More Like this

1. Structure and role for active site lid of lactate monooxygenase from Mycobacterium smegmatis

   https://doi.org/10.1002/pro.3506  

   Kean, Kelsey M. ; Karplus, P. Andrew ( October 2018 , Protein Science)

   Lactate monooxygenase (LMO) catalyzes the FMN‐dependent “coupled” oxidation of lactate and O₂to acetate, carbon dioxide, and water, involving pyruvate and hydrogen peroxide as enzyme‐bound intermediates. Other α‐hydroxy acid oxidase family members follow an “uncoupled pathway,” wherein the α‐keto acid product quickly dissociates before the reduced flavin reacts with oxygen. Here, we report the structures ofMycobacterium smegmatiswild‐type LMO and a wild‐type‐like C203A variant at 2.1 Å and 1.7 Å resolution, respectively. The overall LMO fold and active site organization, including a bound sulfate mimicking substrate, resemble those of other α‐hydroxy acid oxidases. Based on structural similarity, LMO is similarly distant from lactate oxidase, glycolate oxidase, mandelate dehydrogenase, and flavocytochrome b₂and is the first representative enzyme of its type. Comparisons with other α‐hydroxy acid oxidases reveal that LMO has a longer and more compact folded active site loop (Loop 4), which is known in related flavoenzymes to undergo order/disorder transitions to allow substrate/product binding and release. We propose that LMO's Loop 4 has an enhanced stability that is responsible for the slow product release requisite for the coupled pathway. We also note electrostatic features of the LMO active site that promote substrate binding. Whereas the physiological role of LMO remains unknown, we document what can currently be assessed of LMO's distribution in nature, including its unexpected occurrence, presumably through horizontal gene transfer, in halophilic archaea and in a limited group of fungi of the genusBeauveria.

   This first crystal structure of the FMN‐dependent α‐hydroxy acid oxidase family member lactate monooxygenase (LMO) reveals it has a uniquely large active site lid that we hypothesize is stable enough to explain the slow dissociation of pyruvate that leads to its “coupled” oxidation of lactate and O₂to produce acetate, carbon dioxide, and water. Also, the relatively widespread distribution of putative LMOs supports their importance and provides new motivation for their further study.

    

   more » « less
2. Cu( i )–O 2 oxidation reactions in a fluorinated all-O-donor ligand environment

   https://doi.org/10.1039/C8DT05028G  

   Brazeau, Sarah E. ; Doerrer, Linda H. ( April 2019 , Dalton Transactions)

   Investigation of Cu–O 2 oxidation reactivity is important in biological and anthropogenic chemistry. Zeolites are one of the most promising Cu/O based oxidation catalysts for development of industrial-scale CH 4 to CH 3 OH conversion. Their oxidation mechanisms are not well understood, however, highlighting the importance of the investigation of molecular Cu( i )–O 2 reactivity with O-donor complexes. Herein, we give an overview of the synthesis, structural properties, and O 2 reactivity of three different series of O-donor fluorinated Cu( i ) alkoxides: K[Cu(OR) 2 ], [(Ph 3 P)Cu(μ-OR) 2 Cu(PPh 3 )], and K[(R 3 P)Cu(pin F )], in which OR = fluorinated monodentate alkoxide ligands and pin F = perfluoropinacolate. This breadth allowed for the exploration of the influence of the denticity of the ligand, coordination number, the presence of phosphine, and K⋯F/O interactions on their O 2 reactivity. K⋯F/O interactions were required to activate O 2 in the monodentate-ligand-only family, whereas these connections did not affect O 2 activation in the bidentate complexes, potentially due to the presence of phosphine. Both families formed trisanionic, trinuclear cores of the form {Cu 3 (μ 3 -O) 2 } 3− . Intramolecular and intermolecular substrate oxidation were also explored and found to be influenced by the fluorinated ligand. Namely, {Cu 3 (μ 3 -O) 2 } 3− from K[Cu(OR) 2 ] could perform both monooxygenase reactivity and oxidase catalysis, whereas those from K[(R 3 P)Cu(pin F )] could only perform oxidase catalysis. 

   more » « less
3. Dual oxidase/oxygenase reactivity and resonance Raman spectra of {Cu 3 O 2 } moiety with perfluoro- t -butoxide ligands

   https://doi.org/10.1039/c9dt00516a  

   Brazeau, Sarah E. ; Norwine, Emily E. ; Hannigan, Steven F. ; Orth, Nicole ; Ivanović-Burmazović, Ivana ; Rukser, Dieter ; Biebl, Florian ; Grimm-Lebsanft, Benjamin ; Praedel, Gregor ; Teubner, Melissa ; et al ( May 2019 , Dalton Transactions)

   A Cu( i ) fully fluorinated O-donor monodentate alkoxide complex, K[Cu(OC 4 F 9 ) 2 ], was previously shown to form a trinuclear copper–dioxygen species with a {Cu 3 (μ 3 -O) 2 } core, T OC4F9 , upon reactivity with O 2 at low temperature. Herein is reported a significantly expanded kinetic and mechanistic study of T OC4F9 formation using stopped-flow spectroscopy. The T OC4F9 complex performs catalytic oxidase conversion of hydroquinone (H 2 Q) to benzoquinone (Q). T OC4F9 also demonstrated hydroxylation of 2,4-di- tert -butylphenolate (DBP) to catecholate, making T OC4F9 the first trinuclear species to perform tyrosinase (both monooxygenase and oxidase) chemistry. Resonance Raman spectra were also obtained for T OC4F9 , to our knowledge, the first such spectra for any T species. The mechanism and substrate reactivity of T OC4F9 are compared to those of its bidentate counterpart, T pinF , formed from K[Cu(pin F )(PR 3 )]. The monodentate derivative has both faster initial formation and more diverse substrate reactivity. 

   more » « less
4. Electronic structures and spectroscopic signatures of diiron intermediates generated by O 2 activation of nonheme iron( ii )–thiolate complexes

   https://doi.org/10.1039/d1dt02286e  

   Ekanayake, Danushka M. ; Pham, Dao ; Probst, Andrew L. ; Miller, Joshua R. ; Popescu, Codrina V. ; Fiedler, Adam T. ( October 2021 , Dalton Transactions)

   The activation of O 2 at thiolate–ligated iron( ii ) sites is essential to the function of numerous metalloenzymes and synthetic catalysts. Iron–thiolate bonds in the active sites of nonheme iron enzymes arise from either coordination of an endogenous cysteinate residue or binding of a deprotonated thiol-containing substrate. Examples of the latter include sulfoxide synthases, such as EgtB and OvoA, that utilize O 2 to catalyze tandem S–C bond formation and S -oxygenation steps in thiohistidine biosyntheses. We recently reported the preparation of two mononuclear nonheme iron–thiolate complexes (1 and 2) that serve as structural active-site models of substrate-bound EgtB and OvoA ( Dalton Trans. 2020, 49 , 17745–17757). These models feature monodentate thiolate ligands and tripodal N 4 ligands with mixed pyridyl/imidazolyl donors. Here, we describe the reactivity of 1 and 2 with O 2 at low temperatures to give metastable intermediates (3 and 4, respectively). Characterization with multiple spectroscopic techniques (UV-vis absorption, NMR, variable-field and -temperature Mössbauer, and resonance Raman) revealed that these intermediates are thiolate-ligated iron( iii ) dimers with a bridging oxo ligand derived from the four-electron reduction of O 2 . Structural models of 3 and 4 consistent with the experimental data were generated via density functional theory (DFT) calculations. The combined experimental and computational results illuminate the geometric and electronic origins of the unique spectral features of diiron( iii )-μ-oxo complexes with thiolate ligands, and the spectroscopic signatures of 3 and 4 are compared to those of closely-related diiron( iii )-μ-peroxo species. Collectively, these results will assist in the identification of intermediates that appear on the O 2 reaction landscapes of iron–thiolate species in both biological and synthetic environments. 

   more » « less
5. Genomic analysis of siderophore β-hydroxylases reveals divergent stereocontrol and expands the condensation domain family

   https://doi.org/10.1073/pnas.1903161116  

   Reitz, Zachary L. ; Hardy, Clifford D. ; Suk, Jaewon ; Bouvet, Jean ; Butler, Alison ( October 2019 , Proceedings of the National Academy of Sciences)

   Genome mining of biosynthetic pathways streamlines discovery of secondary metabolites but can leave ambiguities in the predicted structures, which must be rectified experimentally. Through coupling the reactivity predicted by biosynthetic gene clusters with verified structures, the origin of the β-hydroxyaspartic acid diastereomers in siderophores is reported herein. Two functional subtypes of nonheme Fe(II)/α-ketoglutarate–dependent aspartyl β-hydroxylases are identified in siderophore biosynthetic gene clusters, which differ in genomic organization—existing either as fused domains (IβH Asp ) at the carboxyl terminus of a nonribosomal peptide synthetase (NRPS) or as stand-alone enzymes (TβH Asp )—and each directs opposite stereoselectivity of Asp β-hydroxylation. The predictive power of this subtype delineation is confirmed by the stereochemical characterization of β-OHAsp residues in pyoverdine GB-1, delftibactin, histicorrugatin, and cupriachelin. The l - threo (2 S , 3 S ) β-OHAsp residues of alterobactin arise from hydroxylation by the β-hydroxylase domain integrated into NRPS AltH, while l - erythro (2 S , 3 R ) β-OHAsp in delftibactin arises from the stand-alone β-hydroxylase DelD. Cupriachelin contains both l - threo and l - erythro β-OHAsp, consistent with the presence of both types of β-hydroxylases in the biosynthetic gene cluster. A third subtype of nonheme Fe(II)/α-ketoglutarate–dependent enzymes (IβH His ) hydroxylates histidyl residues with l - threo stereospecificity. A previously undescribed, noncanonical member of the NRPS condensation domain superfamily is identified, named the interface domain, which is proposed to position the β-hydroxylase and the NRPS-bound amino acid prior to hydroxylation. Through mapping characterized β-OHAsp diastereomers to the phylogenetic tree of siderophore β-hydroxylases, methods to predict β-OHAsp stereochemistry in silico are realized. 

   more » « less