Unprecedented sulfur transfer strategy in ergothioneine and ovothiol biosyntheses

Ergothioneine, a histidine-derived thiol, protects cells against reactive oxygen species and is emerging as a longevity vitamin. Ovothiol, another histidine-derived thiol, is also a potent antioxidant with therapeutic potential due to its anti-inflammatory and anti-proliferative activities. Despite these promising health benefits, the production of ergothioneine is limited by the underlying challenges of its only industrial synthetic method, while ovothiol is not commercially available. Due to these issues, the production of these thiols through metabolic engineering/synthetic biology approaches is appealing. The central steps in the ergothioneine and ovothiol biosynthetic pathways are the oxidative coupling C-S bond formation reaction mediated by non-heme iron sulfoxide synthases, and the pyridoxal-5'-phosphate (PLP)-dependent C-S lyases. This sulfur transfer strategy differs from all other pathways reported. Therefore, these trans-sulfuration reactions in ergothioneine and ovothiol biosyntheses are significant from both basic and translational research perspectives, hence, they were selected as my thesis project. This thesis comprises of five chapters. Sulfur metabolism and the biosynthesis of sulfur-containing natural products are presented in Chapter 1. The computational-guided protein engineering of a thermophilic sulfoxide synthase (EgtB) from Chloracidobacterium thermophiluim is covered in Chapter 2. Chapter 3 describes the mechanistic studies of the reductive C-S lyase (Egt2 from the Neurospora crassa’s ergothioneine biosynthesis), which revealed the involvement of a sulfenic acid intermediate in this reaction. In addition to reconstituting the ergothioneine biosynthetic pathway in vitro presented in Chapter 3, I fully reconstituted the in vitro ovothiol A biosynthetic pathway from Erwinia tasmaniensis, which is described in Chapter 4. In Chapter 5, the mechanistic studies of the ovothiol sulfoxide synthase OvoA using unnatural amino acid incorporation via amber-codon suppression are discussed. The success of this thesis work paves the way for the industrial production of ergothioneine and ovothiol through metabolic engineering/synthetic biology approaches. This study has also laid the foundation for future in-depth mechanistic characterization of these novel enzymes

Hybrid plasmonic photoreactors as visible light-mediated bactericides

Photocatalytic compounds and complexes, such as tris(bipyridine)ruthenium(II), [Ru(bpy)3]2+, have recently attracted attention as light-mediated bactericides that can help to address the need for new antibacterial strategies. We demonstrate in this work that the bactericidal efficacy of [Ru(bpy)3]2+ and the control of its antibacterial function can be significantly enhanced through combination with a plasmonic nanoantenna. We report strong, visible light-controlled bacterial inactivation with a nanocomposite design that incorporates [Ru(bpy)3]2+ as a photocatalyst and a Ag nanoparticle (NP) core as a light-concentrating nanoantenna into a plasmonic hybrid photoreactor. The hybrid photoreactor platform is facilitated by a self-assembled lipid membrane that encapsulates the Ag NP and binds the photocatalyst. The lipid membrane renders the nanocomposite biocompatible in the absence of resonant illumination. Upon illumination, the plasmon-enhanced photoexcitation of the metal-to-ligand charge-transfer band of [Ru(bpy)3]2+ prepares the reactive excited state of the complex that oxidizes the nanocomposite membrane and increases its permeability. The photooxidation induces the release of [Ru(bpy)3]2+, Ag+, and peroxidized lipids into the ambient medium, where they interact synergistically to inactivate bacteria. We measured a 7 order of magnitude decrease in Gram-positive Arthrobacter sp. and a 4 order of magnitude decrease in Gram-negative Escherichia coli colony forming units with the photoreactor bactericides after visible light illumination for 1 h. In both cases, the photoreactor exceeds the bactericidal standard of a log reduction value of 3 and surpasses the antibacterial effect of free Ag NPs or [Ru(bpy)3]2+ by >4 orders of magnitude. We also implement the inactivation of a bacterial thin film in a proof-of-concept study.Accepted manuscrip

Recent examples of α-ketoglutarate-dependent mononuclear non-haem iron enzymes in natural product biosyntheses

Covering: up to 2018 α-Ketoglutarate (αKG, also known as 2-oxoglutarate)-dependent mononuclear non-haem iron (αKG-NHFe) enzymes catalyze a wide range of biochemical reactions, including hydroxylation, ring fragmentation, C-C bond cleavage, epimerization, desaturation, endoperoxidation and heterocycle formation. These enzymes utilize iron(ii) as the metallo-cofactor and αKG as the co-substrate. Herein, we summarize several novel αKG-NHFe enzymes involved in natural product biosyntheses discovered in recent years, including halogenation reactions, amino acid modifications and tailoring reactions in the biosynthesis of terpenes, lipids, fatty acids and phosphonates. We also conducted a survey of the currently available structures of αKG-NHFe enzymes, in which αKG binds to the metallo-centre bidentately through either a proximal- or distal-type binding mode. Future structure-function and structure-reactivity relationship investigations will provide crucial information regarding how activities in this large class of enzymes have been fine-tuned in nature.R01 GM093903 - NIGMS NIH HHSAccepted manuscrip

Chemical modifications of proteins and their applications in metalloenzyme studies

Protein chemical modifications are important tools for elucidating chemical and biological functions of proteins. Several strategies have been developed to implement these modifications, including enzymatic tailoring reactions, unnatural amino acid incorporation using the expanded genetic codes, and recognition-driven transformations. These technologies have been applied in metalloenzyme studies, specifically in dissecting their mechanisms, improving their enzymatic activities, and creating artificial enzymes with non-natural activities. Herein, we summarize some of the recent efforts in these areas with an emphasis on a few metalloenzyme case studies.Published versio

Implications for an imidazol-2-yl carbene intermediate in the rhodanase-catalyzed C-S bond formation reaction of anaerobic ergothioneine biosynthesis

In the anaerobic ergothioneine biosynthetic pathway, a rhodanese domain containing enzyme (EanB) activates tne hercynine's sp2 ε-C-H Dona ana replaces it with a C-S bond to produce ergothioneine. The key intermediate for this trans-sulfuration reaction is the Cys412 persulfide. Substitution of the EanB-Cys412 persulfide with a Cys412 perselenide does not yield the selenium analog of ergothioneine, selenoneine. However, in deuterated buffer, the perselenide-modified EanB catalyzes the deuterium exchange between hercynine's sp2 ε-C-H bond and D2O. Results from QM/MM calculations suggest that the reaction involves a carbene intermediate and that Tyr353 plays a key role. We hypothesize that modulating the pKa of Tyr353 will affect the deuterium-exchange rate. Indeed, the 3,5-difluoro tyrosine containing EanB catalyzes the deuterium exchange reaction with k ex of ~10-fold greater than the wild-type EanB (EanBWT). With regards to potential mechanisms, these results support the involvement of a carbene intermediate in EanB-catalysis, rendering EanB as one of the few carbene-intermediate involving enzymatic systems.R01 GM106443 - NIGMS NIH HHSAccepted manuscrip

OvoAMtht from Methyloversatilis thermotolerans ovothiol biosynthesis is a bifunction enzyme: thiol oxygenase and sulfoxide synthase activities

Mononuclear non-heme iron enzymes are a large class of enzymes catalyzing a wide-range of reactions. In this work, we report that a non-heme iron enzyme in Methyloversatilis thermotolerans, OvoAMtht, has two different activities, as a thiol oxygenase and a sulfoxide synthase. When cysteine is presented as the only substrate, OvoAMtht is a thiol oxygenase. In the presence of both histidine and cysteine as substrates, OvoAMtht catalyzes the oxidative coupling between histidine and cysteine (a sulfoxide synthase). Additionally, we demonstrate that both substrates and the active site iron's secondary coordination shell residues exert exquisite control over the dual activities of OvoAMtht (sulfoxide synthase vs. thiol oxygenase activities). OvoAMtht is an excellent system for future detailed mechanistic investigation on how metal ligands and secondary coordination shell residues fine-tune the iron-center electronic properties to achieve different reactivities.R35 GM136294 - NIGMS NIH HHSPublished versio

Dissecting the mechanism of the nonheme iron endoperoxidase FtmOx1 using substrate analogues

FtmOx1 is a nonheme iron (NHFe) endoperoxidase, catalyzing three disparate reactions, endoperoxidation, alcohol dehydrogenation, and dealkylation, under in vitro conditions; the diversity complicates its mechanistic studies. In this study, we use two substrate analogues to simplify the FtmOx1-catalyzed reaction to either a dealkylation or an alcohol dehydrogenation reaction for structure-function relationship analysis to address two key FtmOx1 mechanistic questions: (1) Y224 flipping in the proposed COX-like model vs α-ketoglutarate (αKG) rotation proposed in the CarC-like mechanistic model and (2) the involvement of a Y224 radical (COX-like model) or a Y68 radical (CarC-like model) in FtmOx1-catalysis. When 13-oxo-fumitremorgin B (7) is used as the substrate, FtmOx1-catalysis changes from the endoperoxidation to a hydroxylation reaction and leads to dealkylation. In addition, consistent with the dealkylation side-reaction in the COX-like model prediction, the X-ray structure of the FtmOx1•CoII•αKG•7 ternary complex reveals a flip of Y224 to an alternative conformation relative to the FtmOx1•FeII•αKG binary complex. Verruculogen (2) was used as a second substrate analogue to study the alcohol dehydrogenation reaction to examine the involvement of the Y224 radical or Y68 radical in FtmOx1-catalysis, and again, the results from the verruculogen reaction are more consistent with the COX-like model.R24 GM134210 - NIGMS NIH HHSPublished versio

Retraction note: endoperoxide formation by an α-ketoglutarate-dependent mononuclear non-haem iron enzyme

Accepted manuscrip

Single-step replacement of an unreactive C-H bond by a C-S bond using polysulfide as the direct sulfur source in anaerobic ergothioneine biosynthesis

Ergothioneine, a natural longevity vitamin and antioxidant, is a thiol-histidine derivative. Recently, two types of biosynthetic pathways were reported. In the aerobic ergothioneine biosynthesis, a non-heme iron enzyme incorporates a sulfoxide to an sp2 C-H bond in trimethyl-histidine (hercynine) through oxidation reactions. In contrast, in the anaerobic ergothioneine biosynthetic pathway in a green sulfur bacterium, Chlorobium limicola, a rhodanese domain containing protein (EanB) directly replaces this unreactive hercynine C-H bond with a C-S bond. Herein, we demonstrate that polysulfide (HSSnSR) is the direct sulfur-source in EanB-catalysis. After identifying EanB's substrates, X-ray crystallography of several intermediate states along with mass spectrometry results provide additional mechanistic details for this reaction. Further, quantum mechanics/molecular mechanics (QM/MM) calculations reveal that protonation of Nπ of hercynine by Tyr353 with the assistance of Thr414 is a key activation step for the hercynine sp2 C-H bond in this trans-sulfuration reaction.R01 GM106443 - NIGMS NIH HHS; R41 AT010878 - NCCIH NIH HHSAccepted manuscrip

Use of a tyrosine analogue to modulate the two activities of a nonheme iron enzyme OvoA in ovothiol biosynthesis, cysteine oxidation versus oxidative C-S bond formation

Ovothiol is a histidine thiol derivative. The biosynthesis of ovothiol involves an extremely efficient trans-sulfuration strategy. The nonheme iron enzyme OvoA catalyzed oxidative coupling between cysteine and histidine is one of the key steps. Besides catalyzing the oxidative coupling between cysteine and histidine, OvoA also catalyzes the oxidation of cysteine to cysteine sulfinic acid (cysteine dioxygenase activity). Thus far, very little mechanistic information is available for OvoA-catalysis. In this report, we measured the kinetic isotope effect (KIE) in OvoA-catalysis using the isotopically sensitive branching method. In addition, by replacing an active site tyrosine (Tyr417) with 2-amino-3-(4-hydroxy-3-(methylthio)phenyl)propanoic acid (MtTyr) through the amber suppressor mediated unnatural amino acid incorporation method, the two OvoA activities (oxidative coupling between cysteine and histidine, and cysteine dioxygenase activity) can be modulated. These results suggest that the two OvoA activities branch out from a common intermediate and that the active site tyrosine residue plays some key roles in controlling the partitioning between these two pathways.R01 GM093903 - NIGMS NIH HHSAccepted manuscrip