Rescue of Deleterious Mutations by the Compensatory Y30F Mutation in Ketosteroid Isomerase

Jin, Hye Jin; Jang, Dogeun; Kim, Yeon Gil; Hong, Bok Sil; Woo, Jae Sung; Kim, Kyong‐Tai; Choi, Kwan Yong

doi:10.1007/s10059-013-0013-1

Cited by 10 publications

(9 citation statements)

References 32 publications

(34 reference statements)

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“…42 Proteins in the NTF2-like superfamily are generally defined into two categories, enzymatically active and non-enzymatically active proteins. The former group includes enzymes with varying activities such as the ketosteroid isomerase, 43 scytalone dehydrogenase, 44 and polyketide cyclase. 45,46 The latter group includes proteins that could play roles as diverse as facilitating protein transport into the nucleus 47 or mediating multimerization of calcium/calmodulin dependent protein kinase II (CaMKII), 48 or may function as a receptor.…”

Section: Resultsmentioning

confidence: 99%

“…This versatile superfamily is a classic example of divergent evolution wherein the proteins have similar overall structures but diverge greatly in their functions. 42,51 Several crystal structures for NTF2-like superfamily proteins were reported, of which the functions have been characterized, including the association domain of CaMKII from mouse (PDB entry 1HKX), 48 NTF2 from rat (PDB entry 1OUN), 52 ketosteroid isomerase (KSI) from Pseudomonas putida (PDB entry 1OPY), 43 scytalone dehydatase ( mg SD) from Magnaporthe grisea (PDB entry 1STD), 44 and polyketide cyclases SnoaL from Streptomyces avidinii (PDB entry 1SJW) 45 and Tcm ARO/CYC from Streptomyces glaucescens (PDB entry 2RER). 46 Despite low amino acid sequence identities, ranging from 9.8% to 17.5%, SgcJ was found to share similar folds with each of the NTF2-like superfamily proteins listed, with rmsds of 3.0, 2.6, 2.7, 2.7, 3.1, and 3.0 Å for the Cα atoms, respectively (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…KSI (isomerase), 43 mg SD (dehydratase), 44 SnoaL (cyclase), 45 and Tcm ARO/CYC (cyclase) 46 are enzymatically active proteins within the NTF2-like superfamily. Although their functions are different, they share a common catalytic mechanism: (i) a general base abstracts a proton from Cα of a carbonyl group to form an enolate intermediate, which is stabilized by a general acid; (ii) the enolate intermediate tautomerizes back to the carbonyl group followed by double bond rearrangement or nucleophilic attack (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Crystal structure of SgcJ, an NTF2-like superfamily protein involved in biosynthesis of the nine-membered enediyne antitumor antibiotic C-1027

et al. 2016

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Comparative analysis of the enediyne biosynthetic gene clusters revealed sets of conserved genes serving as outstanding candidates for the enediyne core. Here we report the crystal structures of SgcJ and its homologue NCS-Orf16, together with gene inactivation and site-directed mutagenesis studies, to gain insight into enediyne core biosynthesis. Gene inactivation in vivo establishes that SgcJ is required for C-1027 production in Streptomyces globisporus. SgcJ and NCS-Orf16 share a common structure with the nuclear transport factor 2-like superfamily of proteins, featuring a putative substrate binding or catalytic active site. Site-directed mutagenesis of the conserved residues lining this site allowed us to propose that SgcJ and its homologues may play a catalytic role in transforming the linear polyene intermediate, along with other enediyne polyketide synthase associated enzymes, into an enzyme-sequestered enediyne core intermediate. These findings will help formulate hypotheses and design experiments to ascertain the function of SgcJ and its homologues in 9-membered enediyne core biosynthesis.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Crystal structure of SgcJ, an NTF2-like superfamily protein involved in biosynthesis of the nine-membered enediyne antitumor antibiotic C-1027

et al. 2016

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“…All mutations were confirmed by sequencing the entire gene of the mutant KSI. Mutant KSIs were overexpressed in Escherichia coli BL21(DE3) (Novagen) harboring an expression vector plasmid containing the mutant KSI gene, and were purified as described previously ( Cha et al, 2013 ; Jang et al, 2004 ). The purity of KSI was confirmed by the presence of only one band in SDS-PAGE.…”

Section: Methodsmentioning

confidence: 99%

Contribution of a Low-Barrier Hydrogen Bond to Catalysis Is Not Significant in Ketosteroid Isomerase

Jang¹,

Choi²,

Jin³

et al. 2015

Molecules and Cells

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Low-barrier hydrogen bonds (LBHBs) have been proposed to have important influences on the enormous reaction rate increases achieved by many enzymes. Δ5-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Δ5-3-ketosteroid to its conjugated Δ4-isomers at a rate that approaches the diffusion limit. Tyr14, a catalytic residue of KSI, has been hypothesized to form an LBHB with the oxyanion of a dienolate steroid intermediate generated during the catalysis. The unusual chemical shift of a proton at 16.8 ppm in the nuclear magnetic resonance spectrum has been attributed to an LBHB between Tyr14 Oη and C3-O of equilenin, an intermediate analogue, in the active site of D38N KSI. This shift in the spectrum was not observed in Y30F/Y55F/D38N and Y30F/Y55F/Y115F/D38N mutant KSIs when each mutant was complexed with equilenin, suggesting that Tyr14 could not form LBHB with the intermediate analogue in these mutant KSIs. The crystal structure of Y30F/Y55F/Y115F/D38N-equilenin complex revealed that the distance between Tyr14 Oη and C3-O of the bound steroid was within a direct hydrogen bond. The conversion of LBHB to an ordinary hydrogen bond in the mutant KSI reduced the binding affinity for the steroid inhibitors by a factor of 8.1–11. In addition, the absence of LBHB reduced the catalytic activity by only a factor of 1.7–2. These results suggest that the amount of stabilization energy of the reaction intermediate provided by LBHB is small compared with that provided by an ordinary hydrogen bond in KSI.

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“…The thermal denaturation experiments were performed using a Jasco J-810 spectropolarimeter equipped with a peltier temperature control system, as described previously [23] . Protein samples were prepared in 20 mM Tris (pH 8.0) in a 2 mm path length cuvette.…”

Section: Methodsmentioning

confidence: 99%

Structure of Putrescine Aminotransferase from Escherichia coli Provides Insights into the Substrate Specificity among Class III Aminotransferases

et al. 2014

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YgjG is a putrescine aminotransferase enzyme that transfers amino groups from compounds with terminal primary amines to compounds with an aldehyde group using pyridoxal-5′-phosphate (PLP) as a cofactor. Previous biochemical data show that the enzyme prefers primary diamines, such as putrescine, over ornithine as a substrate. To better understand the enzyme's substrate specificity, crystal structures of YgjG from Escherichia coli were determined at 2.3 and 2.1 Å resolutions for the free and putrescine-bound enzymes, respectively. Sequence and structural analyses revealed that YgjG forms a dimer that adopts a class III PLP-dependent aminotransferase fold. A structural comparison between YgjG and other class III aminotransferases revealed that their structures are similar. However, YgjG has an additional N-terminal helical structure that partially contributes to a dimeric interaction with the other subunit via a helix-helix interaction. Interestingly, the YgjG substrate-binding site entrance size and charge distribution are smaller and more hydrophobic than other class III aminotransferases, which suggest that YgjG has a unique substrate binding site that could accommodate primary aliphatic diamine substrates, including putrescine. The YgjG crystal structures provide structural clues to putrescine aminotransferase substrate specificity and binding.

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Rescue of Deleterious Mutations by the Compensatory Y30F Mutation in Ketosteroid Isomerase

Cited by 10 publications

References 32 publications

Crystal structure of SgcJ, an NTF2-like superfamily protein involved in biosynthesis of the nine-membered enediyne antitumor antibiotic C-1027

Crystal structure of SgcJ, an NTF2-like superfamily protein involved in biosynthesis of the nine-membered enediyne antitumor antibiotic C-1027

Contribution of a Low-Barrier Hydrogen Bond to Catalysis Is Not Significant in Ketosteroid Isomerase

Structure of Putrescine Aminotransferase from Escherichia coli Provides Insights into the Substrate Specificity among Class III Aminotransferases

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