옥살산 생합성에 관여하는 효소 ObcA 와 Obc1의 구조와 생화학적 기능 분석

학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2017. 2. 이상기.In Burkholderia species, the quorum sensing-dependent production of oxalic acid is an indispensable process for bacterial growth during stationary phase. Oxalic acid produced plays a central role in maintaining the environmental pH, which counteracts inevitable population-collapsing alkaline toxicity in amino acid-based culture medium. In B. glumae, two enzymes are responsible for oxalic acid production. First, the oxalate biosynthetic component (Obc) A catalyzes the formation of a tetrahedral C6-CoA adduct from acetyl-CoA and oxaloacetate. Then the ObcB enzyme liberates three products from the C6-CoA adduct: oxalic acid, acetoacetate, and CoA. Interestingly, these two stepwise reactions are catalyzed by a single bifunctional enzyme, Obc1, from B. thailandensis and B. pseudomallei. Obc1 has an ObcA-like N-terminal domain and shows ObcB activity in its C-terminal domain, despite no sequence homology with ObcB. In this thesis, crystal structure and functional analysis of ObcA and Obc1 are reported, revealing structural and functional insights of oxalogenesis. Overall structure of ObcA and N-terminal domain of Obc1 exhibits (β/α)8-barrel fold, with a metal ion coordinated in its active site. In catalysis, substrate oxaloacetate serves as a nucleophile by forming an enolate intermediate mediated by Tyr residue as a general base, which then attacks the thioester carbonyl carbon of a second substrate acetyl-CoA to yield a tetrahedral adduct. In many reactions involving tetrahedral CoA intermediate, the presence of a negative charge in the intermediate leads to collapse of the intermediate, ejecting CoA moiety form the active site. However, the presence of the metal-coordination shell and absence of general acid(s) could produce an unusual tetrahedral CoA adduct as a stable product. Structure of C-terminal domain of Obc1 has an α/β hydrolase fold that contains a catalytic triad for oxalic acid production and a novel oxyanion hole distinct from the canonical HGGG motif in other α/β hydrolases. Functional analyses through mutagenesis studies suggest that His934 is an additional catalytic acid/base for its lyase activity and liberates two additional products, acetoacetate and CoA. These results provide structural and functional insights into bacterial oxalogenesisan example of the functional diversity of an enzyme to survive and adapt in the environment and divergent evolution of the α/β hydrolase fold, which has both hydrolase and lyase activity.Chapter I. Introduction 1 Chapter II. Structural and Functional Analysis of ObcA 13 Materials and Methods 14 Results 30 Discussion 66 Chapter III. Structural and Functional Analysis of Obc1 69 Materials and Methods 70 Results 82 Discussion 106 References 112 Abstract in Korean 116Docto

Spatio-spectral decomposition of complex eigenmodes in subwavelength nanostructures through transmission matrix analysis

Exploiting multiple near-field optical eigenmodes is an effective means of designing, engineering, and extending the functionalities of optical devices. However, the near-field optical eigenmodes of subwavelength plasmonic nanostructures are often highly multiplexed in both spectral and spatial distributions, making it extremely difficult to extract individual eigenmodes. We propose a novel mode analysis method that can resolve individual eigenmodes of subwavelength nanostructures, which are superimposed in conventional methods. A transmission matrix is constructed for each excitation wavelength by obtaining the near-field distributions for various incident angles, and through singular value decomposition, near-field profiles and energy spectra of individual eigenmodes are effectively resolved. By applying transmission matrix analysis to conventional electromagnetic simulations, we clearly resolved a set of orthogonal eigenmodes of single- and double-slot nanoantennas with a slot width of 20 nm. In addition, transmission matrix analysis leads to solutions that can selectively excite specific eigenmodes of nanostructures, allowing selective use of individual eigenmodes.11Nsciescopu

RNA polymerase II stalls on oxidative DNA damage via a torsion-latch mechanism involving lone pair-π and CH-π interactions.

Oxidation of guanine generates several types of DNA lesions, such as 8-oxoguanine (8OG), 5-guanidinohydantoin (Gh), and spiroiminodihydantoin (Sp). These guanine-derived oxidative DNA lesions interfere with both replication and transcription. However, the molecular mechanism of transcription processing of Gh and Sp remains unknown. In this study, by combining biochemical and structural analysis, we revealed distinct transcriptional processing of these chemically related oxidized lesions: 8OG allows both error-free and error-prone bypass, whereas Gh or Sp causes strong stalling and only allows slow error-prone incorporation of purines. Our structural studies provide snapshots of how polymerase II (Pol II) is stalled by a nonbulky Gh lesion in a stepwise manner, including the initial lesion encounter, ATP binding, ATP incorporation, jammed translocation, and arrested states. We show that while Gh can form hydrogen bonds with adenosine monophosphate (AMP) during incorporation, this base pair hydrogen bonding is not sufficient to hold an ATP substrate in the addition site and is not stable during Pol II translocation after the chemistry step. Intriguingly, we reveal a unique structural reconfiguration of the Gh lesion in which the hydantoin ring rotates ∼90° and is perpendicular to the upstream base pair planes. The perpendicular hydantoin ring of Gh is stabilized by noncanonical lone pair-π and CH-π interactions, as well as hydrogen bonds. As a result, the Gh lesion, as a functional mimic of a 1,2-intrastrand crosslink, occupies canonical -1 and +1 template positions and compromises the loading of the downstream template base. Furthermore, we suggest Gh and Sp lesions are potential targets of transcription-coupled repair

RNA polymerase II trapped on a molecular treadmill: Structural basis of persistent transcriptional arrest by a minor groove DNA binder.

Elongating RNA polymerase II (Pol II) can be paused or arrested by a variety of obstacles. These obstacles include DNA lesions, DNA-binding proteins, and small molecules. Hairpin pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA in a sequence-specific manner and induce strong transcriptional arrest. Remarkably, this Py-Im-induced Pol II transcriptional arrest is persistent and cannot be rescued by transcription factor TFIIS. In contrast, TFIIS can effectively rescue the transcriptional arrest induced by a nucleosome barrier. The structural basis of Py-Im-induced transcriptional arrest and why TFIIS cannot rescue this arrest remain elusive. Here we determined the X-ray crystal structures of four distinct Pol II elongation complexes (Pol II ECs) in complex with hairpin Py-Im polyamides as well as of the hairpin Py-Im polyamides-dsDNA complex. We observed that the Py-Im oligomer directly interacts with RNA Pol II residues, introduces compression of the downstream DNA duplex, prevents Pol II forward translocation, and induces Pol II backtracking. These results, together with biochemical studies, provide structural insight into the molecular mechanism by which Py-Im blocks transcription. Our structural study reveals why TFIIS fails to promote Pol II bypass of Py-Im-induced transcriptional arrest

Structural basis of transcription recognition of a hydrophobic unnatural base pair by T7 RNA polymerase

T7 RNA polymerase (RNAP) is widely used for synthesizing RNA molecules with synthetic modifications and unnatural base pairs (UBPs). Here, authors show the structural basis of how UBPs are recognized as template and substrate, providing mechanistic insights into UBP transcription by T7 RNAP

Time-Variable Chiroptical Vibrational Sum-Frequency Generation Spectroscopy of Chiral Chemical Solution

© 2021 American Chemical Society. All rights reserved.Vibrational sum-frequency generation (VSFG) spectroscopy, a surface-specific technique, was shown to be useful even for characterizing the vibrational optical activity of chiral molecules in isotropic bulk liquids. However, accurately determining the spectroscopic parameters is still challenging because of the spectral congestion of chiroptical VSFG peaks with different amplitudes and phases. Here, we show that a time-variable infrared-visible chiroptical three-wave-mixing technique can be used to determine the spectroscopic parameters of second-order vibrational response signals from chiral chemical liquids. For varying the delay time between infrared and temporally asymmetric visible laser pulses, we measure the chiral VSFG, achiral VSFG, and their interference spectra of bulk R-(+)-limonene liquid and perform a global fitting analysis for those time-variable spectra to determine their spectroscopic parameters accurately. We anticipate that this time-variable VSFG approach will be useful for developing nearly background-free chiroptical characterization techniques with enhanced spectral resolution.11Nsciescopu

3.1 Å structure of yeast RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion solved using streptavidin affinity grids

Despite significant advances in all aspects of single particle cryo-electron microscopy (cryo-EM), specimen preparation still remains a challenge. During sample preparation, macromolecules interact with the air-water interface, which often leads to detrimental effects such as denaturation or adoption of preferred orientations, ultimately hindering structure determination. Randomly biotinylating the protein of interest (for example, at its primary amines) and then tethering it to a cryo-EM grid coated with two-dimensional crystals of streptavidin (acting as an affinity surface) can prevent the protein from interacting with the air-water interface. Recently, this approach was successfully used to solve a high-resolution structure of a test sample, a bacterial ribosome. However, whether this method can be used for samples where interaction with the air-water interface has been shown to be problematic remains to be determined. Here we report a 3.1 Å structure of an RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion (Pol II EC(CPD)) solved using streptavidin grids. Our previous attempt to solve this structure using conventional sample preparation methods resulted in a poor quality cryo-EM map due to Pol II EC(CPD)'s adopting a strong preferred orientation. Imaging the same sample on streptavidin grids improved the angular distribution of its view, resulting in a high-resolution structure. This work shows that streptavidin affinity grids can be used to address known challenges posed by the interaction with the air-water interface

Mechanism of Rad26-assisted rescue of stalled RNA polymerase II in transcription-coupled repair.

Transcription-coupled repair is essential for the removal of DNA lesions from the transcribed genome. The pathway is initiated by CSB protein binding to stalled RNA polymerase II. Mutations impairing CSB function cause severe genetic disease. Yet, the ATP-dependent mechanism by which CSB powers RNA polymerase to bypass certain lesions while triggering excision of others is incompletely understood. Here we build structural models of RNA polymerase II bound to the yeast CSB ortholog Rad26 in nucleotide-free and bound states. This enables simulations and graph-theoretical analyses to define partitioning of this complex into dynamic communities and delineate how its structural elements function together to remodel DNA. We identify an allosteric pathway coupling motions of the Rad26 ATPase modules to changes in RNA polymerase and DNA to unveil a structural mechanism for CSB-assisted progression past less bulky lesions. Our models allow functional interpretation of the effects of Cockayne syndrome disease mutations

A unified Watson-Crick geometry drives transcription of six-letter expanded DNA alphabets by E. coli RNA polymerase.

Artificially Expanded Genetic Information Systems (AEGIS) add independently replicable unnatural nucleotide pairs to the natural G:C and A:T/U pairs found in native DNA, joining the unnatural pairs through alternative modes of hydrogen bonding. Whether and how AEGIS pairs are recognized and processed by multi-subunit cellular RNA polymerases (RNAPs) remains unknown. Here, we show that E. coli RNAP selectively recognizes unnatural nucleobases in a six-letter expanded genetic system. High-resolution cryo-EM structures of three RNAP elongation complexes containing template-substrate UBPs reveal the shared principles behind the recognition of AEGIS and natural base pairs. In these structures, RNAPs are captured in an active state, poised to perform the chemistry step. At this point, the unnatural base pair adopts a Watson-Crick geometry, and the trigger loop is folded into an active conformation, indicating that the mechanistic principles underlying recognition and incorporation of natural base pairs also apply to AEGIS unnatural base pairs. These data validate the design philosophy of AEGIS unnatural basepairs. Further, we provide structural evidence supporting a long-standing hypothesis that pair mismatch during transcription occurs via tautomerization. Together, our work highlights the importance of Watson-Crick complementarity underlying the design principles of AEGIS base pair recognition