Kim, Do Hyeon
(Department of Chemistry, College of Natural Sciences, Soongsil University)
,
Nguyen, Quyet Thang
(Department of Chemistry, College of Natural Sciences, Soongsil University)
,
Ko, Gyeong Soo
(Department of Chemistry, College of Natural Sciences, Soongsil University)
,
Yang, Jin Kuk
(Department of Chemistry, College of Natural Sciences, Soongsil University)
Homoserine dehydrogenase (HSD) catalyzes the reversible conversion of ʟ-aspartate-4-semialdehyde to ʟ-homoserine in the aspartate pathway for the biosynthesis of lysine, methionine, threonine, and isoleucine. HSD has attracted great attention for medical and industrial purposes due to its ...
Homoserine dehydrogenase (HSD) catalyzes the reversible conversion of ʟ-aspartate-4-semialdehyde to ʟ-homoserine in the aspartate pathway for the biosynthesis of lysine, methionine, threonine, and isoleucine. HSD has attracted great attention for medical and industrial purposes due to its recognized application in the development of pesticides and is being utilized in the large scale production of ʟ-lysine. In this study, HSD from Bacillus subtilis (BsHSD) was overexpressed in Escherichia coli and purified to homogeneity for biochemical characterization. We examined the enzymatic activity of BsHSD for ʟ-homoserine oxidation and found that BsHSD exclusively prefers NADP+ to NAD+ and that its activity was maximal at pH 9.0 and in the presence of 0.4 M NaCl. By kinetic analysis, Km values for ʟ-homoserine and NADP+ were found to be 35.08 ± 2.91 mM and 0.39 ± 0.05 mM, respectively, and the Vmax values were 2.72 ± 0.06 μmol/min-1 mg-1 and 2.79 ± 0.11 μmol/min-1 mg-1, respectively. The apparent molecular mass determined with size-exclusion chromatography indicated that BsHSD forms a tetramer, in contrast to the previously reported dimeric HSDs from other organisms. This novel oligomeric assembly can be attributed to the additional C-terminal ACT domain of BsHSD. Thermal denaturation monitoring by circular dichroism spectroscopy was used to determine its melting temperature, which was 54.8℃. The molecular and biochemical features of BsHSD revealed in this study may lay the foundation for future studies on amino acid metabolism and its application for industrial and medical purposes.
Homoserine dehydrogenase (HSD) catalyzes the reversible conversion of ʟ-aspartate-4-semialdehyde to ʟ-homoserine in the aspartate pathway for the biosynthesis of lysine, methionine, threonine, and isoleucine. HSD has attracted great attention for medical and industrial purposes due to its recognized application in the development of pesticides and is being utilized in the large scale production of ʟ-lysine. In this study, HSD from Bacillus subtilis (BsHSD) was overexpressed in Escherichia coli and purified to homogeneity for biochemical characterization. We examined the enzymatic activity of BsHSD for ʟ-homoserine oxidation and found that BsHSD exclusively prefers NADP+ to NAD+ and that its activity was maximal at pH 9.0 and in the presence of 0.4 M NaCl. By kinetic analysis, Km values for ʟ-homoserine and NADP+ were found to be 35.08 ± 2.91 mM and 0.39 ± 0.05 mM, respectively, and the Vmax values were 2.72 ± 0.06 μmol/min-1 mg-1 and 2.79 ± 0.11 μmol/min-1 mg-1, respectively. The apparent molecular mass determined with size-exclusion chromatography indicated that BsHSD forms a tetramer, in contrast to the previously reported dimeric HSDs from other organisms. This novel oligomeric assembly can be attributed to the additional C-terminal ACT domain of BsHSD. Thermal denaturation monitoring by circular dichroism spectroscopy was used to determine its melting temperature, which was 54.8℃. The molecular and biochemical features of BsHSD revealed in this study may lay the foundation for future studies on amino acid metabolism and its application for industrial and medical purposes.
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문제 정의
Thus, BsHSD represents a new class of HSD with features of the three-domain architecture and the tetramer formation. In this study, we investigated this novel BsHSD for its molecular and enzymatic features. We overexpressed the recombinant BsHSD in an E.
가설 설정
The specific activity was measured at varying concentration points of substrate or cofactor to determine the kinetic parameters. (A) L-homoserine concentration was varied with saturated concentration of NADP+. (B) NADP+ concentration was varied with saturated concentration of L-homoserine.
(A) L-homoserine concentration was varied with saturated concentration of NADP+. (B) NADP+ concentration was varied with saturated concentration of L-homoserine.
제안 방법
In addition, this result suggests the structural integrity of the purified BsHSD sample. Next, thermal denaturation experiments were performed using CD spectroscopy to investigate the thermal stability of BsHSD. The spectrum was measured every 30 s at 222 nm, raising the sample temperature from 26℃ to 94℃ and a total of 336 data points were collected.
We investigated the optimal pH, temperature, and NaCl concentration for the enzyme activity and determined the kinetic parameters for the oxidation of L-homoserine. Next, we also examined the oligomeric state of BsHSD in solution through size-exclusion chromatography, and its thermal stability by CD spectroscopic thermal denaturation test. Finally, we built a predicted model for three-dimensional structure of BsHSD.
5 μΜ BsHSD. The cofactor preference between 2 mM NAD+ and 1 mM NADP+ was investigated with 100mM L-HSE and 0.5 μM BsHSD to determine the kinetic parameters for substrate and cofactor. The initial reaction rate was measured for L-HSE and NADP+ at 6, 7 different concentrations, ranging from 1mM to 300mM and 20 mM to 2000 mM, respectively.
The expressed recombinant BsHSD was purified through a series of chromatography columns. The final purified sample showed remarkably high homogeneity, which was assessed with SDS-PAGE (Fig. 2), and it was applied to the subsequent molecular and enzymatic analyses.
Circular dichroism analysis. The fold integrity and the secondary structure composition was checked from the wavelength scan, and the thermal denaturation experiment was carried out to investigate the thermal stability. (A) Wavelength scan (B) Thermal denaturation at 222 nm.
Next, the BsHSD was purified from the supernatants through 3 serial applications of column chromatography using a HisTrapFF, HiPrep 26/10 Desalting and Superdex-200 (GE Healthcare, USA). The protein purity was examined with Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), and the concentration was measured using a NanoDrop 1000 (Thermo Scientific, USA).
org) from the amino acid sequence of BsHSD. The server selected the crystal structure of HSD from Mycolicibacterium hassiacum (PDB - 6DZS; not published) as a template, on which the structure of BsHSD was modeled.
The three-dimensional structure of BsHSD was predicted using the SWISS-MODEL server (http:// swissmodel.expasy.org) based on the crystal structure of HSD from M. hassiacum (PDB - 6DZS; MhHSD, hereafter; not published) as a homology model that was algorithmically selected by the server. MhHSD showed 40.
coli system, and purified it to homogeneity. Then, we carried out the enzyme assay and determined the kinetic parameters for L-HSE, the substrate, and for NADP+, the specific cofactor. In addition, we also performed several molecular analyses including size-exclusion chromatography, circular dichroism spectroscopy, and thermal denaturation.
[2-6]. These studies have revealed features including the dimeric assembly, overall structure, catalytic key residues, and possible reaction mechanism. In particular, the dimeric assembly is a common feature for all the previously reported HSDs.
5 μM BsHSD. To investigate the temperature dependence of the enzyme activity, the enzyme activity was measured at various temperatures from 25oC to 50oC with increments of 5oC in a reaction mixture containing 100 mM L-HSE, 1mM NADP+, 400 mM NaCl, 0.5 μM BsHSD and 100 mM CHES buffer, at pH 9.0. Subsequent kinetic analyses were carried out under the determined optimum condition for NaCl concentration and pH (400 mM NaCl and pH 9.
대상 데이터
Next, thermal denaturation experiments were performed using CD spectroscopy to investigate the thermal stability of BsHSD. The spectrum was measured every 30 s at 222 nm, raising the sample temperature from 26℃ to 94℃ and a total of 336 data points were collected. The melting temperature (Tm) was determined from the sigmoid fitting of these data at 54.
이론/모형
0 software (OriginLab, USA). The Michaelis-Menten equation V=Vmax S/(Km+S) was used as the reference equation to calculate the apparent parameters from the optimal fitting.
성능/효과
3% sequence identity with BsHSD on an alignment covering 430 residues with several short gaps. The model revealed that BsHSD consists of 3 domains: the N-terminal nucleotide-binding domain of a varied Rossman fold, a central substrate-binding domain, and a C-terminal ACT domain (acronym for aspartate kinase, chorismate mutase and TyrA (prephenate dehydrogenase)) of a ferredoxin-like fold (Fig. 7) [5]. The nucleotide-binding domain and the substrate-binding domain are commonly found in all HSDs from any organism, but the Cterminal ACT domain is an additional regulatory domain that is present in only a subset of HSDs [2-6].
3A). The results clearly showed that the presence of NaCl is greatly beneficial for the enzyme activity and even with only 50 mM NaCl, the activity was about 11 times greater than the activity without NaCl. As the NaCl concentration was doubled from 50 mM to 100 mM, and then sequentially to 1, 600 mM, the activity increased to reach its maximum at 400 mM NaCl with a specific activity of 1.
후속연구
Conclusively, the molecular and biochemical features of BsHSD were investigated in this study. Given the importance of HSD as a key enzyme in the aspartate pathway and also as a target for medical and industrial applications, this study will add to the knowledge on the biochemistry of amino acid metabolism and lay the foundation for future efforts in corresponding applications.
참고문헌 (15)
1 Jacques SL Nieman C Bareich D Broadhead G Kinach R Honek JF 2001 Characterization of yeast homoserine dehydrogenase, an antifungal target: the invariant histidine 309 is important for enzyme integrity Biochim. Biophys. Acta 1544 28 41 10.1016/S0167-4838(00)00203-X 11341914
2 Akai S Ikushiro H Sawai T Yano T Kamiya N Miyahara I 2019 The crystal structure of homoserine dehydrogenase complexed with l-homoserine and NADPH in a closed form J. Biochem. 165 185 195 10.1093/jb/mvy094 30423116
4 Hayashi J Inoue S Kim K Yoneda K Kawarabayasi Y Ohshima T 2015 Crystal structures of a hyperthermophilic archaeal homoserine dehydrogenase suggest a novel cofactor binding mode for oxidoreductases Sci. Rep. 5 11674 10.1038/srep11674 26154028
5 Navratna V Reddy G Gopal B 2015 Structural basis for the catalytic mechanism of homoserine dehydrogenase Acta Crystallogr. D Biol. Crystallogr. 71 1216 1225 10.1107/S1399004715004617 25945586
6 Tomonaga Y Kaneko R Goto M Ohshima T Yoshimune K 2015 Structural insight into activation of homoserine dehydrogenase from the archaeon Sulfolobus tokodaii via reduction Biochem. Biophys. Rep. 3 14 17 10.1016/j.bbrep.2015.07.006 29124164
7 Curien G Biou V Mas-Droux C Robert-Genthon M Ferrer JL Dumas R 2008 Amino acid biosynthesis: new architectures in allosteric enzymes Plant Physiol. Biochem. 46 325 339 10.1016/j.plaphy.2007.12.006 18272376
8 Schuller DJ Grant GA Banaszak LJ 1995 The allosteric ligand site in the Vmax-type cooperative enzyme phosphoglycerate dehydrogenase Nat. Struct. Biol. 2 69 76 10.1038/nsb0195-69 7719856
9 Mas-Droux C Curien G Robert-Genthon M Laurencin M Ferrer JL Dumas R 2006 A novel organization of ACT domains in allosteric enzymes revealed by the crystal structure of Arabidopsis aspartate kinase Plant Cell 18 1681 1692 10.1105/tpc.105.040451 16731588
10 Yoshida A Tomita T Kurihara T Fushinobu S Kuzuyama T Nishiyama M 2007 Structural Insight into concerted inhibition of alpha 2 beta 2-type aspartate kinase from Corynebacterium glutamicum J. Mol. Biol. 368 521 536 10.1016/j.jmb.2007.02.017 17350037
11 Kaplun A Vyazmensky M Zherdev Y Belenky I Slutzker A Mendel S 2006 Structure of the regulatory subunit of acetohydroxyacid synthase isozyme III from Escherichia coli J. Mol. Biol. 357 951 963 10.1016/j.jmb.2005.12.077 16458324
12 Gallagher DT Gilliland GL Xiao G Zondlo J Fisher KE Chinchilla D 1998 Structure and control of pyridoxal phosphate dependent allosteric threonine deaminase Structure 6 465 475 10.1016/S0969-2126(98)00048-3 9562556
13 Micsonai A Wien F Kernya L Lee YH Goto Y Refregiers M 2015 Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy Proc. Natl. Acad. Sci. USA 112 E3095 E3103 10.1073/pnas.1500851112 26038575
15 Pucci F Rooman M 2017 Physical and molecular bases of protein thermal stability and cold adaptation Curr. Opin. Struct. Biol. 42 117 128 10.1016/j.sbi.2016.12.007 28040640
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