Structure of the 4-hydroxy-tetrahydrodipicolinate synthase from the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV and the phylogeny of the aminotransferase pathway

The enzyme 4-hydroxy-tetrahydrodipicolinate synthase (DapA) is involved in the production of lysine and precursor molecules for peptidoglycan synthesis. In a multistep reaction, DapA converts pyruvate and L-aspartate-4-semialdehyde to 4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid. In many organisms, lysine binds allosterically to DapA, causing negative feedback, thus making the enzyme an important regulatory component of the pathway. Here, the 2.1 Å resolution crystal structure of DapA from the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV is reported. The enzyme crystallized as a contaminant of a protein preparation from native biomass. Genome analysis reveals that M. fumariolicum SolV utilizes the recently discovered aminotransferase pathway for lysine biosynthesis. Phylogenetic analyses of the genes involved in this pathway shed new light on the distribution of this pathway across the three domains of life.

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Crystal structure of Pistol, a class of self-cleaving ribozyme

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1611191114 ◽

2017 ◽

Vol 114 (5) ◽

pp. 1021-1026 ◽

Cited By ~ 32

Author(s):

Laura A. Nguyen ◽

Jimin Wang ◽

Thomas A. Steitz

Keyword(s):

Crystal Structure ◽

Rna Structure ◽

Tertiary Structure ◽

Genomic Analysis ◽

Comparative Genomic ◽

Nonbridging Oxygen ◽

Close Proximity ◽

Domains Of Life ◽

Guanine Base ◽

A Minor

Small self-cleaving ribozymes have been discovered in all evolutionary domains of life. They can catalyze site-specific RNA cleavage, and as a result, they have relevance in gene regulation. Comparative genomic analysis has led to the discovery of a new class of small self-cleaving ribozymes named Pistol. We report the crystal structure of Pistol at 2.97-Å resolution. Our results suggest that the Pistol ribozyme self-cleavage mechanism likely uses a guanine base in the active site pocket to carry out the phosphoester transfer reaction. The guanine G40 is in close proximity to serve as the general base for activating the nucleophile by deprotonating the 2′-hydroxyl to initiate the reaction (phosphoester transfer). Furthermore, G40 can also establish hydrogen bonding interactions with the nonbridging oxygen of the scissile phosphate. The proximity of G32 to the O5′ leaving group suggests that G32 may putatively serve as the general acid. The RNA structure of Pistol also contains A-minor interactions, which seem to be important to maintain its tertiary structure and compact fold. Our findings expand the repertoire of ribozyme structures and highlight the conserved evolutionary mechanism used by ribozymes for catalysis.

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PPK1 and PPK2 — which polyphosphate kinase is older?

Biologia ◽

10.2478/s11756-013-0324-x ◽

2014 ◽

Vol 69 (3) ◽

Cited By ~ 8

Author(s):

Lucia Achbergerová ◽

Jozef Nahálka

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Amino Acid ◽

Phylogenetic Analyses ◽

Linear Molecule ◽

Polyphosphate Kinase ◽

Hypothetical Proteins ◽

Bacterial Genomes ◽

Metabolic Balance ◽

Domains Of Life

AbstractPolyphosphate kinases (PPKs) catalyse the polymerisation and degradation of polyphosphate chains. As a result of this process, PPK produces or consumes energy in the form of ATP. Polyphosphate is a linear molecule that contains tens to hundreds of phosphate residues connected by macroergic bonds, and it appears to be an easily obtainable and rich source of energy from prebiotic times to the present. Notably, polyphosphate is present in the cells of all three domains of life, but PPKs are widely distributed only in Bacteria, as Archaea and Eucarya use various unrelated or “nonhomologous” proteins for energy and metabolic balance. The present study focuses on PPK1 and PPK2 homologues, which have been described to some extent in Bacteria, and the aim was to determine which homologue group, PPK1 or PPK2, is older. Phylogenetic analyses of 109 sequence homologues of Escherichia coli PPK1 and 109 sequence homologues of Pseudomonas aeruginosa PPK2 from 109 bacterial genomes imply that polyphosphate consumption (PPK2) evolved first and that phosphate polymerisation (PPK1) evolved later. Independently, a theory of the trends in amino acid loss and gain also confirms that PPK2 is older than PPK1. According to the results of this study, we propose 68 hypothetical proteins to mark as PPK2 homologues and 3 hypothetical proteins to mark as PPK1 homologues.

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SMUG2 DNA glycosylase from Pedobacter heparinus as a new subfamily of the UDG superfamily

Biochemical Journal ◽

10.1042/bcj20160934 ◽

2017 ◽

Vol 474 (6) ◽

pp. 923-938 ◽

Cited By ~ 3

Author(s):

Panjiao Pang ◽

Ye Yang ◽

Jing Li ◽

Zhong Wang ◽

Weiguo Cao ◽

...

Keyword(s):

Crystal Structure ◽

Excision Repair ◽

Phylogenetic Analyses ◽

Dna Glycosylase ◽

Amino Acid Residues ◽

Uracil Dna Glycosylase ◽

Important Amino Acid ◽

Repair Mechanisms ◽

Ap Sites ◽

Base Excision

Base deamination is a common type of DNA damage that occurs in all organisms. DNA repair mechanisms are essential to maintain genome integrity, in which the base excision repair (BER) pathway plays a major role in the removal of base damage. In the BER pathway, the uracil DNA glycosylase superfamily is responsible for excising the deaminated bases from DNA and generates apurinic/apyrimidinic (AP) sites. Using bioinformatics tools, we identified a family 3 SMUG1-like DNA glycoyslase from Pedobacter heparinus (named Phe SMUG2), which displays catalytic activities towards DNA containing uracil or hypoxanthine/xanthine. Phylogenetic analyses show that SMUG2 enzymes are closely related to family 3 SMUG1s but belong to a distinct branch of the family. The high-resolution crystal structure of the apoenzyme reveals that the general fold of Phe SMUG2 resembles SMUG1s, yet with several distinct local structural differences. Mutational studies, coupled with structural modeling, identified several important amino acid residues for glycosylase activity. Substitution of G65 with a tyrosine results in loss of all glycosylase activity. The crystal structure of the G65Y mutant suggests a potential misalignment at the active site due to the mutation. The relationship between the new subfamily and other families in the UDG superfamily is discussed. The present study provides new mechanistic insight into the molecular mechanism of the UDG superfamily.

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Crystal structure of the RNA 2′,3′-cyclic phosphodiesterase fromDeinococcus radiodurans

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x17004964 ◽

2017 ◽

Vol 73 (5) ◽

pp. 276-280 ◽

Cited By ~ 1

Author(s):

Wanchun Han ◽

Jiahui Cheng ◽

Congli Zhou ◽

Yuejin Hua ◽

Ye Zhao

Keyword(s):

Crystal Structure ◽

Active Site ◽

Catalytic Mechanism ◽

Deinococcus Radiodurans ◽

Sulfate Ion ◽

Open Conformation ◽

Domains Of Life

2′,3′-Cyclic phosphodiesterase (CPDase) homologues have been found in all domains of life and are involved in diverse RNA and nucleotide metabolisms. The CPDase fromDeinococcus radioduranswas crystallized and the crystals diffracted to 1.6 Å resolution, which is the highest resolution currently known for a CPDase structure. Structural comparisons revealed that the enzyme is in an open conformation in the absence of substrate. Nevertheless, the active site is well formed, and the representative motifs interact with sulfate ion, which suggests a conserved catalytic mechanism.

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Complete genome analysis confirms that the pygmy marmoset adenovirus is a variant of the skunk adenovirus 1 – Short communication

Acta Veterinaria Hungarica ◽

10.1556/004.2020.00039 ◽

2020 ◽

Author(s):

Andor Doszpoly ◽

Ákos Hornyák ◽

Krisztián Bányai

Keyword(s):

Genome Analysis ◽

Genetic Relatedness ◽

Genomic Sequence ◽

Phylogenetic Analyses ◽

Virus Species ◽

Complete Genomic Sequence ◽

Urocyon Cinereoargenteus ◽

Erethizon Dorsatum ◽

Pygmy Marmoset ◽

Complete Genomic

AbstractThe complete genomic sequence along with phylogenetic analyses of an adenovirus (AdV), isolated from a dead captive pygmy marmoset (Callithrix pygmaea) from a Hungarian zoo is reported. Earlier, based on the phylogenetic analysis of the sequence of a PCR-amplified fragment from the DNA polymerase gene, the pygmy marmoset AdV (PMAdV) has been reported to cluster closest to certain chiropteran AdVs. In the following years similar AdVs were discovered in additional mammalian hosts, including a skunk (Mephitis mephitis), African pygmy hedgehogs (Atelerix albiventris), North American porcupines (Erethizon dorsatum) and grey fox (Urocyon cinereoargenteus). After the full genome analysis of the skunk adenovirus (SkAdV-1), a novel species Skunk mastadenovirus A (SkAdV-A) has been established. The AdVs, originating from the African pygmy hedgehogs, have been found to belong to virus species SkAdV-A. Partial gene sequences from the porcupine AdVs have also implied their very close genetic relatedness to SkAdV-A. The complete genomic sequence of PMAdV, examined in this study, was found to share 99.83% nucleotide identity with SkAdV-1, thus unequivocally represents a genomic variant of SkAdV-1. The observation that viruses classifiable as SkAdV-A are able to infect and cause diseases in several, distantly related mammals seems to deserve further studies to elucidate the infection biology of this intriguing AdV.

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The crystal structure of the heme d 1 biosynthesis-associated small c-type cytochrome NirC reveals mixed oligomeric states in crystallo

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798320003101 ◽

2020 ◽

Vol 76 (4) ◽

pp. 375-384

Author(s):

Thomas Klünemann ◽

Steffi Henke ◽

Wulf Blankenfeldt

Keyword(s):

Crystal Structure ◽

Protein Concentration ◽

Opportunistic Pathogen ◽

Nitrite Reduction ◽

Simultaneous Observation ◽

Maximum Resolution ◽

High Protein Concentration ◽

Different Types ◽

Domains Of Life ◽

Sad Phasing

Monoheme c-type cytochromes are important electron transporters in all domains of life. They possess a common fold hallmarked by three α-helices that surround a covalently attached heme. An intriguing feature of many monoheme c-type cytochromes is their capacity to form oligomers by exchanging at least one of their α-helices, which is often referred to as 3D domain swapping. Here, the crystal structure of NirC, a c-type cytochrome co-encoded with other proteins involved in nitrite reduction by the opportunistic pathogen Pseudomonas aeruginosa, has been determined. The crystals diffracted anisotropically to a maximum resolution of 2.12 Å (spherical resolution of 2.83 Å) and initial phases were obtained by Fe-SAD phasing, revealing the presence of 11 NirC chains in the asymmetric unit. Surprisingly, these protomers arrange into one monomer and two different types of 3D domain-swapped dimers, one of which shows pronounced asymmetry. While the simultaneous observation of monomers and dimers probably reflects the interplay between the high protein concentration required for crystallization and the structural plasticity of monoheme c-type cytochromes, the identification of conserved structural motifs in the monomer together with a comparison with similar proteins may offer new leads to unravel the unknown function of NirC.

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Methanococci Use the Diaminopimelate Aminotransferase (DapL) Pathway for Lysine Biosynthesis

Journal of Bacteriology ◽

10.1128/jb.00172-10 ◽

2010 ◽

Vol 192 (13) ◽

pp. 3304-3310 ◽

Cited By ~ 15

Author(s):

Yuchen Liu ◽

Robert H. White ◽

William B. Whitman

Keyword(s):

Specific Activity ◽

Sequence Similarity ◽

Phylogenetic Analyses ◽

Direct Conversion ◽

Maximum Activity ◽

Lysine Biosynthesis ◽

Diaminopimelic Acid ◽

Reading Frame ◽

Cell Extracts ◽

Methanothermobacter Thermautotrophicus

ABSTRACT The pathway of lysine biosynthesis in the methanococci has not been identified previously. A variant of the diaminopimelic acid (DAP) pathway uses diaminopimelate aminotransferase (DapL) to catalyze the direct conversion of tetrahydrodipicolinate (THDPA) to ll-DAP. Recently, the enzyme DapL (MTH52) was identified in Methanothermobacter thermautotrophicus and shown to belong to the DapL1 group. Although the Methanococcus maripaludis genome lacks a gene that can be unambiguously assigned a DapL function based on sequence similarity, the open reading frame MMP1527 product shares 30% amino acid sequence identity with MTH52. A Δmmp1527 deletion mutant was constructed and found to be a lysine auxotroph, suggesting that this DapL homolog in methanococci is required for lysine biosynthesis. In cell extracts of the M. maripaludis wild-type strain, the specific activity of DapL using ll-DAP and α-ketoglutarate as substrates was 24.3 ± 2.0 nmol min−1 mg of protein−1. The gene encoding the DapL homolog in Methanocaldococcus jannaschii (MJ1391) was cloned and expressed in Escherichia coli, and the protein was purified. The maximum activity of MJ1391 was observed at 70°C and pH 8.0 to 9.0. The apparent Km s of MJ1391 for ll-DAP and α-ketoglutarate were 82.8 ± 10 μM and 0.42 ± 0.02 mM, respectively. MJ1391 was not able to use succinyl-DAP or acetyl-DAP as a substrate. Phylogenetic analyses suggested that two lateral gene transfers occurred in the DapL genes, one from the archaea to the bacteria in the DapL2 group and one from the bacteria to the archaea in the DapL1 group. These results demonstrated that the DapL pathway is present in marine methanogens belonging to the Methanococcales.

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Crystal Structure of a Peptidoglycan Synthesis Regulatory Factor (PBP3) fromStreptococcus pneumoniae

Journal of Biological Chemistry ◽

10.1074/jbc.m408446200 ◽

2004 ◽

Vol 280 (16) ◽

pp. 15984-15991 ◽

Cited By ~ 43

Author(s):

Cécile Morlot ◽

Lucile Pernot ◽

Audrey Le Gouellec ◽

Anne Marie Di Guilmi ◽

Thierry Vernet ◽

...

Keyword(s):

Crystal Structure ◽

Cell Growth ◽

Soluble Form ◽

Bacterial Cell Division ◽

Efficient Manner ◽

Penicillin Binding Proteins ◽

Peptidoglycan Synthesis ◽

Division Process ◽

Omega Loop ◽

Β Sheet

Penicillin-binding proteins (PBPs) are membrane-associated enzymes which perform critical functions in the bacterial cell division process. The singled-Ala,d-Ala (d,d)-carboxypeptidase inStreptococcus pneumoniae, PBP3, has been shown to play a key role in control of availability of the peptidoglycal substrate during cell growth. Here, we have biochemically characterized and solved the crystal structure of a soluble form of PBP3 to 2.8 Å resolution. PBP3 folds into an NH2-terminal,d,d-carboxypeptidase-like domain, and a COOH-terminal, elongated β-rich region. The carboxypeptidase domain harbors the classic signature of the penicilloyl serine transferase superfamily, in that it contains a central, five-stranded antiparallel β-sheet surrounded by α-helices. As in other carboxypeptidases, which are present in species whose peptidoglycan stem peptide has a lysine residue at the third position, PBP3 has a 14-residue insertion at the level of its omega loop, a feature that distinguishes it from carboxypeptidases from bacteria whose peptidoglycan harbors a diaminopimelate moiety at this position. PBP3 performs substrate acylation in a highly efficient manner (kcat/Km= 50,500m–1·s–1), an event that may be linked to role in control of pneumococcal peptidoglycan reticulation. A model that places PBP3 poised vertically on the bacterial membrane suggests that its COOH-terminal region could act as a pedestal, placing the active site in proximity to the peptidoglycan and allowing the protein to “skid” on the surface of the membrane, trimming pentapeptides during the cell growth and division processes.

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