19 research outputs found
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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
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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 Ă
resolution crystal structure of ValA in complex with NADâș and ZnÂČâș 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 NADâș 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 â 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 ÎČ pyranose anomer of the substrate
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Structure and proposed mechanism of L-α-glycerophosphate oxidase from Mycoplasma pneumoniae
The formation of hydrogen peroxide (HâOâ) 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 Ă
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 minâ»Âč 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
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De novo synthesis of a sunscreen compound in vertebrates
Ultraviolet-protective compounds, such as mycosporine-like amino acids (MAAs) and related gadusols produced by some bacteria, fungi, algae, and marine invertebrates, are critical for the survival of reef-building corals and other marine organisms exposed to high-solar irradiance. These compounds have also been found in marine fish, where their accumulation is thought to be of dietary or symbiont origin. In this study, we report the unexpected discovery that fish can synthesize gadusol de novo and that the analogous pathways are also present in amphibians, reptiles, and birds. Furthermore, we demonstrate that engineered yeast containing the fish genes can produce and secrete gadusol. The discovery of the gadusol pathway in vertebrates provides a platform for understanding its role in these animals, and the possibility of engineering yeast to efficiently produce a natural sunscreen and antioxidant presents an avenue for its large-scale production for possible use in pharmaceuticals and cosmetics.This is the publisherâs final pdf. The published article is copyrighted by the author(s) and published by eLife Sciences Publications. The published article can be found at: http://elifesciences.org/Supporting information can be found at: http://elifesciences.org/content/4/e0591
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Structural Insights into Novel Sugar Phosphate Cyclases and Flavoenzymes
The function of a protein is defined by its three-dimensional structure, and in understanding the three-dimensional structure of a protein, we gain an understanding of its function and mechanism. Protein structures, especially at high resolution, can provide detailed insights into many elements of enzyme function and catalysis â identifying residues directly involved in binding ligand or in carrying out catalysis, illuminating factors involved in promoting catalysis, and revealing subtleties which allow functionally and/or structurally similar enzymes to carry out distinct chemistries.
This dissertation presents work aimed at the functional and structural characterization of two types of proteins: sedoheptulose 7-phosphate cyclases (SH7PCs) and flavoenzymes. Six chapters of original work are presented in this dissertation and include one review and five primary research reports. The first three chapters (Chapters 2-4) focus on SH7PCs, and the second three chapters (Chapters 5-7) focus on flavoenzymes. All but the last of these chapters are published; the final original research chapter on lactate monooxygenase is at the stage of a manuscript in preparation for submission. These central chapters are bookended by a brief introduction to SH7PCs and flavoenzymes (Chapter 1) and concluding remarks on the main highlights and impacts of this work as well as future directions (Chapter 8).
With regard to the work on SH7PCs, I report the first structures of any SH7PC (Chapters 3 and 4). The crystal structures show these enzymes are structurally similar to each other and to other, closely related sugar phosphate cyclases, in particular dehydroquinate synthase (DHQS). These structures reveal subtle but informative differences between the three members of the SH7PC family â 2-epi-5-epi-valiolone synthase (EEVS), desmethyl-4-deoxygadusol synthase (DDGS), and 2-epi-valiolone synthase (EVS) â and suggest how these enzymes all utilize the same substrate to specifically generate one of three distinct products. The review (Chapter 2) provides an overview of the state of our understanding of SH7PCs, including the structural work reported in this thesis, and gives a broader context for the roles, evolution, and prevalence of these enzymes in nature. Based on structural insights, I propose a novel anomer selection hypothesis to differentiate two of these enzymes, EEVS and DDGS, from the third, EVS, based on which anomer of the substrate, sedoheptulose 7-phosphate, the enzyme selectively binds (Chapter 3). Furthermore, these structures allowed for corrections and additions to the âfingerprintâ used to identify and differentiate SH7PCs and DHQS, which further guided genome mining (Chapter 4).
With regard to the work on flavoenzymes, enzymes utilizing either FAD or FMN to carry out their unique chemistries, I report studies that have advanced our understanding of enzyme families represented by three different proteins: glycerol 3-phosphate oxidase from Mycoplasma pneumoniae (MpGlpO), ferredoxin-NADP+ reductase from corn root (FNR), and lactate monooxygenase from Mycobacterium smegmatis (LMO).
In facilitating glycerol metabolism and producing hydrogen peroxide as a byproduct, glycerol 3-phosphate oxidase (GlpO), which converts glycerol 3-phosphate to dihydroxyacetone phosphate, is implicated as a pathogenicity factor and a potential drug target in pathogens such as Mycoplasma pneuomniae. I report the structure of MpGlpO (Chapter 5). Sequence and structural comparisons of MpGlpO to other structurally known glycerol 3-phosphate oxidases and dehydrogenases (GlpO/DHs) reveal there are two distinct types of GlpO/DHs: Type I GlpO/DHs, including mitochondrial GlpDHs and Streptococcus GlpO, and Type II GlpO/DHs, of which MpGlpO is representative. Guided by a liganded structure of a close homolog for which the structure was solved as part of the Protein Structure Initiative, I proposed the first plausible binding mode of the glycerol 3-phosphate substrate and a detailed catalytic mechanism that arguably will apply to all GlpO/DHs, despite the distinct differences in the composition of their active sites.
The FAD-dependent enzyme FNR catalyzes the transfer of electrons from photoreduced ferredoxin to NADP+ during photosynthesis and serves as a model for a broad superfamily of enzymes including NO synthase, cytochrome P450 reductase, and NADPH oxidases. Using a variant replacing a conserved aromatic amino acid to capture the productive binding mode of the nicotinamide portion of NADP(H), we analyzed a suite of high resolution structures (~1.5 Ă
) in complex with nicotinamide, NADP+, and NADPH. A reinterpretation of previous kinetic data also supports the relevance of these complexes to catalysis. Based on these high resolution structures, we further report insights into factors promoting hydride transfer in FNR and other FNR-like superfamily members. Specifically, we infer that higher anisotropic mobility of the C4 atom of NADP+ compared to NADPH, distortion of FAD geometry from planarity, and a tightly packed active site implicate significant active site compression as a factor promoting hydride transfer. A broadly relevant conclusion of this work is the recognition of active site compression as an important and general â although often overlooked and underappreciated â factor that can promote catalysis.
LMO is an FMN-dependent enzyme that catalyzes the conversion of lactate to acetate, carbon dioxide, and water. LMO is part of a family of α-hydroxy acid oxidases, all of which carry out the same oxidation chemistry, but unlike LMO, proceed through an uncoupled pathway. I report the first structure of an LMO (Chapter 7). This structure reveals details of the LMO active site and provides new insights as to how LMO kinetically and functionally deviates from the other family members by proceeding along a coupled reaction pathway. A highly mobile, variable loop (âloop 4â) known to seal the active site in these α-hydroxy acid oxidases is significantly larger in LMO than in the other enzymes in the family, and has both more compact folding and greater buried surface area. We suggest it is the dynamics of this loop that governs the kinetics of intermediate release (or lack thereof) in these enzymes
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 PHACâs (2001) Project Evaluation to assess success of the
project
Correction to Structure of a Sedoheptulose 7-Phosphate Cyclase: ValA from Streptomyces hygroscopicus
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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 Ă
resolution crystal structure of ValA in complex with NADâș and ZnÂČâș 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 NADâș 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 â 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 ÎČ pyranose anomer of the substrate
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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 Ă
resolution crystal structure of ValA in complex with NADâș and ZnÂČâș 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 NADâș 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 â 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 ÎČ pyranose anomer of the substrate