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Full-Text Articles in Physical Sciences and Mathematics
Identification Of A Histidine Metal Ligand In The Arge-Encoded N-Acetyl-L-Ornithine Deacetylase From Escherichia Coli, Wade C. Mcgregor, Danuta Gillner, Sabina I. Swierczek, Dali Liu, Richard C. Holz
Identification Of A Histidine Metal Ligand In The Arge-Encoded N-Acetyl-L-Ornithine Deacetylase From Escherichia Coli, Wade C. Mcgregor, Danuta Gillner, Sabina I. Swierczek, Dali Liu, Richard C. Holz
Richard C. Holz
The H355A, H355K, H80A, and H80K mutant enzymes of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were prepared, however, only the H355A enzyme was found to be soluble. Kinetic analysis of the Co(II)-loaded H355A exhibited activity levels that were 380-fold less than Co(II)-loaded WT ArgE. Electronic absorption spectra of Co(II)-loaded H355A-ArgE indicate that the bound Co(II) ion resides in a distorted, five-coordinate environment and Isothermal Titration Calorimetry (ITC) data for Zn(II) binding to the H355A enzyme provided a dissociation constant (Kd) of 39 μM. A three-dimensional homology model of ArgE was generated using the X-ray crystal structure of the …
The Active Site Sulfenic Acid Ligand In Nitrile Hydratases Can Function As A Nucleophile, Salette Martinez, Rui Wu, Ruslan Sanishvili, Dali Liu, Richard C. Holz
The Active Site Sulfenic Acid Ligand In Nitrile Hydratases Can Function As A Nucleophile, Salette Martinez, Rui Wu, Ruslan Sanishvili, Dali Liu, Richard C. Holz
Richard C. Holz
Nitrile hydratase (NHase) catalyzes the hydration of nitriles to their corresponding commercially valuable amides at ambient temperatures and physiological pH. Several reaction mechanisms have been proposed for NHase enzymes; however, the source of the nucleophile remains a mystery. Boronic acids have been shown to be potent inhibitors of numerous hydrolytic enzymes due to the open shell of boron, which allows it to expand from a trigonal planar (sp2) form to a tetrahedral form (sp3). Therefore, we examined the inhibition of the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) by boronic acids via kinetics and X-ray crystallography. Both 1-butaneboronic acid …
Analyzing The Catalytic Role Of Asp97 In The Methionine Aminopeptidase From Escherichia Coli, Sanghamitra Mitra, Kathleen M. Job, Lu Meng, Brian Bennett, Richard C. Holz
Analyzing The Catalytic Role Of Asp97 In The Methionine Aminopeptidase From Escherichia Coli, Sanghamitra Mitra, Kathleen M. Job, Lu Meng, Brian Bennett, Richard C. Holz
Richard C. Holz
An active site aspartate residue, Asp97, in the methionine aminopeptidase (MetAPs) from Escherichia coli (EcMetAP-I) was mutated to alanine, glutamate, and asparagine. Asp97 is the lone carboxylate residue bound to the crystallographically determined second metal-binding site in EcMetAP-I. These mutant EcMetAP-I enzymes have been kinetically and spectroscopically characterized. Inductively coupled plasma–atomic emission spectroscopy analysis revealed that 1.0 ± 0.1 equivalents of cobalt were associated with each of the Asp97-mutated EcMetAP-Is. The effect on activity after altering Asp97 to alanine, glutamate or asparagine is, in general, due to a ∼ 9000-fold decrease in kca towards Met-Gly-Met-Met as compared to the wild-type …
13C Nmr Analysis Of Biologically Produced Pyrene Residues By Mycobacterium Sp. Kms In The Presence Of Humic Acid, J. Karl C. Nieman, Richard C. Holz, Ronald C. Sims
13C Nmr Analysis Of Biologically Produced Pyrene Residues By Mycobacterium Sp. Kms In The Presence Of Humic Acid, J. Karl C. Nieman, Richard C. Holz, Ronald C. Sims
Richard C. Holz
Cultures of the pyrene degrading Mycobacterium sp. KMS were incubated with [4-13C]pyrene or [4,5,9,10-14C]pyrene with and without a soil humic acid standard to characterize the chemical nature of the produced residues and evaluate the potential for bonding reactions with humic acid. Cultures were subjected to a “humic acid/humin” separation at acidic pH, a duplicate separation followed by solvent extraction of the humic acid/humin fraction, and a high pH separation. 13C NMR analysis was conducted on the resulting solid extracts. Results indicated that the activity associated with solid extracts did not depend on pH and that approximately 10% of the added …
The Dimerization Domain In Dape Enzymes Is Required For Catalysis, Boguslaw Nocek, Anna Starus, Magdalena Makowska-Grzyska, Blanca Gutierrez, Stephen Sanchez, Robert Jedrzejczak, Jamey C. Mack, Kenneth W. Olsen, Andzrej Joachimiak, Richard C. Holz
The Dimerization Domain In Dape Enzymes Is Required For Catalysis, Boguslaw Nocek, Anna Starus, Magdalena Makowska-Grzyska, Blanca Gutierrez, Stephen Sanchez, Robert Jedrzejczak, Jamey C. Mack, Kenneth W. Olsen, Andzrej Joachimiak, Richard C. Holz
Richard C. Holz
The emergence of antibiotic-resistant bacterial strains underscores the importance of identifying new drug targets and developing new antimicrobial compounds. Lysine and meso-diaminopimelic acid are essential for protein production and bacterial peptidoglycan cell wall remodeling and are synthesized in bacteria by enzymes encoded within dap operon. Therefore dap enzymes may serve as excellent targets for developing a new class of antimicrobial agents. The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) converts N-succinyl-L,L-diaminopimelic acid to L,Ldiaminopimelic acid and succinate. The enzyme is composed of catalytic and dimerization domains, and belongs to the M20 peptidase family. To understand the specific role of each domain of …
The Dape-Encoded N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase From Haemophilus Influenzae Is A Dinuclear Metallohydrolase, Nathaniel J. Cosper, David L. Bienvenue, Jacob E. Shokes, Danuta M. Gilner, Takashi Tsukamoto, Robert A. Scott, Richard C. Holz
The Dape-Encoded N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase From Haemophilus Influenzae Is A Dinuclear Metallohydrolase, Nathaniel J. Cosper, David L. Bienvenue, Jacob E. Shokes, Danuta M. Gilner, Takashi Tsukamoto, Robert A. Scott, Richard C. Holz
Richard C. Holz
The Zn K-edge extended X-ray absorption fine structure (EXAFS) spectra, of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae have been recorded in the presence of one or two equivalents of Zn(II) (i.e. [Zn_(DapE)] and [ZnZn(DapE)]). The Fourier transforms of the Zn EXAFS are dominated by a peak at ca. 2.0 Å, which can be fit for both [Zn_(DapE)] and [ZnZn(DapE)], assuming ca. 5 (N,O) scatterers at 1.96 and 1.98 Å, respectively. A second-shell feature at ca. 3.34 Å appears in the [ZnZn(DapE)] EXAFS spectrum but is significantly diminished in [Zn_(DapE)]. These data show that DapE contains a dinuclear …