scholarly journals Kinetic Crystallography on MutT and its homolog

2014 ◽  
Vol 70 (a1) ◽  
pp. C486-C486
Author(s):  
Teruya Nakamura ◽  
Keisuke Hirata ◽  
Kohei Yoshikawa ◽  
Miyuki Inazato ◽  
Mami Chirifu ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Human Homolog ◽  
Substrate Specificities ◽  
Broad Substrate Specificity ◽  
E Coli ◽  
High Substrate ◽  
Substrate Complex ◽  
Complex Crystals

Oxidized deoxynucleotides cause replicational errors because of their misincorporations into DNA. The MutT and related proteins prevent transversion mutations by hydrolyzing mutagenic oxidized nucleotides such as 8-oxo-dGTP and 2-oxo-dATP, and there is a difference in substrate specificities between them. E. coli MutT hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP with extremely high substrate specificity. On the other hand, its human homolog has broad substrate specificity for oxidized nucleotides and hydrolyzes 8-oxo-dGTP as well as 2-oxo-dATP. In order to understand mechanisms of their substrate specificities, we solved the crystal structures of MutT and its homolog complexed with their substrates and revealed structural basis of the high substrate specificity of E. coli MutT for 8-oxoguanine nucleotide and the broad substrate specificity of its human honolog for oxidized nucleotides. In this paper, we report the hydrolysis mechanisms of both enzymes revealed by kinetic protein crystallography. Both hydrolysis reactions were initiated by soaking the enzyme-substrate complex crystals in divalent metal solution. After incubation under various conditions, the reactions were terminated by freezing the crystals at 100K. X-ray diffraction data were collected at Spring-8 and Photon Factory. In the MutT crystals, the structures of sequential catalytic intermediates showed the activation mechanism of the nucleophilic water molecule synchronized with the coordination of metal ions. Now by using the crystals of its human homolog, the trial of the catching the intermediate state of catalysis is in progress.

scholarly journals Structural basis for the broad substrate specificity of two acyl-CoA dehydrogenases FadE5 from mycobacteria

2020 ◽  
Vol 117 (28) ◽  
pp. 16324-16332
Author(s):  
Xiaobo Chen ◽  
Jiayue Chen ◽  
Bing Yan ◽  
Wei Zhang ◽  
Luke W. Guddat ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Mycobacterium Smegmatis ◽  
Hydrophobic Interactions ◽  
Carbon Chain ◽  
Structural Basis ◽  
Broad Substrate Specificity ◽  
Carbon Chains ◽  
Binding Cavity ◽  
Π Stacking Interaction ◽  
Acyl Coa Dehydrogenases

FadE, an acyl-CoA dehydrogenase, introduces unsaturation to carbon chains in lipid metabolism pathways. Here, we report that FadE5 fromMycobacterium tuberculosis(MtbFadE5) andMycobacterium smegmatis(MsFadE5) play roles in drug resistance and exhibit broad specificity for linear acyl-CoA substrates but have a preference for those with long carbon chains. Here, the structures ofMsFadE5 andMtbFadE5, in the presence and absence of substrates, have been determined. These reveal the molecular basis for the broad substrate specificity of these enzymes. FadE5 interacts with the CoA region of the substrate through a large number of hydrogen bonds and an unusual π–π stacking interaction, allowing these enzymes to accept both short- and long-chain substrates. Residues in the substrate binding cavity reorient their side chains to accommodate substrates of various lengths. Longer carbon-chain substrates make more numerous hydrophobic interactions with the enzyme compared with the shorter-chain substrates, resulting in a preference for this type of substrate.

scholarly journals Substrate specificity and structure of human aminoadipate aminotransferase/kynurenine aminotransferase II

2008 ◽  
Vol 28 (4) ◽  
pp. 205-215 ◽  
Author(s):  
Qian Han ◽  
Tao Cai ◽  
Danilo A. Tagle ◽  
Howard Robinson ◽  
Jianyong Li
Keyword(s):  
Substrate Specificity ◽  
Catalytic Efficiency ◽  
Structural Basis ◽  
Substrate Affinity ◽  
Kinetic Properties ◽  
Amino Group ◽  
Kynurenine Aminotransferase ◽  
Broad Substrate Specificity ◽  
Systematic Assessment ◽  
The Brain

KAT (kynurenine aminotransferase) II is a primary enzyme in the brain for catalysing the transamination of kynurenine to KYNA (kynurenic acid). KYNA is the only known endogenous antagonist of the N-methyl-D-aspartate receptor. The enzyme also catalyses the transamination of aminoadipate to α-oxoadipate; therefore it was initially named AADAT (aminoadipate aminotransferase). As an endotoxin, aminoadipate influences various elements of glutamatergic neurotransmission and kills primary astrocytes in the brain. A number of studies dealing with the biochemical and functional characteristics of this enzyme exist in the literature, but a systematic assessment of KAT II addressing its substrate profile and kinetic properties has not been performed. The present study examines the biochemical and structural characterization of a human KAT II/AADAT. Substrate screening of human KAT II revealed that the enzyme has a very broad substrate specificity, is capable of catalysing the transamination of 16 out of 24 tested amino acids and could utilize all 16 tested α-oxo acids as amino-group acceptors. Kinetic analysis of human KAT II demonstrated its catalytic efficiency for individual amino-group donors and acceptors, providing information as to its preferred substrate affinity. Structural analysis of the human KAT II complex with α-oxoglutaric acid revealed a conformational change of an N-terminal fraction, residues 15–33, that is able to adapt to different substrate sizes, which provides a structural basis for its broad substrate specificity.

scholarly journals Structural Basis for Broad Substrate Specificity in Higher Plant β-d-Glucan Glucohydrolases

2002 ◽  
Vol 14 (5) ◽  
pp. 1033-1052 ◽  
Author(s):  
Maria Hrmova ◽  
Ross De Gori ◽  
Brian J. Smith ◽  
Jon K. Fairweather ◽  
Hugues Driguez ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Structural Basis ◽  
Higher Plant ◽  
Broad Substrate Specificity

Deciphering the structural basis of the broad substrate specificity of myo-inositol monophosphatase (IMP) from Cicer arietinum

2020 ◽  
Vol 151 ◽  
pp. 967-975 ◽  
Author(s):  
Prakarsh K. Yadav ◽  
Prafull Salvi ◽  
Nitin Uttam Kamble ◽  
Bhanu Prakash Petla ◽  
Manoj Majee ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Cicer Arietinum ◽  
Structural Basis ◽  
Inositol Monophosphatase ◽  
Broad Substrate Specificity

scholarly journals Structural basis for the broad substrate specificity of the human tyrosylprotein sulfotransferase-1

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Shinnosuke Tanaka ◽  
Toshiaki Nishiyori ◽  
Hidetaka Kojo ◽  
Reo Otsubo ◽  
Moe Tsuruta ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Structural Basis ◽  
Broad Substrate Specificity

scholarly journals Antimicrobial Peptide Conformation as a Structural Determinant of Omptin Protease Specificity

2015 ◽  
Vol 197 (22) ◽  
pp. 3583-3591 ◽  
Author(s):  
John R. Brannon ◽  
Jenny-Lee Thomassin ◽  
Samantha Gruenheid ◽  
Hervé Le Moual
Keyword(s):  
Substrate Specificity ◽  
Antimicrobial Peptide ◽  
Outer Membrane ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Structural Basis ◽  
Gram Negative ◽  
Content Type ◽  
Substrate Specificities ◽  
Structural Determinant

ABSTRACTBacterial proteases contribute to virulence by cleaving host or bacterial proteins to promote survival and dissemination. Omptins are a family of proteases embedded in the outer membrane of Gram-negative bacteria that cleave various substrates, including host antimicrobial peptides, with a preference for cleaving at dibasic motifs. OmpT, the enterohemorrhagicEscherichia coli(EHEC) omptin, cleaves and inactivates the human cathelicidin LL-37. Similarly, the omptin CroP, found in the murine pathogenCitrobacter rodentium, which is used as a surrogate model to study human-restricted EHEC, cleaves the murine cathelicidin-related antimicrobial peptide (CRAMP). Here, we compared the abilities of OmpT and CroP to cleave LL-37 and CRAMP. EHEC OmpT degraded LL-37 and CRAMP at similar rates. In contrast,C. rodentiumCroP cleaved CRAMP more rapidly than LL-37. The different cleavage rates of LL-37 and CRAMP were independent of the bacterial background and substrate sequence specificity, as OmpT and CroP have the same preference for cleaving at dibasic sites. Importantly, LL-37 was α-helical and CRAMP was unstructured under our experimental conditions. By altering the α-helicity of LL-37 and CRAMP, we found that decreasing LL-37 α-helicity increased its rate of cleavage by CroP. Conversely, increasing CRAMP α-helicity decreased its cleavage rate. This structural basis for CroP substrate specificity highlights differences between the closely related omptins ofC. rodentiumandE. coli. In agreement with previous studies, this difference in CroP and OmpT substrate specificity suggests that omptins evolved in response to the substrates present in their host microenvironments.IMPORTANCEOmptins are recognized as key virulence factors for various Gram-negative pathogens. Their localization to the outer membrane, their active site facing the extracellular environment, and their unique catalytic mechanism make them attractive targets for novel therapeutic strategies. Gaining insights into similarities and variations between the different omptin active sites and subsequent substrate specificities will be critical to develop inhibitors that can target multiple omptins. Here, we describe subtle differences between the substrate specificities of two closely related omptins, CroP and OmpT. This is the first reported example of substrate conformation acting as a structural determinant for omptin activity between OmpT-like proteases.

scholarly journals Escherichia coli Cytosolic Glycerophosphodiester Phosphodiesterase (UgpQ) Requires Mg2+, Co2+, or Mn2+ for Its Enzyme Activity

2007 ◽  
Vol 190 (4) ◽  
pp. 1219-1223 ◽  
Author(s):  
Noriyasu Ohshima ◽  
Saori Yamashita ◽  
Naoko Takahashi ◽  
Chizu Kuroishi ◽  
Yoshitsugu Shiro ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Substrate Specificity ◽  
Phosphate Starvation ◽  
Broad Substrate Specificity ◽  
E Coli ◽  
Glycerophosphodiester Phosphodiesterase

ABSTRACT Escherichia coli cytosolic glycerophosphodiester phosphodiesterase, UgpQ, functions in the absence of other proteins encoded by the ugp operon and requires Mg2+, Mn2+, or Co2+, in contrast to Ca2+-dependent periplasmic glycerophosphodiester phosphodiesterase, GlpQ. UgpQ has broad substrate specificity toward various glycerophosphodiesters, producing sn-glycerol-3-phosphate and the corresponding alcohols. UgpQ accumulates under conditions of phosphate starvation, suggesting that it allows the utilization of glycerophosphodiesters as a source of phosphate. These results clarify how E. coli utilizes glycerophosphodiesters using two homologous enzymes, UgpQ and GlpQ.

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