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Development of inhibitors for histone methyllysine reader proteins is an active area of research due to the importance of reader protein-methyllysine interactions in transcriptional regulation and disease. Optimized peptide-based chemical probes targeting methyllysine readers favor larger alkyllysine residues in place of methyllysine. However, the mechanism by which these larger substituents drive tighter binding is not well understood. This study describes the development of a two-pronged approach combining genetic code expansion (GCE) and structure-activity relationships (SAR) through systematic variation of both the aromatic binding pocket in the protein and the alkyllysine residues in the peptide to probe inhibitor recognition in the CBX5 chromodomain. We demonstrate a novel change in driving force for larger alkyllysines, which weaken cation-π interactions but increases dispersion forces, resulting in tighter binding. This GCE-SAR approach establishes discrete energetic contributions to binding from both ligand and protein, providing a powerful tool to gain mechanistic understanding of SAR trends.

The content and design scheme for a readily adaptable, research-based laboratory course in chemical biology are presented. In this course, students interrogate protein structure and function using the site-specific incorporation of noncanonical amino acids by genetic code expansion. The relatively new field of genetic code expansion enables protein engineering with a diverse array of chemical functional groups. In this quarter or semester research-based undergraduate laboratory experience, student teams design, synthesize, and evaluate the structure-function relationships of proteins containing noncanonical amino acids on the basis of a self-selected hypothesis and then communicate their results in a formal manuscript and research presentation. The flexibility and versatility of genetic code expansion and this course structure empower students to engineer novel biomolecules and highlight the use of organic-chemistry principles for interrogating proteins. Generating noncanonical protein variants that have never been produced previously exposes students to novel research and trains them in essential basic biochemistry skills. This research-based undergraduate laboratory course can easily be adapted, scaled, and implemented on the basis of the interests, demographics, and resources of a given institution.

Lactate monooxygenase (LMO) catalyzes the FMN‐dependent "coupled" oxidation of lactate and O2 to 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 of Mycobacterium smegmatis wild‐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 b2 and 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 genus Beauveria. Broad statement of impact: 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 O2 to 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. PDB Code(s): 6DVI, 6DVH [ABSTRACT FROM AUTHOR]

Ferredoxin: NADP+ reductase ( FNR) is an FAD-containing enzyme best known for catalysing the transfer of electrons from ferredoxin (Fd) to NADP+ to make NADPH during photosynthesis. It is also the prototype for a broad enzyme superfamily, including the NADPH oxidases ( NOXs) that all catalyse similar FAD-enabled electron transfers between NAD(P)H and one-electron carriers. Here, we define further mechanistic details of the NAD(P)H ⇌ FAD hydride-transfer step of the reaction based on spectroscopic studies and high-resolution (~ 1.5 Å) crystallographic views of the nicotinamide-flavin interaction in crystals of corn root FNR Tyr316Ser and Tyr316Ala variants soaked with either nicotinamide, NADP+, or NADPH. The spectra obtained from FNR crystal complexes match those seen in solution and the complexes reveal active site packing interactions and patterns of covalent distortion of the FAD that imply significant active site compression that would favour catalysis. Furthermore, anisotropic B-factors show that the mobility of the C4 atom of the nicotinamide in the FNR: NADP+ complex has a directionality matching that expected for boat-like excursions of the nicotinamide ring thought to enhance hydride transfer. Arguments are made for the relevance of this binding mode to catalysis, and specific consideration is given to how the results extrapolate to provide insight to structure-function relations for the membrane-bound NOX enzymes for which little structural information has been available. Databases Structural data are available in the PDB database under the accession numbers (wild-type), [Y316S:nicotinamide (P3221)], [Y316S:nicotinamide (P3121)], [Y316S:NADP+ (P3221)], [Y316S:NADP+ (P3121)], [Y316S:NADPH (P3221)], [Y316A:nicotinamide (P3221)], [Y316A:NADP+ (P3221)], [Y316A:NADPH (P3221)], [Y316F (P3221)], and [Y316F:NADP+ (P3121)]. Enzyme Commission number: ferredoxin:NADP+ reductase - . [ABSTRACT FROM AUTHOR]

Methylation of arginine (Arg) residues on histones creates a new binding epitope, enabling recognition by aromatic cage binding pockets in Tudor domains; these protein-protein interactions (PPIs) govern gene expression. Despite their biological importance, the molecular details of methylated Arg recognition are poorly understood. While the desolvation, hydrogen bonding, and guanidinium stacking of methylated Arg have been explored in model systems and proposed to contribute to binding, direct interactions between the methyl groups and the aromatic residues in the binding pocket have not previously been investigated. Herein, we mechanistically study the CH 3 -π interactions between the SPIN1 triple Tudor domain and histone asymmetric dimethylarginine. We find that these CH 3 -π interactions are electrostatically tunable, exhibiting cation-π character, albeit attenuated relative to cation-π interactions with quaternary ammonium ions, offering key insight into how methylation of Arg alters its binding epitope to enable new PPIs.

Lactate monooxygenase (LMO) catalyzes the FMN-dependent "coupled" oxidation of lactate and O 2 to 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 of Mycobacterium smegmatis wild-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 b 2 and 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 genus Beauveria. BROAD STATEMENT OF IMPACT: 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 O 2 to 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., (© 2018 The Protein Society.)

2-Epi-5-epi-valiolone synthase (EEVS), a C 7 -sugar phosphate cyclase (SPC) homologous to 3-dehydroquinate synthase (DHQS), was discovered during studies of the biosynthesis of the C 7 N-aminocyclitol family of natural products. EEVS was originally thought to be present only in certain actinomycetes, but analyses of genome sequences showed that it is broadly distributed in both prokaryotes and eukaryotes, including vertebrates. Another SPC, desmethyl-4-deoxygadusol synthase (DDGS), was later discovered as being involved in the biosynthesis of mycosporine-like amino acid sunscreen compounds. Current database annotations are quite unreliable, with many EEVSs reported as DHQS, and most DDGSs reported as EEVS, DHQS, or simply hypothetical proteins. Here, we identify sequence features useful for distinguishing these enzymes, report a crystal structure of a representative DDGS showing the high similarity of the EEVS and DDGS enzymes, identify notable active site differences, and demonstrate the importance of two of these active site residues for catalysis by point mutations. Further, we functionally characterized two representatives of a distinct clade equidistant from known EEVS and known DDGS groups and show them to be authentic EEVSs. Moreover, we document and discuss the distribution of genes that encode EEVS and DDGS in various prokaryotes and eukaryotes, including pathogenic bacteria, plant symbionts, nitrogen-fixing bacteria, myxobacteria, cyanobacteria, fungi, stramenopiles, and animals, suggesting their broad potential biological roles in nature.