Unique anticodon loop conformation with the flipped-out wobble nucleotide in the crystal structure of unbound tRNAVal

ABSTRACTIMP-type metallo-β-lactamases (MBLs) are exogenous zinc metalloenzymes that hydrolyze a broad range of β-lactams, including carbapenems. Here we report the crystal structure of IMP-18, an MBL cloned fromPseudomonas aeruginosa, at 2.0-Å resolution. The overall structure of IMP-18 resembles that of IMP-1, with an αβ/βα “folded sandwich” configuration, but the loop that covers the active site has a distinct conformation. The relationship between IMP-18's loop conformation and its kinetic properties was investigated by replacing the amino acid residues that can affect the loop conformation (Lys44, Thr50, and Ile69) in IMP-18 with those occupying the corresponding positions in the well-described enzyme IMP-1. The replacement of Thr50 with Pro considerably modified IMP-18's kinetic properties, specifically those pertaining to meropenem, with thekcat/Kmvalue increased by an order of magnitude. The results indicate that this is a key residue that defines the kinetic properties of IMP-type β-lactamases.

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Crystal Structure of a Phospholipase D from the Plant-Associated Bacteria Serratia plymuthica Strain AS9 Reveals a Unique Arrangement of Catalytic Pocket

International Journal of Molecular Sciences ◽

10.3390/ijms22063219 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3219

Author(s):

Fanghua Wang ◽

Siyu Liu ◽

Xuejing Mao ◽

Ruiguo Cui ◽

Bo Yang ◽

...

Keyword(s):

Crystal Structure ◽

Rational Design ◽

Structural Information ◽

Plant Protection ◽

Serratia Plymuthica ◽

Associated Bacteria ◽

Loop Region ◽

C Terminus ◽

Loop Conformation

Phospholipases D (PLDs) play important roles in different organisms and in vitro phospholipid modifications, which attract strong interests for investigation. However, the lack of PLD structural information has seriously hampered both the understanding of their structure–function relationships and the structure-based bioengineering of this enzyme. Herein, we presented the crystal structure of a PLD from the plant-associated bacteria Serratia plymuthica strain AS9 (SpPLD) at a resolution of 1.79 Å. Two classical HxKxxxxD (HKD) motifs were found in SpPLD and have shown high structural consistence with several PLDs in the same family. While comparing the structure of SpPLD with the previous resolved PLDs from the same family, several unique conformations on the C-terminus of the HKD motif were demonstrated to participate in the arrangement of the catalytic pocket of SpPLD. In SpPLD, an extented loop conformation between β9 and α9 (aa228–246) was found. Moreover, electrostatic surface potential showed that this loop region in SpPLD was positively charged while the corresponding loops in the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9) were neutral. The shortened loop between α10 and α11 (aa272–275) made the SpPLD unable to form the gate-like structure which existed specically in the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9) and functioned to stabilize the substrates. In contrast, the shortened loop conformation at this corresponding segment was more alike to several nucleases (Nuc, Zuc, mZuc, NucT) within the same family. Moreover, the loop composition between β11 and β12 was also different from the two Streptomyces originated PLDs (PDB ID: 1F0I, 2ZE4/2ZE9), which formed the entrance of the catalytic pocket and were closely related to substrate recognition. So far, SpPLD was the only structurally characterized PLD enzyme from Serratia. The structural information derived here not only helps for the understanding of the biological function of this enzyme in plant protection, but also helps for the understanding of the rational design of the mutant, with potential application in phospholipid modification.

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Asymmetry of the Active Site Loop Conformation between Subunits of Glutamate-1-semialdehyde Aminomutase in Solution

BioMed Research International ◽

10.1155/2013/353270 ◽

2013 ◽

Vol 2013 ◽

pp. 1-10 ◽

Cited By ~ 12

Author(s):

Barbara Campanini ◽

Stefano Bettati ◽

Martino Luigi di Salvo ◽

Andrea Mozzarelli ◽

Roberto Contestabile

Keyword(s):

Crystal Structure ◽

Fluorescence Quenching ◽

Potassium Iodide ◽

Active Site ◽

Catalytic Site ◽

Crystallographic Data ◽

Closed Conformation ◽

Common Precursor ◽

Coenzyme B ◽

Loop Conformation

Glutamate-1-semialdehyde aminomutase (GSAM) is a dimeric, pyridoxal 5′-phosphate (PLP)- dependent enzyme catalysing in plants and some bacteria the isomerization of L-glutamate-1-semialdehyde to 5-aminolevulinate, a common precursor of chlorophyll, haem, coenzyme B12, and other tetrapyrrolic compounds. During the catalytic cycle, the coenzyme undergoes conversion from pyridoxamine 5′-phosphate (PMP) to PLP. The entrance of the catalytic site is protected by a loop that is believed to switch from an open to a closed conformation during catalysis. Crystallographic studies indicated that the structure of the mobile loop is related to the form of the cofactor bound to the active site, allowing for asymmetry within the dimer. Since no information on structural and functional asymmetry of the enzyme in solution is available in the literature, we investigated the active site accessibility by determining the cofactor fluorescence quenching of PMP- and PLP-GSAM forms. PLP-GSAM is partially quenched by potassium iodide, suggesting that at least one catalytic site is accessible to the anionic quencher and therefore confirming the asymmetry observed in the crystal structure. Iodide induces release of the cofactor from PMP-GSAM, apparently from only one catalytic site, therefore suggesting an asymmetry also in this form of the enzyme in solution, in contrast with the crystallographic data.

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Initiator tRNAs have a unique anticodon loop conformation.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.76.7.3289 ◽

1979 ◽

Vol 76 (7) ◽

pp. 3289-3293 ◽

Cited By ~ 58

Author(s):

P. Wrede ◽

N. H. Woo ◽

A. Rich

Keyword(s):

Anticodon Loop ◽

Loop Conformation

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Crystal structure of N6-(δ2-isopentenyl)adenine, a base in the anticodon loop of some tRNA's

Biochemical and Biophysical Research Communications ◽

10.1016/s0006-291x(72)80208-0 ◽

1972 ◽

Vol 46 (2) ◽

pp. 779-784 ◽

Cited By ~ 34

Author(s):

Charles E. Bugg ◽

Ulf Thewalt

Keyword(s):

Crystal Structure ◽

Anticodon Loop ◽

Isopentenyl Adenine

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Stability of the unique anticodon loop conformation of E.coli tRNAfMet

Nucleic Acids Research ◽

10.1093/nar/7.6.1457 ◽

1979 ◽

Vol 7 (6) ◽

pp. 1457-1467 ◽

Cited By ~ 15

Author(s):

Paul Wrede ◽

Alexander Rich

Keyword(s):

Anticodon Loop ◽

Loop Conformation

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Comments on the recent crystal structure of TsaBDE complex of bacterial t6A biosynthesis system and its significance for understanding TC-AMP processing

10.1101/563171 ◽

2019 ◽

Author(s):

Boguslaw Stec

Keyword(s):

Crystal Structure ◽

Active Sites ◽

Structural Model ◽

Thermotoga Maritima ◽

Biosynthesis Pathway ◽

Binding Modes ◽

Anticodon Loop ◽

Structural Details ◽

Unstable Intermediate ◽

Trna Binding

ABSTRACTThe N(6)-threonylcarbamoyl adenosine (t6A) modification at position 37 of a tRNA of the anticodon loop is universal and central to the translational fidelity of all known organisms. The ternary complex of TsaBDE is the central and essential workstation for t6A biosynthesis in bacteria. The recently published crystal structure of Thermotoga maritima (T.maritima) TsaBDE complex (Missoury et al., 2018) has ~15% incorrectly-placed, misplaced/mistraced, or missing residues. These structural errors have precipitated incorrect conclusions about the disordering of the active site and inferred action of the TsaE element. In this report, we rectify the published structural model of the T.maritima TsaBDE complex. In stark contrast, a corrected structural model of TsaBDE shows that both active sites of the TsaD element are fully occupied with threonylcarbamoyladenosine (TC-AMP), an unstable intermediate chemical moiety of the t6A biosynthesis pathway. This observation has profound implications for understanding the funneling of intermediates in the t6A pathway and also in helping to elucidate tRNA binding modes. Based on the structural details described in here we propose a unifying principle for binding the tRNA to the TsaD subunit of the complex which is universally required in all known t6A modification pathways.

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