Richard C. Holz

A cobalt-containing eukaryotic nitrile hydratase.

Nitrile hydratase (NHase), an industrially important enzyme that catalyzes the hydration of nitriles to their corresponding amides, has only been characterized from prokaryotic microbes. The putative NHase from the eukaryotic unicellular choanoflagellate organism Monosiga brevicollis (MbNHase) was heterologously expressed in Escherichia coli. The resulting enzyme expressed as a single polypeptide with fused α- and β-subunits linked by a seventeen-histidine region. Size-exclusion chromatography indicated that MbNHase exists primarily as an (αβ)2 homodimer in solution, analogous to the α2β2 homotetramer architecture observed for prokaryotic NHases. The NHase enzyme contained its full complement of Co(III) and was fully functional without the co-expression of an activator protein or E. coli GroES/EL molecular chaperones. The homology model of MbNHase was developed identifying Cys400, Cys403, and Cys405 as active site ligands. The results presented here provide the first experimental data for a mature and active eukaryotic NHase with fused subunits. Since this new member of the NHase family is expressed from a single gene without the requirement of an activator protein, it represents an alternative biocatalyst for industrial syntheses of important amide compounds.

The Dimerization Domain in DapE Enzymes Is required for Catalysis.

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,L-diaminopimelic 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 enzyme we engineered dimerization domain deletion mutants of DapEs from Haemophilus influenzae and Vibrio cholerae, and characterized these proteins structurally and biochemically. No activity was observed for all deletion mutants. Structural comparisons of wild-type, inactive monomeric DapE enzymes with other M20 peptidases suggest that the dimerization domain is essential for DapE enzymatic activity. Structural analysis and molecular dynamics simulations indicate that removal of the dimerization domain increased the flexibility of a conserved active site loop that may provide critical interactions with the substrate.

Identification of a Histidine Metal Ligand in the argE-Encoded N-Acetyl-L-Ornithine Deacetylase from Escherichia coli

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 dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae confirming the assignment of H355 as well as H80 as active site ligands.

The Iron-Type Nitrile Hydratase Activator Protein Is A GTPase

The Fe-type nitrile hydratase activator protein from Rhodococcus equi TG328-2 (ReNHase TG328-2) was successfully expressed and purified. Sequence analysis and homology modeling suggest that it is a G3E P-loop guanosine triphosphatase (GTPase) within the COG0523 subfamily. Kinetic studies revealed that the Fe-type activator protein is capable of hydrolyzing GTP to GDP with a kcat value of 1.2u2009×u200910-3u2005s-1 and a Km value of 40u2005μM in the presence of 5u2005mM MgCl2 in 50u2005mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid at a pH of 8.0. The addition of divalent metal ions, such as Co(II), which binds to the ReNHase TG328-2 activator protein with a Kd of 2.9u2005μM, accelerated the rate of GTP hydrolysis, suggesting that GTP hydrolysis is potentially connected to the proposed metal chaperone function of the ReNHase TG328-2 activator protein. Circular dichroism data reveal a significant conformational change upon the addition of GTP, which may be linked to the interconnectivity of the cofactor binding sites, resulting in an activator protein that can be recognized and can bind to the NHase α-subunit. A combination of these data establishes, for the first time, that the ReNHase TG328-2 activator protein falls into the COG0523 subfamily of G3E P-loop GTPases, many of which play a role in metal homeostasis processes.

Analyzing the Catalytic Role of Active Site Residues in the Fe-type Nitrile Hydratase from Comamonas testosteroni Ni1

AbstractA strictly conserved active site arginine residue (αR157) and two histidine residues (αH80 and αH81) located near the active site of the Fe-type nitrile hydratase from Comamonas testosteroni Ni1 (CtNHase), were mutated. These mutant enzymes were examined for their ability to bind iron and hydrate acrylonitrile. For the αR157A mutant, the residual activity (kcatxa0=xa010xa0±xa02xa0s−1) accounts for less than 1xa0% of the wild-type activity (kcatxa0=xa01100xa0±xa030xa0s−1) while the Km value is nearly unchanged at 205xa0±xa010xa0mM. On the other hand, mutation of the active site pocket αH80 and αH81 residues to alanine resulted in enzymes with kcat values nof 220xa0±xa040 and 77xa0±xa013xa0s−1, respectively, and Km values of 187xa0±xa011 and 179xa0±xa018xa0mM. The double mutant (αH80A/αH81A) was also prepared and provided an enzyme with a kcat value of 132xa0±xa03xa0s−1 and a Km value of 213xa0±xa061xa0mM. These data indicate that all three residues are catalytically important, but not essential. X-ray crystal structures of the αH80A/αH81A, αH80W/αH81W, and αR157A mutant CtNHase enzymes were solved to 2.0, 2.8, and 2.5xa0Å resolutions, respectively. In each mutant enzyme, hydrogen-bonding interactions crucial for the catalytic function of the αCys104-SOH ligand are disrupted. Disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion.

Analyzing the function of the insert region found between the α and β-subunits in the eukaryotic nitrile hydratase from Monosiga brevicollis

The functional roles of the (His)17 region and an insert region in the eukaryotic nitrile hydratase (NHase, EC 4.2.1.84) from Monosiga brevicollis (MbNHase), were examined. Two deletion mutants, MbNHaseΔ238-257 and MbNHaseΔ219-272, were prepared in which the (His)17 sequence and the entire insert region were removed. Each of these MbNHase enzymes provided an α2β2 heterotetramer, identical to that observed for prokaryotic NHases and contains their full complement of cobalt ions. Deletion of the (His)17 motif provides an MbNHase enzyme that is ∼55% as active as the WT enzyme when expressed in the absence of the Co-type activator (ε) protein from Pseudonocardia thermophila JCM 3095 (PtNHaseact) but ∼28% more active when expressed in the presence of PtNHaseact. MbNHaseΔ219-272 exhibits ∼55% and ∼89% of WT activity, respectively, when expressed in the absence or presence of PtNHaseact. Proteolytic cleavage of MbNHase provides an α2β2 heterotetramer that is modestly more active compared to WT MbNHase (kcatu202f=u202f163u202f±u202f4 vs 131u202f±u202f3 s-1). Combination of these data establish that neither the (His)17 nor the insert region are required for metallocentre assembly and maturation, suggesting that Co-type eukaryotic NHases utilize a different mechanism for metal ion incorporation and post-translational activation compared to prokaryotic NHases.