The structure of the colorectal cancer-associated enzyme GalNAc-T12 reveals how nonconserved residues dictate its function

Polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts) initiate mucin type O-glycosylation by catalyzing the transfer of N-acetylgalactosamine (GalNAc) to Ser or Thr on a protein substrate. Inactive and partially active variants of the isoenzyme GalNAc-T12 are present in subsets of patients with colorectal cancer, and several of these variants alter nonconserved residues with unknown functions. While previous biochemical studies have demonstrated that GalNAc-T12 selects for peptide and glycopeptide substrates through unique interactions with its catalytic and lectin domains, the molecular basis for this distinct substrate selectivity remains elusive. Here we examine the molecular basis of the activity and substrate selectivity of GalNAc-T12. The X-ray crystal structure of GalNAc-T12 in complex with a di-glycosylated peptide substrate reveals how a nonconserved GalNAc binding pocket in the GalNAc-T12 catalytic domain dictates its unique substrate selectivity. In addition, the structure provides insight into how colorectal cancer mutations disrupt the activity of GalNAc-T12 and illustrates how the rules dictating GalNAc-T12 function are distinct from those for other GalNAc-Ts.

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Insight into the binding affinity of thiourea in the calcium binding pocket of proteinase K, through high resolution X-ray crystallography

Bioorganic Chemistry ◽

10.1016/j.bioorg.2019.103443 ◽

2020 ◽

Vol 94 ◽

pp. 103443

Author(s):

Malik Shoaib Ahmad ◽

Zeeshan Akbar ◽

M. Iqbal Choudhary

Keyword(s):

High Resolution ◽

Binding Affinity ◽

Calcium Binding ◽

Binding Pocket ◽

Proteinase K ◽

X Ray ◽

X Ray Crystallography ◽

Insight Into

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The X-Ray Structure of the Haloalcohol Dehalogenase HheA from Arthrobacter sp. Strain AD2: Insight into Enantioselectivity and Halide Binding in the Haloalcohol Dehalogenase Family

Journal of Bacteriology ◽

10.1128/jb.01866-05 ◽

2006 ◽

Vol 188 (11) ◽

pp. 4051-4056 ◽

Cited By ~ 30

Author(s):

René M. de Jong ◽

Kor H. Kalk ◽

Lixia Tang ◽

Dick B. Janssen ◽

Bauke W. Dijkstra

Keyword(s):

Tertiary Structure ◽

Halogen Bond ◽

Environmental Pollutants ◽

Binding Pocket ◽

Catalytic Triad ◽

Side Chain ◽

Structural Determinants ◽

Substrate Binding Pocket ◽

X Ray ◽

Insight Into

ABSTRACT Haloalcohol dehalogenases are bacterial enzymes that cleave the carbon-halogen bond in short aliphatic vicinal haloalcohols, like 1-chloro-2,3-propanediol, some of which are recalcitrant environmental pollutants. They use a conserved Ser-Tyr-Arg catalytic triad to deprotonate the haloalcohol oxygen, which attacks the halogen-bearing carbon atom, producing an epoxide and a halide ion. Here, we present the X-ray structure of the haloalcohol dehalogenase HheAAD2 from Arthrobacter sp. strain AD2 at 2.0-Å resolution. Comparison with the previously reported structure of the 34% identical enantioselective haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1 shows that HheAAD2 has a similar quaternary and tertiary structure but a much more open substrate-binding pocket. Docking experiments reveal that HheAAD2 can bind both enantiomers of the haloalcohol substrate 1-p-nitrophenyl-2-chloroethanol in a productive way, which explains the low enantiopreference of HheAAD2. Other differences are found in the halide-binding site, where the side chain amino group of Asn182 is in a position to stabilize the halogen atom or halide ion in HheAAD2, in contrast to HheC, where a water molecule has taken over this role. These results broaden the insight into the structural determinants that govern reactivity and selectivity in the haloalcohol dehalogenase family.

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Tipiracil binds to uridine site and inhibits Nsp15 endoribonuclease NendoU from SARS-CoV-2

10.1101/2020.06.26.173872 ◽

2020 ◽

Cited By ~ 2

Author(s):

Youngchang Kim ◽

Jacek Wower ◽

Natalia Maltseva ◽

Changsoo Chang ◽

Robert Jedrzejczak ◽

...

Keyword(s):

Colorectal Cancer ◽

Active Site ◽

Catalytic Domain ◽

Binding Pocket ◽

Rapid Identification ◽

Innate Immune ◽

Manganese Ions ◽

Antiviral Compounds ◽

Sense Strand ◽

Cell Assays

ABSTRACTSARS-CoV-2 Nsp15 is a uridylate-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family. It degrades the polyuridine extensions in (−) sense strand of viral RNA and some non-translated RNA on (+) sense strand. This activity seems to be responsible for the interference with the innate immune response and evasion of host pattern recognition. Nsp15 is highly conserved in coronaviruses suggesting that its activity is important for virus replication. Here we report first structures with bound nucleotides and show that SARS-CoV-2 Nsp15 specifically recognizes U in a pattern previously predicted for EndoU. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. Inhibitors of Nsp15 have been reported but not actively pursued into therapeutics. The current COVID-19 pandemic brought to attention the repurposing of existing drugs and the rapid identification of new antiviral compounds. Tipiracil is an FDA approved drug that is used with trifluridine in the treatment of colorectal cancer. Here, we combine crystallography, biochemical and whole cell assays, and show that this compound inhibits SARS-CoV-2 Nsp15 and interacts with the uridine binding pocket of the enzyme’s active site, providing basis for the uracil scaffold-based drug development.

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Molecular basis for catabolism of the abundant metabolite trans-4-hydroxy-L-proline by a microbial glycyl radical enzyme

eLife ◽

10.7554/elife.51420 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Lindsey RF Backman ◽

Yolanda Y Huang ◽

Mary C Andorfer ◽

Brian Gold ◽

Ronald T Raines ◽

...

Keyword(s):

Active Site ◽

Metabolic Pathways ◽

Molecular Basis ◽

Radical Mechanism ◽

Human Gut ◽

Clostridioides Difficile ◽

Glycyl Radical ◽

Biochemical Studies ◽

Glycyl Radical Enzyme ◽

Insight Into

The glycyl radical enzyme (GRE) superfamily utilizes a glycyl radical cofactor to catalyze difficult chemical reactions in a variety of anaerobic microbial metabolic pathways. Recently, a GRE, trans-4-hydroxy-L-proline (Hyp) dehydratase (HypD), was discovered that catalyzes the dehydration of Hyp to (S)-Δ1-pyrroline-5-carboxylic acid (P5C). This enzyme is abundant in the human gut microbiome and also present in prominent bacterial pathogens. However, we lack an understanding of how HypD performs its unusual chemistry. Here, we have solved the crystal structure of HypD from the pathogen Clostridioides difficile with Hyp bound in the active site. Biochemical studies have led to the identification of key catalytic residues and have provided insight into the radical mechanism of Hyp dehydration.

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Molecular basis for the broad substrate selectivity of a peptide prenyltransferase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1609869113 ◽

2016 ◽

Vol 113 (49) ◽

pp. 14037-14042 ◽

Cited By ~ 23

Author(s):

Yue Hao ◽

Elizabeth Pierce ◽

Daniel Roe ◽

Maho Morita ◽

John A. McIntosh ◽

...

Keyword(s):

Molecular Basis ◽

Structural Basis ◽

Substrate Selectivity ◽

Peptide Substrate ◽

Peptide Substrates ◽

Recognition Motif ◽

Substrate Tolerance ◽

Computational Analyses ◽

Macrocyclic Peptide ◽

Large Peptide

The cyanobactin prenyltransferases catalyze a series of known or unprecedented reactions on millions of different substrates, with no easily observable recognition motif and exquisite regioselectivity. Here we define the basis of broad substrate tolerance for the otherwise uncharacterized TruF family. We determined the structures of the Tyr-prenylating enzyme PagF, in complex with an isoprenoid donor analog and a panel of linear and macrocyclic peptide substrates. Unexpectedly, the structures reveal a truncated barrel fold, wherein binding of large peptide substrates is necessary to complete a solvent-exposed hydrophobic pocket to form the catalytically competent active site. Kinetic, mutational, chemical, and computational analyses revealed the structural basis of selectivity, showing a small motif within peptide substrates that is sufficient for recognition by the enzyme. Attaching this 2-residue motif to two random peptides results in their isoprenylation by PagF, demonstrating utility as a general biocatalytic platform for modifications on any peptide substrate.

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Quantitative Analysis Of X-Ray Images From Geological Materials

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010013482x ◽

1990 ◽

Vol 48 (2) ◽

pp. 244-245

Author(s):

J. M. Paque ◽

R. Browning ◽

P. L. King ◽

P. Pianetta

Keyword(s):

Quantitative Analysis ◽

Bulk Composition ◽

Principal Component ◽

Analytical Description ◽

Bulk Sample ◽

Geological Samples ◽

X Ray ◽

Software And Hardware ◽

And Storage ◽

Insight Into

Geological samples typically contain many minerals (phases) with multiple element compositions. A complete analytical description should give the number of phases present, the volume occupied by each phase in the bulk sample, the average and range of composition of each phase, and the bulk composition of the sample. A practical approach to providing such a complete description is from quantitative analysis of multi-elemental x-ray images.With the advances in recent years in the speed and storage capabilities of laboratory computers, large quantities of data can be efficiently manipulated. Commercial software and hardware presently available allow simultaneous collection of multiple x-ray images from a sample (up to 16 for the Kevex Delta system). Thus, high resolution x-ray images of the majority of the detectable elements in a sample can be collected. The use of statistical techniques, including principal component analysis (PCA), can provide insight into mineral phase composition and the distribution of minerals within a sample.

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Characterization of Recombinant Human Coagulation Factor XFriuli

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650267 ◽

1996 ◽

Vol 75 (02) ◽

pp. 313-317 ◽

Cited By ~ 8

Author(s):

D J Kim ◽

A Girolami ◽

H L James

Keyword(s):

Catalytic Domain ◽

Coagulation Factor ◽

Molecular Size ◽

Binding Pocket ◽

Site Directed Mutagenesis ◽

Bleeding Tendency ◽

Factor X ◽

Amino Terminal ◽

Normal Plasma ◽

Plasma Factor

SummaryNaturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational y-carboxy-glutamic acid (Gla) and (β-hydroxy aspartic acid (β-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel (3-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.

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Copper–oxygen Dynamics in Tyrosinase

10.26434/chemrxiv.9255251 ◽

2019 ◽

Author(s):

Nobutaka Fujieda ◽

Sachiko Yanagisawa ◽

Minoru Kubo ◽

Genji Kurisu ◽

Shinobu Itoh

Keyword(s):

Catalytic Mechanism ◽

Substrate Binding ◽

Active Form ◽

Copper Ion ◽

Atomic Resolution ◽

Oxygen Species ◽

X Ray ◽

Oxygen Dynamics ◽

Insight Into ◽

Copper Centre

To unveil the activation of dioxygen on the copper centre (Cu2O2core) of tyrosinase, we performed X-ray crystallograpy with active-form tyrosinase at near atomic resolution. This study provided a novel insight into the catalytic mechanism of the tyrosinase, including the rearrangement of copper-oxygen species as well as the intramolecular migration of copper ion induced by substrate-binding.

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