Structural analysis of substrate recognition by glucose isomerase in Mn²⁺ binding mode at M2 site in <i>S. rubiginosus</i>

Bae, Ji-Eun,Hwang, Kwang Yeon,Nam, Ki Hyun Elsevier 2018 Biochemical and biophysical research communication Vol.503 No.2

<P><B>Abstract</B></P> <P>Glucose isomerase (GI) catalyzes the reversible enzymatic isomerization of <SMALL>D</SMALL>-glucose and <SMALL>D</SMALL>-xylose to <SMALL>D</SMALL>-fructose and <SMALL>D</SMALL>-xylulose, respectively. This is one of the most important enzymes in the production of high-fructose corn syrup (HFCS) and biofuel. We recently determined the crystal structure of GI from <I>S. rubiginosus</I> (SruGI) complexed with a xylitol inhibitor in one metal binding mode. Although we assessed inhibitor binding at the M1 site, the metal binding at the M2 site and the substrate recognition mechanism for SruGI remains the unclear. Here, we report the crystal structure of the two metal binding modes of SruGI and its complex with glucose. This study provides a snapshot of metal binding at the SruGI M2 site in the presence of Mn<SUP>2+</SUP>, but not in the presence of Mg<SUP>2+</SUP>. Metal binding at the M2 site elicits a configuration change at the M1 site. Glucose molecule can only bind to the M1 site in presence of Mn<SUP>2+</SUP> at the M2 site. Glucose and Mn<SUP>2+</SUP> at the M2 site were bridged by water molecules using a hydrogen bonding network. The metal binding geometry of the M2 site indicates a distorted octahedral coordination with an angle of 55–110°, whereas the M1 site has a relatively stable octahedral coordination with an angle of 85–95°. We suggest a two-step sequential process for SruGI substrate recognition, in Mn<SUP>2+</SUP> binding mode, at the M2 site. Our results provide a better understanding of the molecular role of the M2 site in GI substrate recognition.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A snapshot of metal binding at the SruGI M2 site in the presence of Mn<SUP>2+</SUP>. </LI> <LI> Metal binding at the M2 site elicits a configuration change at the M1 site. </LI> <LI> Glucose molecule can only bind to the M1 site in presence of Mn<SUP>2+</SUP> at the M2 site. </LI> <LI> Two-step sequential process for SruGI substrate recognition, in Mn<SUP>2+</SUP> at M2 site mode. </LI> </UL> </P>

Glutathione S-transferase의 기질결합부위에 관한 연구

박희중,김용태,공광훈 中央大學校 遺傳工學硏究所 1998 遺傳工學硏究論集 Vol.11 No.1

Glutathione S-transferase(GST, EC 2.5. 1.18)의 기질 결합부위에 대한 연구를 위하여 human GST P 1-1의 6개의 잔기에 대하여 부위특이적 변이법을 이용하여 F8A, V10A, N204A, G205A, G205I 그리고 G205V의 새로운 변이체 6개를 얻었다. 대장균을 이용한 대량발현과 affinity column에 의해 정제된 변이체 효소에 대하여 GSH와 CDNB, DCNB 또는 ETA와의 포합반응 활성을 측정하였다. 야생형과의 활성비교에 있어서 G205V 변이체의 경우 CDNB나 ETA에 대한 활성이 현저히 감소함을 알 수 있었다. 이러한 결과 Gly205 잔기는 기질결합부위의 중요한 잔기로 생각되어진다. In order to study on the substrate binding site of glutathione S-transferase(GST), six residues in human GST P 1-1 were individually replaced with alanine, isoleucine or valine by site-directed mutagenesis to obtain mutants F8A, V10A, N204A, G205A, G205I and G205V. The specific activities were determined by measuring the initial rates of the enzymes-catalyzed conjugation of GSH with CDNB, DCNB or ETA. The replacement of Gly205 with valine resulted in the drastic decreases in the specific activities toward CDNB and ETA. These results suggest that Gly205 is an important residue in the binding site of the xenobiotic substrate.

Structural insights into conserved l-arabinose metabolic enzymes reveal the substrate binding site of a thermophilic l-arabinose isomerase

Lee, Y.J.,Lee, S.J.,Kim, S.B.,Lee, S.J.,Lee, S.H.,Lee, D.W. North-Holland Pub ; Elsevier Science Ltd 2014 FEBS letters Vol.588 No.6

Structural genomics demonstrates that despite low levels of structural similarity of proteins comprising a metabolic pathway, their substrate binding regions are likely to be conserved. Herein based on the 3D-structures of the α/β-fold proteins involved in the ara operon, we attempted to predict the substrate binding residues of thermophilic Geobacillus stearothermophilusl-arabinose isomerase (GSAI) with no 3D-structure available. Comparison of the structures of l-arabinose catabolic enzymes revealed a conserved feature to form the substrate-binding modules, which can be extended to predict the substrate binding site of GSAI (i.e., D195, E261 and E333). Moreover, these data implicated that proteins in the l-arabinose metabolic pathway might retain their substrate binding niches as the modular structure through conserved molecular evolution even with totally different structural scaffolds.

O-Methyltransferases from Arabidopsis thaliana

Kim, Bong-Gyu,Kim, Dae-Hwan,Hur, Hor-Gil,Lim, Jun,Lim, Yoong-Ho,Ahn, Joong-Hoon The Korean Society for Applied Biological Chemistr 2005 Journal of Applied Biological Chemistry (J. Appl. Vol.48 No.3

O-methylation mediated by O-methyltransferases (OMTs) is a common modification in natural product biosynthesis and contributes to diversity of secondary metabolites. OMTs use phenylpropanoids, flavonoids, other phenolics and alkaloids as substrates, and share common domains for S-adenosyl-L-methionine (AdoMet) and substrate binding. We searched Arabiposis genome and found 17 OMTs genes (AtOMTs). AdoMet- and substrate-binding sites were predicted. AdoMet binding domain of AtOMTs is highly conserved, while substrate-binding domain is diverse, indicating use of different substrates. In addition, expressions of six AtOMT genes in response to UV and in different tissues were investigated using real-time quantitative reverse transcriptase-polymerase chain reaction. All the AtOMTs investigated were expressed under normal growth condition and most, except AtOMT10, were induced after UV illumination. AtOMT1 and AtOMT8 were expressed in all the tissues, whereas AtOMT10 showed flower-specific expression. Analysis of these AtOMT gene expressions could provide some clues on AtOMT involvement in the cellular processes.

Human Glutathione S-transferase의 Tyr108 잔기의 역할에 관한 연구

박희중,공광훈 中央大學校 遺傳工學硏究所 1999 遺傳工學硏究論集 Vol.12 No.1

Glutathione S-transferase(GST, EC 2.5. 1.18)의 기질 결합부위에 대한 연구를 위하여 human GST P 1-1의 친전자성 기질 결합부위 (H-site)로 알려지고 있는 Tyr108 잔기에 대하여 부위특이적 변이법을 이용하여 Y108A, Y108F 그리고 Y108W의 새로운 변이체 3개를 얻었다. 대장균을 이용한 대량발현과 affinity column에 의해 정제된 변이체 효소에 대하여 GSH와 CDNB, DCNB, ETA, EPNP 또는 steroid와의 포합반응 활성을 측정하여 야생형과의 활성비교를 하였다. 야생형과의 활성비교에 있어서 각 변이체들의 변화를 볼 수 있었으며, 특히 입체적 장애를 준 Y108W 변이체의 경우 CDNB에 대한 활성이 다른 변이체에 비해 현저히 감소한 반면 ETA에 대한 활성은 증가하였다. 이러한 결과 Tyr108 잔기는 친전자성 기질 결합부위에 관련되는 잔기로 생각되어진다. In order to study the role of residue in the active site of glutathione S-transferase (GST), Tyr108 residue in human GST P 1-1 was replaced with alanine, phenylalanine and tryptophan by site-directed mutagenesis to obtain mutants Y108A, Y108F and Y108W. The specific activities were determined by measuring the initial rates of the enzymes-catalyzed conjugation of GSH with CDNB, DCNB, ETA, EPNP or steroid. The Y108W mutant slightly increased the conjugating activity toward ethacrynic acid(ETA)., but it decreased in specific activity toward 1-chloro-2,4-nitrobenzene(CDNB). On the other hand, the Y108A and Y108F mutants had negligible effect on the activity toward CDNB, ETA and DCNB of the wild type. These results indicated that Tyr108 in human GST P1-1 may contribute to the binding of electrophilic substrate.

Structural and functional analysis of substrate recognition by the 250s loop in amylomaltase from <i>Thermus brockianus</i>

Jung, Jong‐,Hyun,Jung, Tae‐,Yang,Seo, Dong‐,Ho,Yoon, Sei‐,Mee,Choi, Hyun‐,Chang,Park, Byoung Chul,Park, Cheon‐,Seok,Woo, Eui‐,Jeon Wiley Subscription Services, Inc., A Wiley Company 2011 Proteins Vol.79 No.2

<P><B>Abstract</B></P><P>Amylomaltase, or 4‐α‐glucanotransferase (EC 2.4.1.25), is involved in glycogen and maltooligosaccharide metabolism in microorganisms, catalyzing both the hydrolysis and transfer of an α‐1,4‐oligosacchraride to other sugar molecules. In this study, we determined the crystal structure of amylomaltase from <I>Thermus brockianus</I> at a resolution of 2.3 Å and conducted a biochemical study to understand the detailed mechanism for its activity. Careful comparison with previous amylomaltase structures showed a pattern of conformational flexibility in the 250s loop with higher B‐factor. Amylomaltase from <I>T. brockianus</I> exhibited a high transglycosylation factor for glucose and a lower value for maltose. Mutation of Gln256 resulted in increased <I>K</I><SUB>m</SUB> for maltotriose and a sharp decrease of the transglycosylation factor for maltose, suggesting the involvement of Gln 256 in substrate binding between subsites +1 and +2. Mutation of Phe251 resulted in significantly lower glucose production but increased maltose production from maltopentose substrates, showing an altered substrate‐binding affinity. The mutational data suggest the conformational flexibility of the loop may be involved in substrate binding in the GH77 family. Here, we present an action model of the 250s loop providing the molecular basis for the involvement of residues Phe251, Gln256, and Trp258 in the hydrolysis and transglycosylation activities in amylomaltase. Proteins 2011. © 2010 Wiley‐Liss, Inc.</P>

The Importance of Tyr-475 and Glu-506 in β-Galactosidase from L. lactis ssp. lactis 7962

( Eun Ju Yang ),( Jung Min Lee ),( Hyong Joo Lee ),( Jeong Hwan Kim ),( Dae Kyun Chung ),( Jong Moon Lee ),( Hae Choon Chang ) 한국미생물생명공학회 2003 Journal of microbiology and biotechnology Vol.13 No.1

Biochemistry,Molecular Biology ; O-Methyltransferases from Arabidopsis thaliana

Bong Gyu Kim,Dae Hwan Kim,Hor Gil Hur,Jun Lim,Yoong Ho Lim,Joong Hoon Ahn 한국응용생명화학회 2005 Journal of Applied Biological Chemistry (J. Appl. Vol.48 No.3