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

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 alpha-hydroxy acid oxidase family members follow an "uncoupled pathway," wherein the alpha-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 angstrom and 1.7 angstrom resolution, respectively. The overall LMO fold and active site organization, including a bound sulfate mimicking substrate, resemble those of other alpha-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 alpha-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 alpha-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

Structure of a Sedoheptulose 7-Phosphate Cyclase: ValA from Streptomyces hygroscopicus

Sedoheptulose 7-phosphate cyclases (SH7PCs) encompass three enzymes involved in producing the core cyclitol structures of pseudoglycosides and similar bioactive natural products. One such enzyme is ValA from Streptomyces hygroscopicus subsp. jinggangensis 5008 which makes 2-epi-5-epi-valiolone as part of the biosynthesis of the agricultural antifungal agent validamycin A. We present, as the first SH7PC structure, the 2.1 Å resolution crystal structure of ValA in complex with NAD⁺ and Zn²⁺ cofactors. ValA has a fold and active site organization resembling the sugar phosphate cyclase dehydroquinate synthase (DHQS) and contains two notable, previously unrecognized interactions between NAD⁺ and Asp side chains conserved in all sugar phosphate cyclases that may influence catalysis. Because the domains of ValA adopt a nearly closed conformation even though no sugar substrate is present, comparisons with a ligand-bound DHQS provide a model for aspects of substrate binding. One striking active site difference is a loop that adopts a distinct conformation as a result of an Asp → Asn change with respect to DHQS and alters the identity and orientation of a key Arg residue. This and other active site differences in ValA are mostly localized to areas where the ValA substrate differs from that of DHQS. Sequence comparisons with a second SH7PC making a product with distinct stereochemistry lead us to postulate that the product stereochemistry of a given SH7PC is not the result of events taking place during catalysis, but is accomplished by selective binding of either the α or β pyranose anomer of the substrate

Structure and proposed mechanism of L-α-glycerophosphate oxidase from Mycoplasma pneumoniae

The formation of hydrogen peroxide (H₂O₂) by the FAD-dependent α-glycerophosphate oxidase (GlpO), is important for the pathogenesis of Streptococcus pneumoniae and Mycoplasma pneumoniae. The structurally known GlpO from Streptococcus sp. (SspGlpO) is similar to the pneumococcal protein (SpGlpO) and provides a guide for drug design against that target. However, M. pneumoniae GlpO (MpGlpO), having <20% sequence identity with structurally known GlpOs, appears to represent a second type of GlpO we designate as Type II GlpOs. Here, the recombinant His-tagged MpGlpO structure is described at ~2.5 Å resolution, solved by molecular replacement using as a search model the Bordetella pertussis protein 3253 (Bp3253) a protein of unknown function solved by structural genomics efforts. Recombinant MpGlpO is an active oxidase with a turnover number of ~580 min⁻¹ while Bp3253 showed no GlpO activity. No substantial differences exist between the oxidized and dithionite-reduced MpGlpO structures. Although, no liganded structures were determined, a comparison with the tartrate-bound Bp3253 structure and consideration of residue conservation patterns guided the construction of a model for α-glycerophosphate (Glp) recognition and turnover by MpGlpO. The predicted binding mode also appears relevant for the type I GlpOs (such as SspGlpO) despite differences in substrate recognition residues, and it implicates a histidine conserved in type I and II Glp oxidases and dehydrogenases as the catalytic acid/base. This work provides a solid foundation for guiding further studies of the mitochondrial Glp dehydrogenases as well as for continued studies of M. pneumoniae and S. pneumoniae glycerol metabolism and the development of novel therapeutics targeting MpGlpO and SpGlpO.Keywords: drug design, flavoenzyme, protein evolution, GlpA, hydride transfe

Optimizing well-pregnancy care on Prince Edward Island

While pregnancy is a normal, transitional life event many women experience, the need exists for competent, holistic health care during this time in their lives. On Prince Edward Island, 90% of perinatal care is administered by obstetricians following a medical model of care; this is 30% greater than the national average (Public Health Agency of Canada [PHAC], 2009). Pregnancy outcomes on P.E.I., in regards to folic acid supplementation, high pre-pregnancy and pregnancy body mass indexes (BMI), overweight or obese expectant mothers, substance use, and breastfeeding consistently fall outside national averages (P.E.I. Reproductive Care Program [P.E.I. RCP], 2008; PHAC, 2009, 2012). An initiative to implement nurse practitioner-led perinatal care for healthy pregnant women can help to address these outcomes by ensuring appropriate ongoing physical assessment, addressing psychosocial needs, and increasing perinatal education to women and their families during the preconception, prenatal, and postpartum periods. The initiative will include communication to key stakeholders, and a timeline of planned activities and will follow the PHAC’s (2001) Project Evaluation to assess success of the project

KarplusPAndrewBiochemistryBiophysicsStructureSedoheptulose7-Phosphate.pdf

Sedoheptulose 7-phosphate cyclases (SH7PCs) encompass three enzymes involved in producing the core cyclitol structures of pseudoglycosides and similar bioactive natural products. One such enzyme is ValA from Streptomyces hygroscopicus subsp. jinggangensis 5008 which makes 2-epi-5-epi-valiolone as part of the biosynthesis of the agricultural antifungal agent validamycin A. We present, as the first SH7PC structure, the 2.1 Å resolution crystal structure of ValA in complex with NAD⁺ and Zn²⁺ cofactors. ValA has a fold and active site organization resembling the sugar phosphate cyclase dehydroquinate synthase (DHQS) and contains two notable, previously unrecognized interactions between NAD⁺ and Asp side chains conserved in all sugar phosphate cyclases that may influence catalysis. Because the domains of ValA adopt a nearly closed conformation even though no sugar substrate is present, comparisons with a ligand-bound DHQS provide a model for aspects of substrate binding. One striking active site difference is a loop that adopts a distinct conformation as a result of an Asp → Asn change with respect to DHQS and alters the identity and orientation of a key Arg residue. This and other active site differences in ValA are mostly localized to areas where the ValA substrate differs from that of DHQS. Sequence comparisons with a second SH7PC making a product with distinct stereochemistry lead us to postulate that the product stereochemistry of a given SH7PC is not the result of events taking place during catalysis, but is accomplished by selective binding of either the α or β pyranose anomer of the substrate

KarplusPAndrewBiochemistryBiophysicsStructureSedoheptulose7-Phosphate(AdditionCorrection).pdf

Sedoheptulose 7-phosphate cyclases (SH7PCs) encompass three enzymes involved in producing the core cyclitol structures of pseudoglycosides and similar bioactive natural products. One such enzyme is ValA from Streptomyces hygroscopicus subsp. jinggangensis 5008 which makes 2-epi-5-epi-valiolone as part of the biosynthesis of the agricultural antifungal agent validamycin A. We present, as the first SH7PC structure, the 2.1 Å resolution crystal structure of ValA in complex with NAD⁺ and Zn²⁺ cofactors. ValA has a fold and active site organization resembling the sugar phosphate cyclase dehydroquinate synthase (DHQS) and contains two notable, previously unrecognized interactions between NAD⁺ and Asp side chains conserved in all sugar phosphate cyclases that may influence catalysis. Because the domains of ValA adopt a nearly closed conformation even though no sugar substrate is present, comparisons with a ligand-bound DHQS provide a model for aspects of substrate binding. One striking active site difference is a loop that adopts a distinct conformation as a result of an Asp → Asn change with respect to DHQS and alters the identity and orientation of a key Arg residue. This and other active site differences in ValA are mostly localized to areas where the ValA substrate differs from that of DHQS. Sequence comparisons with a second SH7PC making a product with distinct stereochemistry lead us to postulate that the product stereochemistry of a given SH7PC is not the result of events taking place during catalysis, but is accomplished by selective binding of either the α or β pyranose anomer of the substrate

Systematic Variation of Both the Aromatic Cage and Dialkyllysine via GCE-SAR Reveal Mechanistic Insights in CBX5 Reader Protein Binding

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

Dataset for: High resolution studies of hydride transfer in the ferredoxin:NADP+ reductase superfamily

Ferredoxin-NADP+ reductase (FNR) is an FAD-containing enzyme best known for catalyzing 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 catalyze 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 favor 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