Substrate spectrum of mandelate racemase

ABSTRACTWe describe a deletion mutation in a class A β-lactamase, PenA, ofBurkholderia thailandensisthat extended the substrate spectrum of the enzyme to include ceftazidime. Glu168del was located in a functional domain called the omega loop causing expansion of the space in the loop, which in turn increased flexibility at the active site. This deletion mutation represents a rare but significant alternative mechanical path to substrate spectrum extension in PenA besides more common substitution mutations.

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Structural and functional insights into esterase-mediated macrolide resistance

Nature Communications ◽

10.1038/s41467-021-22016-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Michał Zieliński ◽

Jaeok Park ◽

Barry Sleno ◽

Albert M. Berghuis

Keyword(s):

Active Site ◽

Pathogenic Bacteria ◽

Catalytic Mechanism ◽

Three Dimensional ◽

Resistance Mechanisms ◽

Docking Studies ◽

Macrolide Resistance ◽

Flexible Docking ◽

Sequence Identity ◽

Substrate Spectrum

AbstractMacrolides are a class of antibiotics widely used in both medicine and agriculture. Unsurprisingly, as a consequence of their exensive usage a plethora of resistance mechanisms have been encountered in pathogenic bacteria. One of these resistance mechanisms entails the enzymatic cleavage of the macrolides’ macrolactone ring by erythromycin esterases (Eres). The most frequently identified Ere enzyme is EreA, which confers resistance to the majority of clinically used macrolides. Despite the role Eres play in macrolide resistance, research into this family enzymes has been sparse. Here, we report the first three-dimensional structures of an erythromycin esterase, EreC. EreC is an extremely close homologue of EreA, displaying more than 90% sequence identity. Two structures of this enzyme, in conjunction with in silico flexible docking studies and previously reported mutagenesis data allowed for the proposal of a detailed catalytic mechanism for the Ere family of enzymes, labeling them as metal-independent hydrolases. Also presented are substrate spectrum assays for different members of the Ere family. The results from these assays together with an examination of residue conservation for the macrolide binding site in Eres, suggests two distinct active site archetypes within the Ere enzyme family.

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Profiling PRMT methylome reveals roles of hnRNPA1 arginine methylation in RNA splicing and cell growth

Nature Communications ◽

10.1038/s41467-021-21963-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Wen-juan Li ◽

Yao-hui He ◽

Jing-jing Yang ◽

Guo-sheng Hu ◽

Yi-an Lin ◽

...

Keyword(s):

Alternative Splicing ◽

Cell Growth ◽

Rna Splicing ◽

Synergistic Effects ◽

Arginine Methylation ◽

Type I ◽

Prostate Cancer Cells ◽

Cancer Cell Growth ◽

Cancer Mutations ◽

Substrate Spectrum

AbstractNumerous substrates have been identified for Type I and II arginine methyltransferases (PRMTs). However, the full substrate spectrum of the only type III PRMT, PRMT7, and its connection to type I and II PRMT substrates remains unknown. Here, we use mass spectrometry to reveal features of PRMT7-regulated methylation. We find that PRMT7 predominantly methylates a glycine and arginine motif; multiple PRMT7-regulated arginine methylation sites are close to phosphorylations sites; methylation sites and proximal sequences are vulnerable to cancer mutations; and methylation is enriched in proteins associated with spliceosome and RNA-related pathways. We show that PRMT4/5/7-mediated arginine methylation regulates hnRNPA1 binding to RNA and several alternative splicing events. In breast, colorectal and prostate cancer cells, PRMT4/5/7 are upregulated and associated with high levels of hnRNPA1 arginine methylation and aberrant alternative splicing. Pharmacological inhibition of PRMT4/5/7 suppresses cancer cell growth and their co-inhibition shows synergistic effects, suggesting them as targets for cancer therapy.

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Preliminary x-ray data on crystals of mandelate racemase.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)76534-x ◽

1988 ◽

Vol 263 (19) ◽

pp. 9268-9270

Author(s):

D J Neidhart ◽

V M Powers ◽

G L Kenyon ◽

A Y Tsou ◽

S C Ransom ◽

...

Keyword(s):

X Ray ◽

Mandelate Racemase

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Perturbing the Hydrophobic Pocket of Mandelate Racemase To Probe Phenyl Motion during Catalysis†

Biochemistry ◽

10.1021/bi0473096 ◽

2005 ◽

Vol 44 (25) ◽

pp. 9013-9021 ◽

Cited By ~ 19

Author(s):

Ferhan Siddiqi ◽

Jennifer R. Bourque ◽

Haiyan Jiang ◽

Marieke Gardner ◽

Martin St. Maurice ◽

...

Keyword(s):

Hydrophobic Pocket ◽

Mandelate Racemase

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Investigating the dynamic nature of the ABC transporters: ABCB1 and MsbA as examples for the potential synergies of MD theory and EPR applications

Biochemical Society Transactions ◽

10.1042/bst20150138 ◽

2015 ◽

Vol 43 (5) ◽

pp. 1023-1032 ◽

Cited By ~ 10

Author(s):

Thomas Stockner ◽

Anna Mullen ◽

Fraser MacMillan

Keyword(s):

Crystal Structures ◽

Conformational Changes ◽

Abc Transporters ◽

Epr Spectroscopy ◽

Structural Information ◽

Dynamic Properties ◽

Molecular System ◽

Synergistic Effects ◽

Md Simulations ◽

Substrate Spectrum

ABC transporters are primary active transporters found in all kingdoms of life. Human multidrug resistance transporter ABCB1, or P-glycoprotein, has an extremely broad substrate spectrum and confers resistance against chemotherapy drug treatment in cancer cells. The bacterial ABC transporter MsbA is a lipid A flippase and a homolog to the human ABCB1 transporter, with which it partially shares its substrate spectrum. Crystal structures of MsbA and ABCB1 have been solved in multiple conformations, providing a glimpse into the possible conformational changes the transporter could be going through during the transport cycle. Crystal structures are inherently static, while a dynamic picture of the transporter in motion is needed for a complete understanding of transporter function. Molecular dynamics (MD) simulations and electron paramagnetic resonance (EPR) spectroscopy can provide structural information on ABC transporters, but the strength of these two methods lies in the potential to characterise the dynamic regime of these transporters. Information from the two methods is quite complementary. MD simulations provide an all atom dynamic picture of the time evolution of the molecular system, though with a narrow time window. EPR spectroscopy can probe structural, environmental and dynamic properties of the transporter in several time regimes, but only through the attachment sites of an exogenous spin label. In this review the synergistic effects that can be achieved by combining the two methods are highlighted, and a brief methodological background is also presented.

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The effect of amino acid substitution at position 219 of Citrobacter freundii cephalosporinase on extension of its substrate spectrum

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1992.tb17150.x ◽

1992 ◽

Vol 207 (3) ◽

pp. 1123-1127 ◽

Cited By ~ 9

Author(s):

Kikuo TSUKAMOTO ◽

Reiko OHNO ◽

Michiyoshi NUKAGA ◽

Tetsuo SAWAI

Keyword(s):

Amino Acid ◽

Amino Acid Substitution ◽

Citrobacter Freundii ◽

Substrate Spectrum

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The structure of the metallo-β-lactamase VIM-2 in complex with a triazolylthioacetamide inhibitor

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x16016113 ◽

2016 ◽

Vol 72 (11) ◽

pp. 813-819 ◽

Cited By ~ 16

Author(s):

Tony Christopeit ◽

Ke-Wu Yang ◽

Shao-Kang Yang ◽

Hanna-Kirsti S. Leiros

Keyword(s):

Public Health ◽

Crystal Structure ◽

Active Site ◽

Drug Target ◽

Zinc Ions ◽

Clinical Use ◽

Global Public Health ◽

Promising Strategy ◽

Conserved Residue ◽

Substrate Spectrum

The increasing number of pathogens expressing metallo-β-lactamases (MBLs), and in this way achieving resistance to β-lactam antibiotics, is a significant threat to global public health. A promising strategy to treat such resistant pathogens is the co-administration of MBL inhibitors together with β-lactam antibiotics. However, an MBL inhibitor suitable for clinical use has not yet been identified. Verona integron-encoded metallo-β-lactamase 2 (VIM-2) is a widespread MBL with a broad substrate spectrum and hence is an interesting drug target for the treatment of β-lactam-resistant infections. In this study, three triazolylthioacetamides were tested as inhibitors of VIM-2. One of the tested compounds showed clear inhibition of VIM-2, with an IC50of 20 µM. The crystal structure of the inhibitor in complex with VIM-2 was obtained by DMSO-free co-crystallization and was solved at a resolution of 1.50 Å. To our knowledge, this is the first structure of a triazolylthioacetamide inhibitor in complex with an MBL. Analysis of the structure shows that the inhibitor binds to the two zinc ions in the active site of VIM-2 and revealed detailed information on the interactions involved. Furthermore, the crystal structure showed that binding of the inhibitor induced a conformational change of the conserved residue Trp87.

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