The structure of L-lactate oxidase from Mycobacterium smegmatis.
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ABSTRACT:
SUBMITTER: Sullivan PA
PROVIDER: S-EPMC1164910 | biostudies-other | 1977 Aug
REPOSITORIES: biostudies-other
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Project description:1. An original method was devised for purifying and crystallizing l-lactate oxidase from Mycobacterium smegmatis. 2. The crystalline enzyme exhibited a single protein component (S(0) (20,w) 14.7s) in the ultracentrifuge and also on electrophoresis on cellulose acetate strips. 3. The enzyme has a typical flavoprotein spectrum, and it was confirmed that the prosthetic group is FMN. 4. Preliminary studies indicated that the molecular weight is in the range 300000-400000. Since the minimum molecular weight was found to be 50000+/-3000, it was concluded that l-lactate oxidase contains 6-8moles of flavine/mole. 5. Other properties of the enzyme reported include the substrate specificity, an apparent K(m) for l-lactate, the effect of several inhibitors on the enzyme activity and the pH-activity curve.
| S-EPMC1187213 | biostudies-other
Project description: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 ?-hydroxy acid oxidase family members follow an "uncoupled pathway," wherein the ?-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 ?-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 ?-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 ?-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.
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Project description:The cydAB genes from Mycobacterium smegmatis have been cloned and characterized. The cydA and cydB genes encode the two subunits of a cytochrome bd oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes. The cydD and cydC genes located immediately downstream of cydB encode a putative ATP-binding cassette-type transporter. At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type M. smegmatis displayed spectral features that are characteristic of the gamma-proteobacterial type cytochrome bd oxidase. Inactivation of cydA or cydB by insertion of a kanamycin resistance marker resulted in loss of d-heme absorbance at 631 nm. The d-heme could be restored by transformation of the M. smegmatis cyd mutants with a replicating plasmid carrying the highly homologous cydABDC gene cluster from Mycobacterium tuberculosis. Inactivation of cydA had no effect on the ability of M. smegmatis to exit from stationary phase at 37 or 42 degrees C. The growth rate of the cydA mutant was tested under oxystatic conditions. Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 x 10(2) Pa of pO(2) or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 x 10(2) Pa of pO(2) or 0.5 to 1% air saturation). These observations were in accordance with the two- to threefold increase in cydAB gene expression observed upon reduction of the pO(2) of the growth medium from 21 to 0.5% air saturation and with the concomitant increase in d-heme absorbance in spectra of membranes isolated from wild-type M. smegmatis cultured at 1% air saturation. Finally, the cydA mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat. In conjunction with these findings, our results suggest that cytochrome bd is an important terminal oxidase in M. smegmatis.
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Project description:Arabinosyltransferase B (EmbB) belongs to a family of membrane-bound glycosyltransferases that build the lipidated polysaccharides of the mycobacterial cell envelope, and are targets of anti-tuberculosis drug ethambutol. We present the 3.3 Å resolution single-particle cryo-electron microscopy structure of Mycobacterium smegmatis EmbB, providing insights on substrate binding and reaction mechanism. Mutations that confer ethambutol resistance map mostly around the putative active site, suggesting this to be the location of drug binding.
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Project description:Encapsulins containing dye-decolorizing peroxidase (DyP)-type peroxidases are ubiquitous among prokaryotes, protecting cells against oxidative stress. However, little is known about how they interact and function. Here, we have isolated a native cargo-packaging encapsulin from Mycobacterium smegmatis and determined its complete high-resolution structure by cryogenic electron microscopy (cryo-EM). This encapsulin comprises an icosahedral shell and a dodecameric DyP cargo. The dodecameric DyP consists of two hexamers with a twofold axis of symmetry and stretches across the interior of the encapsulin. Our results reveal that the encapsulin shell plays a role in stabilizing the dodecameric DyP. Furthermore, we have proposed a potential mechanism for removing the hydrogen peroxide based on the structural features. Our study also suggests that the DyP is the primary cargo protein of mycobacterial encapsulins and is a potential target for antituberculosis drug discovery.
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Project description:The Rv2416c gene of Mycobacterium tuberculosis (Mtb) encodes the enhanced intracellular survival (Eis) protein that enhances intracellular survival of the pathogen in host macrophages during infection. The Mtb Eis protein is released into the cytoplasm of the phagocyte during intracellular infection and modulates the host immune response. It also contributes to drug resistance by acetylating multiple amine groups of aminoglycosides. Interestingly, the nonpathogenic M. smegmatis (Msm) contains a homologous eis gene (MSMEG_3513). The overall structures of Mtb Eis and Msm Eis are highly similar to each other, reflecting the high level (58%) of amino-acid sequence identity between them. Both Mtb Eis and Msm Eis are active as aminoglycoside acetyltransferases, while only Mtb Eis functions as an N(ℇ)-acetyltransferase to acetylate Lys55 of dual-specificity protein phosphatase 16 (DUSP16)/mitogen-activated protein kinase phosphatase 7 (MKP-7), leading to the suppression of host immune responses. Here, the crystal structure of Msm Eis in the paromomycin-bound form is reported, revealing detailed interactions between an aminoglycoside antibiotic and Msm Eis. The crystal structure of Msm Eis in the paromomycin-bound form has been determined at 3.3 Å resolution. This work provides potentially useful information for structure-guided discovery of Eis inhibitors as a novel antituberculosis drug against drug-resistant Mtb.
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Project description:Polynucleotide phosphorylase (PNPase) catalyzes stepwise phosphorolysis of the 3'-terminal phosphodiesters of RNA chains to yield nucleoside diphosphate products. In the reverse reaction PNPase acts as a polymerase, using NDPs as substrates to add NMPs to the 3'-OH terminus of RNA chains while expelling inorganic phosphate. The apparent essentiality of PNPase for growth of M. tuberculosis militates for mycobacterial PNPase as a potential drug target. A cryo-EM structure of Mycobacterium smegmatis PNPase (MsmPNPase) reveals a characteristic ring-shaped homotrimer in which each protomer consists of two RNase PH-like domains and an intervening α-helical module on the inferior surface of the ring. The C-terminal KH and S1 domains, which impart RNA specificity to MsmPNPase, are on the opposite face of the core ring and are conformationally mobile. Single particle reconstructions of MsmPNPase in the act of poly(A) synthesis highlight a 3'-terminal (rA)4 oligonucleotide and two magnesium ions in the active site and an adenine nucleobase in the central tunnel. We identify amino acids that engage the 3' segment of the RNA chain (Phe68, Arg105, Arg112, Arg430, Arg431) and the two metal ions (Asp526, Asp532, Gln546, Asp548) and we infer those that bind inorganic phosphate (Thr470, Ser471, His435, Lys534). Alanine mutagenesis pinpointed RNA and phosphate contacts as essential (Arg105, Arg431, Lys534, Thr470+Ser471), important (Arg112, Arg430), or unimportant (Phe68) for PNPase activity. Severe phosphorolysis and polymerase defects accompanying alanine mutations of the enzymic metal ligands suggest a two-metal mechanism of catalysis by MsmPNPase.
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Project description:The MSMEG_4306 gene from Mycobacterium smegmatis encodes a protein of unknown function with 242 amino-acid residues that contains a conserved zinc-ribbon domain at its C-terminus. Here, the crystal structure of MSMEG_4306 determined by the single-wavelength anomalous dispersion method using just one zinc ion co-purified with the protein is reported. The crystal structure of MSMEG_4306 shows a coiled-coil helix domain in the N-terminal region and a zinc-ribbon domain in the C-terminal region. A structural similarity search against the Protein Data Bank using MSMEG_4306 as a query revealed two similar structures, namely CT398 from Chlamydia trachomatis and HP0958 from Helicobacter pylori, although they share only ∼15% sequence identity with MSMEG_4306. Based on comparative analysis, it is predicted that MSMEG_4306 may be involved in secretion systems, possibly by interacting with multiple proteins or nucleic acids.
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Project description:Mycobacterium tuberculosis produces glycogen (also known as α-glucan) to help evade human immunity. This pathogen uses the GlgE pathway to generate glycogen rather than the more well known glycogen synthase GlgA pathway, which is absent in this bacterium. Thus, the building block for this glucose polymer is α-maltose-1-phosphate rather than an NDP-glucose donor. One of the routes to α-maltose-1-phosphate is now known to involve the GlgA homologue GlgM, which uses ADP-glucose as a donor and α-glucose-1-phosphate as an acceptor. To help compare GlgA (a GT5 family member) with GlgM enzymes (GT4 family members), the X-ray crystal structure of GlgM from Mycobacterium smegmatis was solved to 1.9 Å resolution. While the enzymes shared a GT-B fold and several residues responsible for binding the donor substrate, they differed in some secondary-structural details, particularly in the N-terminal domain, which would be expected to be largely responsible for their different acceptor-substrate specificities.
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Project description:The crystal structure of L-lactate oxidase (LOX) from Aerococcus viridans has been determined at 2.1 A resolution. LOX catalyzes the flavin mononucleotide (FMN) dependent oxidation of lactate to pyruvate and hydrogen peroxide. LOX belongs to the alpha-hydroxy-acid oxidase flavoenzyme family; members of which bind similar substrates and to some extent have conserved catalytic properties and structural motifs. LOX crystallized as two tightly packed tetramers in the asymmetric unit, each having fourfold symmetry. The present structure shows a conserved FMN coordination, but also reveals novel residues involved in substrate binding compared with other family members.
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