The role of OXA-1 β-lactamase Asp66 in the stabilization of the active-site carbamate group and in substrate turnover

2008 ◽  
Vol 410 (3) ◽  
pp. 455-462 ◽  
Author(s):  
David A. Leonard ◽  
Andrea M. Hujer ◽  
Brian A. Smith ◽  
Kyle D. Schneider ◽  
Christopher R. Bethel ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Fold Increase ◽  
Luria Bertani ◽  
Saturation Mutagenesis ◽  
Substrate Affinity ◽  
Luria Bertani Broth ◽  
Class D ◽  
Carbamate Group

The OXA-1 β-lactamase is one of the few class D enzymes that has an aspartate residue at position 66, a position that is proximal to the active-site residue Ser67. In class A β-lactamases, such as TEM-1 and SHV-1, residues adjacent to the active-site serine residue play a crucial role in inhibitor resistance and substrate selectivity. To probe the role of Asp66 in substrate affinity and catalysis, we performed site-saturation mutagenesis at this position. Ampicillin MIC (minimum inhibitory concentration) values for the full set of Asp66 mutants expressed in Escherichia coli DH10B ranged from ≤8 μg/ml for cysteine, proline and the basic amino acids to ≥256 μg/ml for asparagine, leucine and the wild-type aspartate. Replacement of aspartic acid by asparagine at position 66 also led to a moderate enhancement of extended-spectrum cephalosporin resistance. OXA-1 shares with other class D enzymes a carboxylated residue, Lys70, that acts as a general base in the catalytic mechanism. The addition of 25 mM bicarbonate to Luria–Bertani-broth agar resulted in a ≥16-fold increase in MICs for most OXA-1 variants with amino acid replacements at position 66 when expressed in E. coli. Because Asp66 forms hydrogen bonds with several other residues in the OXA-1 active site, we propose that this residue plays a role in stabilizing the CO2 bound to Lys70 and thereby profoundly affects substrate turnover.

scholarly journals Sequence Determinants of Substrate Ambiguity in a HAD Phosphosugar Phosphatase of Arabidopsis Thaliana

Biology ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 77
Author(s):  
Caparrós-Martín ◽  
McCarthy-Suárez ◽  
Culiáñez-Macià
Keyword(s):  
Arabidopsis Thaliana ◽  
Active Site ◽  
Phosphate Ester ◽  
Substrate Affinity ◽  
Evolutionary Selection ◽  
Functional Studies ◽  
Additional Sequence ◽  
Signature Sequence ◽  
Wide Family

The Arabidopsis thaliana broad-range sugar phosphate phosphatase AtSgpp (NP_565895.1, locus AT2G38740) and the specific DL-glycerol-3-phosphatase AtGpp (NP_568858.1, locus AT5G57440) are members of the wide family of magnesium-dependent acid phosphatases subfamily I with the C1-type cap domain haloacid dehalogenase-like hydrolase proteins (HAD). Although both AtSgpp and AtGpp have a superimporsable α/β Rossmann core active site, they differ with respect to the loop-5 of the cap domain, accounting for the differences in substrate specificity. Recent functional studies have demonstrated the essential but not sufficient role of the signature sequence within the motif-5 in substrate discrimination. To better understand the mechanism underlying the control of specificity, we explored additional sequence determinants underpinning the divergent evolutionary selection exerted on the substrate affinity of both enzymes. The most evident difference was found in the loop preceding the loop-5 of the cap domain, which is extended in three additional residues in AtSgpp. To determine if the shortening of this loop would constrain the substrate ambiguity of AtSgpp, we deleted these three aminoacids. The kinetic analyses of the resulting mutant protein AtSgpp3Δ (ΔF53, ΔN54, ΔN55) indicate that promiscuity is compromised. AtSgpp3Δ displays highest level of discrimination for D-ribose-5-phosphate compared to the rest of phosphate ester metabolites tested, which may suggest a proper orientation of D-ribose-5-phosphate in the AtSgpp3Δ active site.

scholarly journals GlcNAcstatins are nanomolar inhibitors of human O-GlcNAcase inducing cellular hyper-O-GlcNAcylation

2009 ◽  
Vol 420 (2) ◽  
pp. 221-227 ◽  
Author(s):  
Helge C. Dorfmueller ◽  
Vladimir S. Borodkin ◽  
Marianne Schimpl ◽  
Daan M. F. van Aalten
Keyword(s):  
Active Site ◽  
Cell Biology ◽  
Rational Design ◽  
Catalytic Mechanism ◽  
Conserved Residues ◽  
Cellular Processes ◽  
Acetyl Group ◽  
Nanomolar Range ◽  
Insight Into

O-GlcNAcylation is an essential, dynamic and inducible post-translational glycosylation of cytosolic proteins in metazoa and can show interplay with protein phosphorylation. Inhibition of OGA (O-GlcNAcase), the enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, is a useful strategy to probe the role of this modification in a range of cellular processes. In the present study, we report the rational design and evaluation of GlcNAcstatins, a family of potent, competitive and selective inhibitors of human OGA. Kinetic experiments with recombinant human OGA reveal that the GlcNAcstatins are the most potent human OGA inhibitors reported to date, inhibiting the enzyme in the sub-nanomolar to nanomolar range. Modification of the GlcNAcstatin N-acetyl group leads to up to 160-fold selectivity against the human lysosomal hexosaminidases which employ a similar substrate-assisted catalytic mechanism. Mutagenesis studies in a bacterial OGA, guided by the structure of a GlcNAcstatin complex, provides insight into the role of conserved residues in the human OGA active site. GlcNAcstatins are cell-permeant and, at low nanomolar concentrations, effectively modulate intracellular O-GlcNAc levels through inhibition of OGA, in a range of human cell lines. Thus these compounds are potent selective tools to study the cell biology of O-GlcNAc.

scholarly journals Role of the sRNA GcvB in regulation of cycA in Escherichia coli

Microbiology ◽  
2009 ◽  
Vol 155 (1) ◽  
pp. 106-114 ◽  
Author(s):  
Sarah C. Pulvermacher ◽  
Lorraine T. Stauffer ◽  
George V. Stauffer
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Minimal Medium ◽  
Luria Bertani ◽  
Cell Physiology ◽  
Rt Pcr ◽  
Luria Bertani Broth ◽  
Increased Sensitivity ◽  
Transcriptional Fusions

In Escherichia coli, the gcvB gene encodes a small non-translated RNA that regulates several genes involved in transport of amino acids and peptides (including sstT, oppA and dppA). Microarray analysis identified cycA as an additional regulatory target of GcvB. The cycA gene encodes a permease for the transport of glycine, d-alanine, d-serine and d-cycloserine. RT-PCR confirmed that GcvB and the Hfq protein negatively regulate cycA mRNA in cells grown in Luria–Bertani broth. In addition, deletion of the gcvB gene resulted in increased sensitivity to d-cycloserine, consistent with increased expression of cycA. A cycA : : lacZ translational fusion confirmed that GcvB negatively regulates cycA expression in Luria–Bertani broth and that Hfq is required for the GcvB effect. GcvB had no effect on cycA : : lacZ expression in glucose minimal medium supplemented with glycine. However, Hfq still negatively regulated the fusion in the absence of GcvB. A set of transcriptional fusions of cycA to lacZ identified a sequence in cycA necessary for regulation by GcvB. Analysis of GcvB identified a region complementary to this region of cycA mRNA. However, mutations predicted to disrupt base-pairing between cycA mRNA and GcvB did not alter expression of cycA : : lacZ. A model for GcvB function in cell physiology is discussed.

scholarly journals Structural Characterization of an S-enantioselective Imine Reductase from Mycobacterium Smegmatis

Biomolecules ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1130
Author(s):  
Timo Meyer ◽  
Nadine Zumbrägel ◽  
Christina Geerds ◽  
Harald Gröger ◽  
Hartmut H. Niemann
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Mycobacterium Smegmatis ◽  
Catalytic Mechanism ◽  
Building Blocks ◽  
Secondary Amines ◽  
Hydrophobic Amino Acids ◽  
High Degree

NADPH-dependent imine reductases (IREDs) are enzymes capable of enantioselectively reducing imines to chiral secondary amines, which represent important building blocks in the chemical and pharmaceutical industry. Since their discovery in 2011, many previously unknown IREDs have been identified, biochemically and structurally characterized and categorized into families. However, the catalytic mechanism and guiding principles for substrate specificity and stereoselectivity remain disputed. Herein, we describe the crystal structure of S-IRED-Ms from Mycobacterium smegmatis together with its cofactor NADPH. S-IRED-Ms belongs to the S-enantioselective superfamily 3 (SFam3) and is the first IRED from SFam3 to be structurally described. The data presented provide further evidence for the overall high degree of structural conservation between different IREDs of various superfamilies. We discuss the role of Asp170 in catalysis and the importance of hydrophobic amino acids in the active site for stereospecificity. Moreover, a separate entrance to the active site, potentially functioning according to a gatekeeping mechanism regulating access and, therefore, substrate specificity is described.

scholarly journals Evolutionary dynamics of asexual hypermutators adapting to a novel environment

2021 ◽  
Author(s):  
Wei-Chin Ho ◽  
Megan G. Behringer ◽  
Samuel F. Miller ◽  
Jadon Gonzales ◽  
Amber Nguyen ◽  
...  
Keyword(s):  
Adaptive Evolution ◽  
Evolutionary Biology ◽  
Evolutionary Dynamics ◽  
Fold Increase ◽  
Luria Bertani ◽  
Mutation Rates ◽  
Wild Type ◽  
Novel Environment ◽  
Luria Bertani Broth ◽  
Nucleotide Mutation

How microbes adapt to a novel environment is a central question in evolutionary biology. While adaptive evolution must be fueled by beneficial mutations, whether higher mutation rates facilitate the rate of adaptive evolution remains unclear. To address this question, we cultured Escherichia coli hypermutating populations, in which a defective methyl-directed mismatch repair pathway causes a 140-fold increase in single-nucleotide mutation rates. In parallel with wild-type E. coli, populations were cultured in tubes containing Luria-Bertani broth, a complex medium known to promote the evolution of subpopulation structure. After 900 days of evolution, in three transfer schemes with different population-size bottlenecks, hypermutators always exhibited similar levels of improved fitness as controls. Fluctuation tests revealed that the mutation rates of hypermutator lines converged evolutionarily on those of wild-type populations, which may have contributed to the absence of fitness differences. Further genome-sequence analysis revealed that, although hypermutator populations have higher rates of genomic evolution, this largely reflects the effects of genetic draft under strong linkage. Despite these linkage effects, the evolved populations exhibit parallelism in fixed mutations, including those potentially related to biofilm formation, transcription regulation, and mutation-rate evolution. Together, these results generally negate the presumed relationship between high mutation rates and high adaptive speed of evolution, providing insight into how clonal adaptation occurs in novel environments.

Role of the Escherichia coli Hfq protein in GcvB regulation of oppA and dppA mRNAs

Microbiology ◽  
2009 ◽  
Vol 155 (1) ◽  
pp. 115-123 ◽  
Author(s):  
Sarah C. Pulvermacher ◽  
Lorraine T. Stauffer ◽  
George V. Stauffer
Keyword(s):  
Escherichia Coli ◽  
Small Rnas ◽  
Target Genes ◽  
Luria Bertani ◽  
Transport Systems ◽  
Regulation Of Transcription ◽  
Luria Bertani Broth ◽  
The Stability ◽  
Dipeptide Transport

The gcvB gene encodes a small non-translated RNA (referred to as GcvB) that regulates oppA and dppA, two genes that encode periplasmic binding proteins for the oligopeptide and dipeptide transport systems. Hfq, an RNA chaperone protein, binds many small RNAs and is required for the small RNAs to regulate expression of their respective target genes. We showed that repression by GcvB of dppA : : lacZ and oppA : : phoA translational fusions is dependent upon Hfq. Double mutations in gcvB and hfq yielded similar expression levels of dppA : : lacZ and oppA : : phoA compared with gcvB or hfq single mutations, suggesting that GcvB and Hfq repress by the same mechanism. The effect of Hfq is not through regulation of transcription of gcvB. Hfq is known to increase the stability of some small RNAs and to facilitate the interactions between small RNAs and specific mRNAs. In the absence of Hfq, there is a marked decrease in the half-life of GcvB in cells grown in both Luria–Bertani broth and glucose minimal medium with glycine, suggesting that part of the role of Hfq is to stabilize GcvB. Overproduction of GcvB in wild-type Escherichia coli results in superrepression of a dppA : : lacZ fusion, but overproduction of GcvB in an hfq mutant does not result in significant repression of the dppA : : lacZ fusion. These results suggest that Hfq also is likely required for GcvB–mRNA pairing.

scholarly journals Structure-Based Mechanism for Early PLP-Mediated Steps of Rabbit Cytosolic Serine Hydroxymethyltransferase Reaction

2013 ◽  
Vol 2013 ◽  
pp. 1-13 ◽  
Author(s):  
Martino L. Di Salvo ◽  
J. Neel Scarsdale ◽  
Galina Kazanina ◽  
Roberto Contestabile ◽  
Verne Schirch ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Serine Hydroxymethyltransferase ◽  
Active Site Residue ◽  
Wild Type ◽  
Hydroxymethyl Group ◽  
New Energy ◽  
Mutant Forms

Serine hydroxymethyltransferase catalyzes the reversible interconversion of L-serine and glycine with transfer of one-carbon groups to and from tetrahydrofolate. Active site residue Thr254 is known to be involved in the transaldimination reaction, a crucial step in the catalytic mechanism of all pyridoxal 5′-phosphate- (PLP-) dependent enzymes, which determines binding of substrates and release of products. In order to better understand the role of Thr254, we have expressed, characterized, and determined the crystal structures of rabbit cytosolic serine hydroxymethyltransferase T254A and T254C mutant forms, in the absence and presence of substrates. These mutants accumulate a kinetically stablegem-diamine intermediate, and their crystal structures show differences in the active site with respect to wild type. The kinetic and crystallographic data acquired with mutant enzymes permit us to infer that conversion ofgem-diamine to external aldimine is significantly slowed because intermediates are trapped into an anomalous position by a misorientation of the PLP ring, and a new energy barrier hampers the transaldimination reaction. This barrier likely arises from the loss of the stabilizing hydrogen bond between the hydroxymethyl group of Thr254 and theε-amino group of active site Lys257, which stabilizes the external aldimine intermediate in wild type SHMTs.

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