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Seo, Hogyun,Kim, Kyung-Jin Elsevier 2018 Biochemical and biophysical research communication Vol.499 No.3
<P><B>Abstract</B></P> <P> <I>Streptomyces coelicolor</I> A3 contains <I>Sc5140</I>, a gene coding for poorly understood bacterial LOG-like protein. In this study, we determined the crystal structure of <I>Sc</I>5140 and found it resembles the overall structure of other type-II LOGs. In addition, <I>Sc</I>5140 exhibited phosphoribohydrolase activity against adenosine monophosphate (AMP), indicating that it had the same function as known type-II LOGs. Based on these results, we designated <I>Sc</I>5140 as <I>Sc</I>LOGII. We performed docking calculations of AMP into the <I>Sc</I>LOGII structure, which suggested the mode of binding for type-II LOG with their AMP substrate. The <I>Sc</I>LOGII structure uniquely exhibited a long tail-like structure at the N-terminus that was involved in hexamerization of the protein; the disordered N-terminal region (DNR). Truncation of DNR in <I>Sc</I>LOGII negatively affected both the phosphoribohydrolase activity and the oligomerization of the protein, suggesting that this region functioned in enzyme stabilization. However, results from truncation experiments using <I>Sc</I>LOGII and <I>Cg</I>LOGII, a type-II LOG homologue from <I>Corynebacterium glutamicum</I>, were quite different, leaving uncertainty regarding the general functions of DNR in type-II LOGs. Overall, the current structural work may help in understand the significance of type-II LOG protein at the molecular level.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Crystal structure of type-II LOG from <I>Streptomyces coelicolor</I> A3 (<I>Sc</I>LOGII) was determined. </LI> <LI> The substrate binding mode of type-II LOG is suggested by docking calculations of AMP into the <I>Sc</I>LOGII. </LI> <LI> <I>Sc</I>LOGII has a long tail-like structure at the N-terminus that is involved in hexamerization. </LI> </UL> </P>
Seo, Hogyun,Kim, Seongmin,Son, Hyeoncheol Francis,Sagong, Hye-Young,Joo, Seongjoon,Kim, Kyung-Jin Elsevier 2019 Biochemical and biophysical research communication Vol.508 No.1
<P><B>Abstract</B></P> <P>Poly(ethylene terephthalate) (PET) is the most commonly used polyester polymer resin in fabrics and storage materials, and its accumulation in the environment is a global problem. The ability of PET hydrolase from <I>Ideonella sakaiensis</I> 201-F6 (<I>Is</I>PETase) to degrade PET at moderate temperatures has been studied extensively. However, due to its low structural stability and solubility, it is difficult to apply standard laboratory-level <I>Is</I>PETase expression and purification procedures in industry. To overcome this difficulty, the expression of <I>Is</I>PETase can be improved by using a secretion system. This is the first report on the production of an extracellular <I>Is</I>PETase, active against PET film, using Sec-dependent translocation signal peptides from <I>E. coli</I>. In this work, we tested the effects of fusions of the Sec-dependent and SRP-dependent signal peptides from <I>E. coli</I> secretory proteins into <I>Is</I>PETase, and successfully produced the extracellular enzyme using pET22b-SP<SUB>MalE</SUB>:<I>IsPETase</I> and pET22b-SP<SUB>LamB</SUB>:<I>IsPETase</I> expression systems. We also confirmed that the secreted <I>Is</I>PETase has PET-degradation activity. The work will be used for development of a new <I>E. coli</I> strain capable of degrading and assimilating PET in its culture medium.</P> <P><B>Highlights</B></P> <P> <UL> <LI> PETase from <I>Ideonella sakaiensis</I> (<I>Is</I>PETase) was successfully produced using the protein secretory expression system. </LI> <LI> Extracellular production of <I>Is</I>PETase is achieved by sec-dependent secretion system. </LI> <LI> The extracellularly produced <I>Is</I>PETase shows a PET degradation activity. </LI> </UL> </P>
Hogyun Seo,Kyung-Jin Kim 한국생물공학회 2019 Biotechnology and Bioprocess Engineering Vol.24 No.1
Although enoyl-CoA hydratase/isomerase superfamily proteins are functionally diverse and extremely abundant in microbial and higher organism’s genome, they still have been elusively annotated. The genome of Cupriavidus necator H16 contains at least 54 enoyl-CoA hydratase/isomerase superfamily proteins that might influence on polyhydroxyalkanoate synthesis, but most of them are uncharacterized. Among them, we first determined crystal structure of H16_B0756 at a 2.0 Å resolution. The protein exhibits unique amino acid sequences compared to the other isoforms with identity lower than 36%. The structure of H16_B0756 forms a trimeric architecture and showed canonical disk-shape. Interestingly, H16_B0756 has only one glutamate residue at the active site while other enoyl- CoA hydratases have two nucleophilic glutamate at the catalytic site. We found that the active site conformation of H16_B0756 is quite similar to that of 1,2-epoxyphenylacetyl- CoA isomerase (PaaG) rather than those of other enoyl-CoA hydratases. In addition to the structural comparison, gene neighborhoods analysis suggested that H16_B0756 might function in the ring compound degradation.
Hong, Hwaseok,Seo, Hogyun,Park, Woojin,Kim, Kyung‐,Jin BLACKWELL SCIENCE 2020 ENVIRONMENTAL MICROBIOLOGY Vol.22 No.1
<P><B>Summary</B></P><P>Fumarylacetoacetate hydrolase (FAH) superfamily proteins are found ubiquitously in microbial pathways involved in the catabolism of aromatic substances. Although extensive bioinformatic data on these proteins have been acquired, confusion caused by problems with the annotation of these proteins hinders research into determining their physiological functions. Here we classify 606 FAH superfamily proteins using a maximum likelihood (ML) phylogenetic tree, comparative gene‐neighbourhood patterns and <I>in vitro</I> enzyme assays. The FAH superfamily proteins used for the analyses are divided into five distinct subfamilies, and two of them, FPH‐A and FPH‐B, contain the majority of the proteins of undefined function. These subfamilies include clusters designated FPH‐I and FPH‐II, respectively, which include two distinct types of fumarylpyruvate hydrolase (FPH), an enzyme involved in the final step of the gentisate pathway. We determined the crystal structures of these FPH enzymes at 2.0 Å resolutions and investigate the substrate binding mode by which these types of enzymes can accommodate fumarylpyruvate as a substrate. Consequentially, we identify the molecular signatures of the two types of FPH enzymes among the broadly conserved FAH superfamily proteins. Our studies allowed us to predict the relationship of unknown FAH superfamily proteins using their sequence information.</P>
Hong, Hwaseok,Seo, Hogyun,Kim, Kyung-Jin Elsevier 2019 Biochemical and biophysical research communication Vol.514 No.3
<P><B>Abstract</B></P> <P> <I>Pseudomonas aeruginosa</I> PAO1 can utilize various aromatic hydrocarbons as a carbon source. Among the three genes involved in the gentisate pathway of <I>P. aeruginosa</I>, the gene product of <I>PA2473</I> belongs to the ζ-class glutathione S-transferase and is predicted to be a maleylpyruvate isomerase. In this study, we determined the crystal structure of maleylpyruvate isomerase from <I>Pseudomonas aeruginosa</I> PAO1 (<I>Pa</I>MPI) at a resolution of 1.8 Å. <I>Pa</I>MPI functions as a dimer and shows the glutathione S-transferase fold. The structure comparison with other glutathione S-transferase structures enabled us to predict the glutathione cofactor binding site and suggests that <I>Pa</I>MPI has differences in residues that make up the putative substrate binding site. Biochemical study of <I>Pa</I>MPI showed that the protein has an MPI activity. Interestingly, unlike the reported glutathione S-transferases so far, the purified <I>Pa</I>MPI showed isomerase activity without the addition of the reduced glutathione, although the protein showed much higher activity when the glutathione cofactor was added to the reaction mixture. Taken together, our studies reveal that the gene product of <I>PA2473</I> functions as a maleylpyruvate isomerase and might be involved in the gentisate pathway.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Crystal structure of maleylpyruvate isomerase from <I>Pseudomonas aeruginosa</I> PAO1 was determined. </LI> <LI> The gene product of <I>PA2473</I> functions as a maleylpyruvate isomerase and might be involved in the gentisate pathway. </LI> <LI> Based on the structural comparisons, we suggested the formation of the cofactor and substrate binding site of <I>Pa</I>MPI. </LI> </UL> </P>
Hong, Hwaseok,Seo, Hogyun,Kim, Kyung-Jin Elsevier 2019 Biochemical and biophysical research communication Vol.514 No.3
<P><B>Abstract</B></P> <P> <I>Sphingobium sp.</I> strain SYK-6, an aerobic gram-negative bacillus found in soil, is known for utilizing lignin-derived monoaryls and biaryls as carbon sources and degrading aromatic compounds. The <I>Sphingobium sp.</I> strain SYK-6 genome contains three genes involved in salicylate catabolism: <I>SLG_11260</I>, <I>SLG_11270,</I> and <I>SLG_11280</I>. Here, we report that the gene product of <I>SLG_11280</I> functions as a maleylpyruvate hydrolase (<I>Ss</I>MPH) with <I>K</I> <SUB> <I>m</I> </SUB> and <I>K</I> <SUB> <I>cat</I> </SUB> values of 166.2 μM and 3.76 min<SUP>−1</SUP>, respectively. This study also reveals the crystal structures of both the apo and pyruvate-manganese ion-bound <I>Ss</I>MPH, which revealed that like other fumarylacetoacetate hydrolases, <I>Ss</I>MPH dimerizes and has nine unique 3<SUB>10</SUB>-helices. Molecular docking studies of maleylpyruvate also revealed the likely binding mode of <I>Ss</I>MPH and its substrate.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The gene product of SLG_11280 functions as a maleylpyruvate hydrolase. </LI> <LI> The Km and Kcat values of SsMPH for maleylpyruvate were 166.2 μM and 3.76 min-1, respectively. </LI> <LI> The first crystal structure of maleylpyruvate hydrolase was determined. </LI> </UL> </P>