °.
The present invention provides a kind of people source 5, and the crystalline crystallization method of 10-anhydroleucovorin synthetic enzyme comprises: with said people source 5, the solution of 10-anhydroleucovorin synthetic enzyme mixes with pond liquid; Utilize sessile drop method or the seat method of dripping to carry out crystallization; After 1-2 days, collect crystal; It is characterized in that pond liquid is: contain 0.01-0.03mol/L MgCl in 5 to 25 degrees centigrade, 50% to 80% humidity range
2, 0.01-0.04mol/L NiCl
2, 20%-30%PEG3000 to 4000,0.1mol/L N-2-hydroxyethyl piperazine-N-2-ethyl sulfonic acid or the morpholino b acid damping fluid of pH=6.4-7.0.
The present invention provides a kind of people source 5; The crystalline crystallization method of the mixture of 10-anhydroleucovorin synthetic enzyme and ADP; Comprise: to said people source 5, add ATP solution in the solution of 10-anhydroleucovorin synthetic enzyme, add reductive agent and mg ion again; Obtain people source 5 after the mixing, the mixed solution of 10-anhydroleucovorin synthetic enzyme and ATP; With said people source 5,10-anhydroleucovorin synthetic enzyme mixes with pond liquid with the mixed solution of ATP; Utilize sessile drop method or the seat method of dripping to carry out crystallization; After 1-2 days, collect crystal; It is characterized in that pond liquid is: contain 0.01-0.03mol/LMgCl in 5 to 25 degrees centigrade, 50% to 80% humidity range
2, 0.01-0.04mol/L NiCl
2, 20%-30%PEG3000 to 4000,0.1mol/L N-2-hydroxyethyl piperazine-N-2-ethyl sulfonic acid or the morpholino b acid damping fluid of pH=6.4-7.0.
The present invention provides a kind of people source 5; The crystalline crystallization method of the mixture of 10-anhydroleucovorin synthetic enzyme and L-glutamic acid; Comprise: to said people source 5; Add glutamic acid solution in the solution of 10-anhydroleucovorin synthetic enzyme, obtain people source 5 after the mixing, the mixed solution of 10-anhydroleucovorin synthetic enzyme and L-glutamic acid; With said people source 5,10-anhydroleucovorin synthetic enzyme mixes with pond liquid with the mixed solution of L-glutamic acid; Utilize sessile drop method or the seat method of dripping to carry out crystallization; After 1-2 days, collect crystal; It is characterized in that pond liquid is: contain 0.01-0.03mol/L MgCl in 5 to 25 degrees centigrade, 50% to 80% humidity range
2, 0.01-0.04mol/L NiCl
2, 20%-30%PEG3000 to 4000,0.1mol/L N-2-hydroxyethyl piperazine-N-2-ethyl sulfonic acid or the morpholino b acid damping fluid of pH=6.4-7.0.
The present invention provides a kind of people source 5; The crystalline crystallization method of the mixture of 10-anhydroleucovorin synthetic enzyme and N5-imines phosphoric acid transition state; Comprise: to said people source 5; Add ATP, 5-LEUCOVORIN ACETATE, mercaptoethanol, magnesium ion solution in the solution of 10-anhydroleucovorin synthetic enzyme, obtain people source 5 after the mixing, the mixed solution of 10-anhydroleucovorin synthetic enzyme and N5-imines phosphoric acid; With said people source 5,10-anhydroleucovorin synthetic enzyme mixes with pond liquid with the mixed solution of N5-imines phosphoric acid; Utilize sessile drop method or the seat method of dripping to carry out crystallization; After 1-2 days, collect crystal; It is characterized in that pond liquid is: contain 0.01-0.03mol/L MgCl in 5 to 25 degrees centigrade, 50% to 80% humidity range
2, 0.01-0.04mol/LNiCl
2, 20%-30%PEG3000 to 4000,0.1mol/L N-2-hydroxyethyl piperazine-N-2-ethyl sulfonic acid or the morpholino b acid damping fluid of pH=6.4-7.0.
The present invention provides a kind of people source 5; The crystalline crystallization method of the mixture of 10-anhydroleucovorin synthetic enzyme and 10-LEUCOVORIN ACETATE; Comprise: to said people source 5; Add ATP, 5-LEUCOVORIN ACETATE, mercaptoethanol, magnesium ion solution in the solution of 10-anhydroleucovorin synthetic enzyme, obtain people source 5 after the mixing, the mixed solution of 10-anhydroleucovorin synthetic enzyme and N5-imines phosphoric acid; With said people source 5,10-anhydroleucovorin synthetic enzyme mixes with pond liquid with the mixed solution of N5-imines phosphoric acid; Utilize sessile drop method or the seat method of dripping to carry out crystallization; After 4-14 days, collect crystal; It is characterized in that pond liquid is: contain 0.01-0.03mol/L MgCl in 5 to 25 degrees centigrade, 50% to 80% humidity range
2, 0.01-0.04mol/LNiCl
2, 20%-30%PEG3000 to 4000,0.1mol/L N-2-hydroxyethyl piperazine-N-2-ethyl sulfonic acid or the morpholino b acid damping fluid of pH=6.4-7.0.
The present invention provides a kind of people source 5; The crystalline F55 of 10-anhydroleucovorin synthetic enzyme, M58, E61, M90, Y152, Y153 amino acids formed structural domain in the space is characterized in that said structural domain combines with pterin group in the substrate 5-LEUCOVORIN ACETATE.
The present invention provides a kind of people source 5; The crystalline F85 of 10-anhydroleucovorin synthetic enzyme, W109, R148 amino acids formed structural domain in the space is characterized in that said structural domain combines with para-amino benzoic acid group in the substrate 5-LEUCOVORIN ACETATE.
The present invention provides a kind of people source 5, and the crystalline K10 of 10-anhydroleucovorin synthetic enzyme, R14, R145, D154 amino acids formed structural domain in the space is characterized in that said structural domain combines with ATP.
The present invention provides a kind of people source 5; The 152-161 amino acids of α 5 formed structural domain in the space in the crystal of the mixture of 10-anhydroleucovorin synthetic enzyme and ADP is characterized in that said structural domain is and the phosphate group bonded structural domain that gets off from the ATP hydrolysis.
The present invention provides a kind of people source 5; Amino acid in the crystal of the mixture of 10-anhydroleucovorin synthetic enzyme and L-glutamic acid between β 3 and the β 4 i.e. amino acid formed structural domain in the space between the 89th and 135 amino acids; It is characterized in that said structural domain forms a loop ring structure, the amino acid in this structure combines with L-glutamic acid.
The present invention provides a kind of people source 5, amino-acid residue F55 in the crystal of the mixture of 10-anhydroleucovorin synthetic enzyme and N5-imines phosphoric acid transition state, L56, S57; M58, E61, P81, Y83; M90, W109, P135, R148; K150, the formed structural domain of Y152 and Y153 is characterized in that said structural domain forms a spherical pocket, the pterin ring of N5-imines phosphoric acid transition state is positioned at the centre of said spherical pocket.
The present invention provides a kind of people source 5; The structural domain of pterin ring in the space in the crystal of the mixture of 10-anhydroleucovorin synthetic enzyme and N5-imines phosphoric acid transition state; It is characterized in that said pterin ring serves as that axle has taken place to reverse with the key between C4a and the C8a, rearrangement has taken place in the electronics on the said petrin ring.
The present invention provides a kind of people source 5; The structural domain of Y83 in the space in the crystal of the mixture of 10-anhydroleucovorin synthetic enzyme and 10-LEUCOVORIN ACETATE; The position that it is characterized in that Y83 with compare in the described crystalline of claim 5 position, variation has taken place.
The present invention provides the compsn of one or more in similar or bonded micromolecular compound, small peptide, polypeptide, nucleic acid, antibody or the immune conjugate with above-mentioned arbitrary structural domain.
Description of drawings
Fig. 1 shows 5, position and the effect of 10-methylene tetrahydrofolate synthetic enzyme in single carbon pathways metabolism.TS: thymidylate synthetase (thymidylate synthase is called for short TS); MTHFS:5,10-anhydroleucovorin synthetic enzyme (5,10-methenyltetrahydrofolatesynthetase is called for short MTHFS); SHMT: serine hydroxymethylase (serinehydroxymethyrltransferase is called for short SHMT); DHFR: Tetrahydrofolate dehydrogenase (dihydrofolate reductase is called for short DHFR); MTHFR: MTHFR (Methylenetetrahydrofolate reductase is called for short MTHFR); MTHFD: methylenetetrahydrofolate dehydrogenase (methylenetetrahydrofolate dehydrogenase is called for short MTHFD)
Fig. 2 shows MTHFS catalytic reaction products 5, and the 10-anhydroleucovorin is converted into the 10-LEUCOVORIN ACETATE, and 5, the 10-anhydroleucovorin is the intermediate product of folic acid metabolism, can be converted into the 10-LEUCOVORIN ACETATE with the enzyme bonding state.A:5, the 10-anhydroleucovorin; The B:10-LEUCOVORIN ACETATE.
Fig. 3 shows 5,10-methylene tetrahydrofolate synthetic enzyme crystalline stereoscopic three-dimensional structural representation.
Fig. 4 shows 5, the crystalline three-dimensional structure synoptic diagram of the mixture of 10-methylene tetrahydrofolate synthetic enzyme and ADP.
Fig. 5 shows 5, the crystalline three-dimensional structure synoptic diagram of the mixture of 10-methylene tetrahydrofolate synthetic enzyme and L-glutamic acid.
Fig. 6 shows 5, the crystalline three-dimensional structure synoptic diagram of the mixture of 10-methylene tetrahydrofolate synthetic enzyme and N5-imines phosphoric acid transition state.
Fig. 7 shows 5, the crystalline three-dimensional structure synoptic diagram of the mixture of 10-methylene tetrahydrofolate synthetic enzyme and 10-LEUCOVORIN ACETATE.
Embodiment
People source 5, the expression and purification of 10-anhydroleucovorin synthetic enzyme (MTHFS):
The aminoacid sequence of MTHFS is following: MAAAAVSSAKRSLRGELKQRLRAMSAEERLRQSRVLSQKVIAHSEYQKSKRISIFL SMQDEIETEEIIKDIFQRGKICFIPRYRFQSNHM DMVRIESPEEISLLPKTSWNIPQPGEGDVREEALSTGGLDLIFMPGLGFDKHGNRL GRGKGYYDAYLKRCLQHQEVKPYTLALAFKEQICLQVPVNENDMKVDEVLYEDSST A
Transcribe the gene that PCR clones the people MTHFS that obtains total length the library from the commercialization people.The primer that uses is following:
The BC019921 upper reaches:
TACTTCCAATCCAATGCTATGGCGGCGGCAGCGGT
The BC019921 downstream:
TTATCCACTTCCAATGTTAAGCTGTTGACGAGTCT
The gene that the clone obtains is cloned in the commercialization PET21b carrier through ligation, and this carrier has the expression label of 6 Histidines at proteinic nitrogen end.Use BL21 (DE3) host bacterium express recombinant protein, 37 degrees centigrade, when bacteria concentration reaches OD
600nm=0.8 o'clock, adding final concentration was the IPTG of 0.2mM.Then, expressed 24 hours for 16 degrees centigrade, 4000rpm collects and expresses bacterium.
The expression bacterium of collecting is resuspended in phosphate buffered saline buffer (50mM Na
2HPO
4, 10mMKH
2PO
4, 137mM NaCl, 2.7mM KCl; PH 7.4), use 100W ultrasonic disruption cell, resuspended bacterium in phosphate buffered saline buffer; 16000rpm 30 minutes, gets supernatant; Can obtain the albumen of preliminary purification through the Ni-NTA of QIAGEN affinity column, use marmor erodens (TEV) proteolytic cleavage to dezymotize 6 Histidines that the nitrogen end has and express labels.Then, TEV proteolytic enzyme and 6 Histidines of removing in the albumen through the Ni-NTA affinity column of QIAGEN are expressed label again.The purifying protein that obtains uses the condensed prod Amicon of Milipore company, molecular weight cut-off to be 5kDa, protein concentrate to 1 milliliter, use GE Corporation's Super Dex G75 molecular sieve purification.
Using molecular sieve purification MTHFS is the single peak that washes out; The albumen damping fluid is changed to simultaneously: 20mM Tris-HCl, and pH 8.0,200mM NaCl; 0.5mM EDTA (EDTA Disodium) and 5mM beta-mercaptoethanol (β-mercaptoethanol); Use the condensed prod Amicon of Milipore company, molecular weight cut-off is 5kDa, is concentrated to 10mg/ml.
The crystalline growth:
Crystal can adopt methods such as batch crystallization, liquid-liquid diffusion, vapor diffusion, dialysis to obtain.What adopt among the present invention is the method for liquid-liquid diffusion, comprises sessile drop method or sits the method for dripping.In following experiment, adopt the crystal screening reagent box of Hampton company, the condition of crystal growth is that pond liquid is: contain 0.01-0.03mol/L MgCl in 5 to 25 degrees centigrade, 50% to 80% humidity range
2, 0.01-0.04mol/L NiCl
2, 20%-30%PEG3000 to 4000,0.1mol/L N-2-hydroxyethyl piperazine-N-2-ethyl sulfonic acid or the morpholino b acid damping fluid of pH=6.4-7.0.Before crystallization, with people source 5,10-anhydroleucovorin synthetic enzyme mixes with pond liquid or with people source 5, the mixed solution of 10-anhydroleucovorin synthetic enzyme and various substrates mixes with pond liquid.
Disclose some preferred embodiment below:
People source 5, the crystalline crystallization method of 10-anhydroleucovorin synthetic enzyme, at first with said people source 5, the liquid concentrator of 10-anhydroleucovorin synthetic enzyme mixes with pond liquid; Above-mentioned zymoprotein solution is centrifugal 14 at the Eppendorf refrigerated centrifuge, 000rpm, 15 minutes; Adopt the crystal screening reagent box crystallization of Hampton company, crystalline pond liquid consist of 0.02MMgCl
2, 0.02M NiCl
2, (0.1M pH6.6) and 20%PEG3350, collects the crystal that obtains to HEPES approximately two days later.
People source 5; The crystalline crystallization method of the mixture of 10-anhydroleucovorin synthetic enzyme and ADP; At first to said people source 5, it is the ATP solution of 5mM that the solution of 10-anhydroleucovorin synthetic enzyme adds final concentration, adds reductive agent and mg ion again; Obtain people source 5 after the mixing, the mixed solution of 10-anhydroleucovorin synthetic enzyme and ATP; With said people source 5,10-anhydroleucovorin synthetic enzyme mixed 1 hour with pond liquid with the mixed solution of ATP; Said protein solution is centrifugal 14 at the Eppendorf refrigerated centrifuge before the crystallization, 000rpm, 15 minutes (main purpose is in order to remove impurity) adopts the crystal screening reagent box crystallization of Hampton company, crystalline pond liquid consist of 0.02M MgCl
2, 0.02M NiCl
2, (0.1M pH6.6) and 20%PEG3350, collected crystal and carries out data gathering HEPES in 1-2 days.
People source 5, the crystalline crystallization method of the mixture of 10-anhydroleucovorin synthetic enzyme and L-glutamic acid, in the 5-10mg/ml protein solution, adding final concentration is the glutamic acid solution of 3mM; Mixed 1 hour; Said protein solution is centrifugal 14 at the Eppendorf refrigerated centrifuge before the crystallization, 000rpm, 15 minutes; Adopt the crystal screening reagent box crystallization of Hampton company, crystalline pond liquid consist of 0.02M MgCl
2, 0.02M NiCl
2, (0.1M pH6.6) and 20%PEG3350, collected crystal and carries out data gathering HEPES in 1-2 days.
People source 5, the crystalline crystallization method of the mixture of 10-anhydroleucovorin synthetic enzyme and N5-imines phosphoric acid transition state, in the 5-10mg/ml protein solution, adding final concentration is 5mM ATP, 5mM 5-LEUCOVORIN ACETATE, 2mM mercaptoethanol, 5mM mg ion; Mixed 1 hour; Said protein solution is centrifugal 14 at the Eppendorf refrigerated centrifuge before the crystallization, 000rpm, 15 minutes; Adopt the crystal screening reagent box crystallization of Hampton company, crystalline pond liquid consist of 0.02M MgCl
2, 0.02M NiCl
2, (0.1M pH6.6) and 20%PEG3350, collected crystal and carries out data gathering HEPES in 1-2 days.
People source 5; The crystalline crystallization method of the mixture of 10-anhydroleucovorin synthetic enzyme and 10-LEUCOVORIN ACETATE, adding final concentration in the 5-10mg/ml protein solution is 5mM ATP, 5mM 5-LEUCOVORIN ACETATE, 2mM mercaptoethanol, 5mM mg ion, mixes 1 hour; Before the crystallization with said protein solution at Eppendorf refrigerated centrifuge centrifugal 14; 000rpm, 15 minutes, crystalline pond liquid consist of 0.02M MgCl
2, 0.02M NiCl
2, (0.1M pH6.6) and 20%PEG3350, collected crystal and carries out data gathering HEPES in 4-14 days.
The crystalline detailed process is that crystal growth is in 16 ℃; Carry out the crystallization of hanging drop vapor diffusion or sit dripping the method crystallization experiment; To comprise the mixing of mixed solution of mixing pit liquid 2 microlitres and 2 microlitre protein solutions or protein solution and substrate or product, spread, comprise 0.02MMgCl at 300 μ l
2, 0.02M NiCl
2, HEPES (0.1M; PH6.6) and the pond liquid of 20%PEG3350 exist down; Through finding that overall dimension reaches the tabular crystal of 300-500 μ m after a while; With crystal be transferred to comprise 0.01M MgCl2,0.01M NiCl2, HEPES (0.1M, pH6.6), among the 10%PEG3350, the solution of 20% glycerine as frostproofer.Quick freezing sample before collecting the X-ray data then.
Crystalline is resolved
The compound crystal that obtains is transferred in the pond liquid that contains 10-15% glycerine, chooses monocrystalline, and quick freezing after preliminary X diffraction screening, is confirmed the crystal data that will collect to liquid nitrogen.The collection of diffraction data: use synchrotron radiation; Wavelength is that HKL2000 (Otwinowski and Minor, 1997) is used in
data rough handling.The wild-type structure elucidation uses the molecular replacement method, uses software to be Balbes (Long et.al., 2008), with the mpMTHFS in the Protein Data Bank (PDB code 1U3G) modeling.The compound crystal data gathering use in-house FRE+copper rotating anode and a R-AXIS IV++detector (Rigaku, USA) and synchrotron radiation.The parsing of composite structure uses software to be Phaser (McCoy et.al., 2007), is the template modeling with the structure of people source hMTHFS.Software Phenix refine (Adams et.al., 2002) and Refmac (Murshudov et.al., 1997) are used in the correction of structural models.Come monitoring model quality and round-off error with the R factor and the Rfree factor.
People source 5; The crystal of 10-anhydroleucovorin synthetic enzyme; Spacer is: C2221; Unit cell parameters is
α=90.00 °; β=90.00 °, γ=90.00 °.
People source 5; The crystal of the mixture of 10-anhydroleucovorin synthetic enzyme and ADP; Spacer is: C2221; Unit cell parameters is
α=90.00 °; β=90.00 °, γ=90.00 °.
People source 5; The crystal of the mixture of 10-anhydroleucovorin synthetic enzyme and ADP; Spacer is: P21212; Unit cell parameters is
α=90.00 °; β=90.00 °, γ=90.00 °.
People source 5; The crystal of the mixture of 10-anhydroleucovorin synthetic enzyme and L-glutamic acid; Spacer is: C2221; Unit cell parameters is
α=90.00 °; β=90.00 °, γ=90.00 °.
People source 5; The crystal of the mixture of 10-anhydroleucovorin synthetic enzyme and N5-imines phosphoric acid transition state; Spacer is: C2221; Unit cell parameters is
α=90.00 °; β=90.00 °, γ=90.00 °.
People source 5; The crystal of the mixture of 10-anhydroleucovorin synthetic enzyme and 10-LEUCOVORIN ACETATE; Spacer is: C2221; Unit cell parameters is
α=90.00 °; β=90.00 °, γ=90.00 °.
Table 2 is people source 5, the crystalline coordinate of 10-anhydroleucovorin synthetic enzyme
Table 2
Table 3 is people source 5, the crystalline coordinate of the mixture of 10-anhydroleucovorin synthetic enzyme and L-glutamic acid:
Table 3
Table 4 is people source 5, the crystal coordinates of the mixture of 10-anhydroleucovorin synthetic enzyme and ADP.
Table 4
Table 5 is people source 5, the crystalline coordinate of the mixture of 10-anhydroleucovorin synthetic enzyme and N5-imines phosphoric acid transition state.
Table 5
Table 6 is people source 5, the crystalline coordinate of the mixture of 10-anhydroleucovorin synthetic enzyme and 10-LEUCOVORIN ACETATE.
Table 6
The enzyme assay of various MTHFS two mutants and people's wild-type MTHFS
Through to the biological chemistry of various MTHFS homologous proteins and the research of zymologic property, the contriver has selected 15 sites and has carried out rite-directed mutagenesis research.
The principle that sudden change is used is: the method through using rite-directed mutagenesis is suddenlyd change the potential critical sites that analysis obtains; The method of sudden change is used STRATAGENE company rite-directed mutagenesis test kit; Obtain two mutants through PCR one step, verify whether success of sudden change through dna sequencing.Potential key amino acid residue all is mutated into and is L-Ala (A), because L-Ala has the simplest amino acid side chain, again proteinic secondary structure is produced slight influence simultaneously, is widely used in the rite-directed mutagenesis so this method has become.
With the 5-methyl tetrahydrofolate is substrate, adds any MTHFS two mutants or people's wild-type MTHFS and cofactor mg ion, ATP (Triphosaden).Room temperature reaction 2 minutes through the variation of uv absorption spectrum detection 360nm place absorb light intensity, is confirmed resultant 5, the content of 10-methylene tetrahydrofolate, and then the power of definite enzymic activity.The activity of people's wild-type MTHFS is made as 100%, and the relative reactivity of mutant enzyme is to obtain with people's wild-type enzyme specific activity.
The relative reactivity of table 7.MTHFS two mutants
People source 5, the sterie configuration of 10-anhydroleucovorin synthetic enzyme and mixture thereof
Obtain the atomic coordinate of people source MTHFS through structural modifications, the three-dimensional structure of MTHFS is seen Fig. 3, wherein comprises α spiral and β lamella in the three-dimensional structure of this enzyme, and its site is following: α 1 2-18; α 2 26-42; α 3 44-48; β 1 50-54; α 4 65-73; β 2 77-84; β 3 89-95; β 4 131-134; β 5 138-140; β 6 145-146; α 5 152-162; β 7169-173; β 8 194-196.
Can know from the result of mutating experiment and tomograph: following site is crucial for enzyme performance activity: F55, M58, E61, M90, Y152, Y153 through with substrate 5-methyl tetrahydrofolate the pterin group combine to bring into play enzymic activity; F85, W109, R148 through with substrate 5-methyl tetrahydrofolate in the para-amino benzoic acid group combine to bring into play enzymic activity; K10, R14, R145, D154 are through combining to bring into play enzymic activity with ATP.
People source 5,10-anhydroleucovorin synthetic enzyme and ADP mixture: after the protein binding phosphate group, part that originally can't modeling in structure, E187-D189 has obtained good structure in people source MTHFS combines the composite structure of ADP.E187-D189 is positioned at the combining site of ATP, and the O1 Sauerstoffatom of the phosphoric acid that D189 and ATP hydrolysis are got off forms a hydrogen bond, and has combined a mg ion.In addition, D189 and K10 form a salt bridge.The formation of the chemical action key that these are new, make in the apoprotein structure can't modeling part (A174-D193) obtained stable.Because two phosphate groups and one that in three-dimensional structure, can only show ADP is the phosphate group under the hydrolysis from the ATP, wherein the phosphoric acid that gets off of hydrolysis will be participated in enzyme reaction as nucleophilic group.This phosphate group is positioned at a section of α 5 (152-161 amino acids), and 151-153 amino acids residue has interaction with it.Use the ATP analogue, for example use sulfur analogs to substitute phosphate group design small-molecule drug and can suppress the enzyme of phosphate group entering here molecule, thus inhibitory enzyme activity.
People source 5; 10-anhydroleucovorin synthetic enzyme and L-glutamic acid mixture: be combined near the neutral charge crack of a 111-116 amino acids residue; This crack is the big Loop ring of between β 3 (89-95 amino acid section) and the β 4 (131-135 amino acid section) zone; Wherein there is the interaction within 4 dusts G115 and E116 and it, and the L-glutamic acid part is through being positioned at protein surface beyond the outstanding binding pocket of a circular port.This species specific combination can be used to design peptide inhibitor, is used for the enzyme inhibitors of designs specificity.
People source 5, the mixture of 10-anhydroleucovorin synthetic enzyme and N5-imines phosphoric acid transition state: amino-acid residue F55, L56, S57; M58, E61, P81, Y83; M90, W109, P135, R148; In the big spherical pocket that K150, Y152 and Y153 surround, the pterin of 5-imine phosphate acid-respons intermediate state ring is located in the middle of the spheric.4-spiral and 3,4-are folding, and the long loop between them, the structure that has formed a hat-shaped cover the active site above.W109 be positioned at pterin ring above; And look it is the lid that is used for closing the active site, W109 and pterin ring distance be
explain between the two less than interaction.The conformation of pterin ring is not a plane, and it stretches along vertical axis in the C6 position.The N3 and the amino nitrogen of 61 carboxyl oxygen and pterin ring interact.E61 is a conserved residues in lineal homology MTHFS.The N8 of M90 and pterin ring forms a hydrogen bond.At pterin ring and F55, Y83, P135, Y152 exists a lot of hydrophobic Van der Waals forces and interacts between the aromatic side chain of Y153.Y83 distinguishes maximum in the crystal of wild-type and mixture, and the side chain of Y83 rotates and forms a plumbness, makes the pterin ring get into binding pocket.Moving of Y83 side chain, the side chain that makes Y85 conversely is away from binding pocket.Described amino-acid residue is the crucial group of participating in enzyme reaction, can structural information be provided for the design small-molecule drug.In this N5-imines phosphoric acid transition state, the pterin ring serves as that axle has taken place to reverse with the key between C4a and the C8a, and rearrangement has taken place the electronics on the said petrin ring.
People source 5; The mixture of 10-anhydroleucovorin synthetic enzyme and resultant 10-LEUCOVORIN ACETATE: with people source 5; 10-anhydroleucovorin synthetic enzyme is identical generally with the mixture spatial configuration of N5-imines phosphoric acid transition state, has only the shift position of Y83 side chain different.Y83 can be used for its small molecules that moves of design with suppressed, and then the design small-molecule drug.
Top structural information can be used for design or makes medicine.
The present invention also provides 5, the suppressor factor of 10-anhydroleucovorin synthetic enzyme or its mixture, comprise with above-mentioned structural domain similar or with above-mentioned structural domain bonded micromolecular compound, small peptide, polypeptide, nucleic acid, antibody or immune conjugate.
The present invention also provides the pharmaceutical composition that is used to transplant cancer, comprises 5, the suppressor factor of 10-anhydroleucovorin synthetic enzyme or its mixture, and pharmaceutical carrier or inert matter.
Sequence table
< 110>Institute of Biophysics, Academia Sinica
< 120>the people source 5, the crystallization method of 10-anhydroleucovorin synthetic enzyme and mixture thereof, crystal and application
<130>P25377IBP
<160>1
<170>PatentIn version 3.2
<210>1
<211>203
<212>PRT
< 213>mankind
<400>1
Met Ala Ala Ala Ala Val Ser Ser Ala Lys Arg Ser Leu Arg Gly Glu
1 5 10 15
Leu Lys Gln Arg Leu Arg Ala Met Ser Ala Glu Glu Arg Leu Arg Gln
20 25 30
Ser Arg Val Leu Ser Gln Lys Val Ile Ala His Ser Glu Tyr Gln Lys
35 40 45
Ser Lys Arg Ile Ser Ile Phe Leu Ser Met Gln Asp Glu Ile Glu Thr
50 55 60
Glu Glu Ile Ile Lys Asp Ile Phe Gln Arg Gly Lys Ile Cys Phe Ile
65 70 75 80
Pro Arg Tyr Arg Phe Gln Ser Asn His Met Asp Met Val Arg Ile Glu
85 90 95
Ser Pro Glu Glu Ile Ser Leu Leu Pro Lys Thr Ser Trp Asn Ile Pro
100 105 110
Gln Pro Gly Glu Gly Asp Val Arg Glu Glu Ala Leu Ser Thr Gly Gly
115 120 125
Leu Asp Leu Ile Phe Met Pro Gly Leu Gly Phe Asp Lys His Gly Asn
130 135 140
Arg Leu Gly Arg Gly Lys Gly Tyr Tyr Asp Ala Tyr Leu Lys Arg Cys
145 150 155 160
Leu Gln His Gln Glu Val Lys Pro Tyr Thr Leu Ala Leu Ala Phe Lys
165 170 175
Glu Gln Ile Cys Leu Gln Val Pro Val Asn Glu Asn Asp Met Lys Val
180 185 190
Asp Glu Val Leu Tyr Glu Asp Ser Ser Thr Ala
195 200
Claims (5)
1. people source 5; The crystal of 10-anhydroleucovorin synthetic enzyme; It is characterized in that said crystalline spacer is: C2221; Unit cell parameters is
α=90.00 °; β=90.00 °, γ=90.00 °.
2. people according to claim 1 source 5, the crystalline crystallization method of 10-anhydroleucovorin synthetic enzyme comprises:
With said people source 5, the solution of 10-anhydroleucovorin synthetic enzyme mixes with pond liquid;
Utilize sessile drop method or the seat method of dripping to carry out crystallization;
After 1-2 days, collect crystal;
It is characterized in that said crystalline growth conditions is that pond liquid is: contain 0.01-0.03mol/L MgCl in 5 to 25 degrees centigrade, 50% to 80% humidity range
2, 0.01-0.04mol/L NiCl
2, 20%-30%PEG3000 to 4000,0.1mol/L N-2-hydroxyethyl piperazine-N-2-ethyl sulfonic acid or the morpholino b acid damping fluid of pH=6.4-7.0.
3. people according to claim 1 source 5, the crystal of 10-anhydroleucovorin synthetic enzyme, wherein F55, M58, E61, M90, Y152, Y153 amino acids combine with pterin group in the substrate 5-LEUCOVORIN ACETATE.
4. people according to claim 1 source 5, the crystal of 10-anhydroleucovorin synthetic enzyme, wherein F85, W109, R148 amino acids combine with para-amino benzoic acid group in the substrate 5-LEUCOVORIN ACETATE.
5. people according to claim 1 source 5, the crystal of 10-anhydroleucovorin synthetic enzyme, wherein K10, R14, R145, D154 amino acids combine with ATP.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5389516A (en) * | 1993-03-19 | 1995-02-14 | Universite De Montreal | CDNA probes and antibodies for human methenyltetrahydrofolate synthetase |
| CA2235094A1 (en) * | 1998-05-13 | 1999-11-13 | Charron, Guy | Overexpressing human methenyltetrahydrofolate synthetase to sensitize cancer cells to 5-fluorouracil cytotoxicity |
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2009
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5389516A (en) * | 1993-03-19 | 1995-02-14 | Universite De Montreal | CDNA probes and antibodies for human methenyltetrahydrofolate synthetase |
| CA2235094A1 (en) * | 1998-05-13 | 1999-11-13 | Charron, Guy | Overexpressing human methenyltetrahydrofolate synthetase to sensitize cancer cells to 5-fluorouracil cytotoxicity |
Non-Patent Citations (2)
| Title |
|---|
| Christoph Meier等.Structure of 5-formyltetrahydrofolate cycloligase from Bacillus anthracis.《Acta Cryst》.2007,第63卷168-172. * |
| Shengfeng Chen等.Crystal structure of methenyltetrahydrofolate synthetase from Mycoplasma pneumoniae (GI:13508087) at 2.2 A resolution.《PROTEINS: Structure, Function, and Bioinformatics》.2004,第56卷839-843. * |
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