Mammalian cysteine dioxygenase (CDO) is a non-heme iron metalloenzyme that catalyzes the first committed step in oxidative cysteine catabolism. The active site coordination of CDO comprises a mononuclear iron ligated by the Nepsilon atoms of three protein-derived histidines, thus representing a new variant on the 2-histidine-1-carboxylate (2H1C) facial triad motif. Nitric oxide was used as a spectroscopic probe in investigating the order of substrate-O2 binding by EPR spectroscopy. In these experiments, CDO exhibits an ordered binding of l-cysteine prior to NO (and presumably O2) similar to that observed for the 2H1C class of non-heme iron enzymes. Moreover, the CDO active site is essentially unreactive toward NO in the absence of substrate, suggesting an obligate ordered binding of l-cysteine prior to NO. Typically, addition of NO to a mononuclear non-heme iron center results in the formation of an {FeNO}7 (S = 3/2) species characterized by an axial EPR spectrum with gx, gy, and gz values of approximately 4, approximately 4, and approximately 2, respectively. However, upon addition of NO to CDO in the presence of substrate l-cysteine, a low-spin {FeNO}7 (S = 1/2) signal that accounts for approximately 85% of the iron within the enzyme develops. Similar {FeNO}7 (S = 1/2) EPR signals have been observed for a variety of octahedral mononuclear iron-nitrosyl synthetic complexes; however, this type of iron-nitrosyl species is not commonly observed for non-heme iron enzymes. Substitution of l-cysteine with isosteric substrate analogues cysteamine, 3-mercaptopropionic acid, and propane thiol did not produce any analogous {FeNO}7 signals (S = 1/2 or 3/2), thus reflecting the high substrate specificity of the enzyme observed by a number of researchers. The unusual {FeNO}7 (S = 1/2) electronic configuration adopted by the substrate-bound iron-nitrosyl CDO (termed {ES-NO}7) is a result of the bidentate thiol/amine coordination of l-cysteine in the NO-bound CDO active site. DFT computations were performed to further characterize this species. The DFT-predicted geometric parameters for {ES-NO}7 are in good agreement with the crystallographically determined substrate-bound active site configuration of CDO and are consistent with known iron-nitrosyl model complexes. Moreover, the computed EPR parameters (g and A values) are in excellent agreement with experimental results for this CDO species and those obtained from comparable synthetic {FeNO}7 (S = 1/2) iron-nitrosyl complexes.

Cysteine dioxygenase (CDO) catalyzes the oxidation of L-cysteine to cysteine sulfinic acid. Deficiencies in this enzyme have been linked to autoimmune diseases and neurological disorders. The x-ray crystal structure of CDO from Mus musculus was solved to a nominal resolution of 1.75 Å. The sequence is 91% identical to that of a human homolog. The structure reveals that CDO adopts the typical ␤-barrel fold of the cupin superfamily. The NE2 atoms of His-86, -88, and -140 provide the metal binding site. The structure further revealed a covalent linkage between the side chains of Cys-93 and Tyr-157, the cysteine of which is conserved only in eukaryotic proteins. Metal analysis showed that the recombinant enzyme contained a mixture of iron, nickel, and zinc, with increased iron content associated with increased catalytic activity. Details of the predicted active site are used to present and discuss a plausible mechanism of action for the enzyme.cupin ͉ cysteine metabolism ͉ O2-activation M ouse cysteine dioxygenase (CDO) catalyzes the initial step in the biochemical pathway used for oxidation of cysteine to sulfate (1), namely the oxidation of L-cysteine to cysteine sulfinic acid as shown in Fig. 1. The enzyme activity has important medical implications because elevated cysteine levels have been associated with Parkinson's and Alzheimer's diseases (2). High cysteine-to-sulfate ratios have been observed in patients suffering from systemic lupus erthematosus and rheumatoid arthritis (3, 4). Moreover, the Hallervorden-Spatz syndrome, a neurological disorder associated with iron accumulation, has been linked to a decline in CDO activity (5).CDO displays significant sequence identity with some members of the cupin superfamily (6), which have a conserved ␤-barrel fold and share two conserved sequence motifs: G(X) 5 HXH(X) 3,4 E(X) 6 G and G(X) 5 PXG(X) 2 H(X) 3 N (6-8).The two His and Glu residues from the first motif and the His from the second motif coordinate the metal ion in germin, the superfamily archetype (9). The Mus musculus CDO sequence contains the first motif with the exception of the glutamate, which is replaced by cysteine. This substitution is conserved in other eukaryotic CDOs. The second motif is less conserved, and only the His and Asn residues are present in the mouse CDO.CDO does not require an external reductant ( Fig. 1) and incorporates both oxygen atoms from O 2 (10), which justifies the dioxygenase classification, but relatively little else is known about the reaction mechanism. The recombinant enzyme from Rattus norvegicus has been purified and characterized by steadystate kinetics (11); the mouse enzyme investigated here has an identical sequence. Reconstitution of the rat apoenzyme with various transition metals confirmed that iron was required for activity, in accord with the earlier conclusions (1). Moreover, the recombinant rat enzyme was active without a second interacting factor, despite previous reports suggesting that additional components were required (12, 13).Here, we describe the x-ra...

Carboxylate-bridged diiron hydroxylases are multicomponent enzyme complexes responsible for the catabolism of a wide range of hydrocarbons and as such have drawn attention for their mechanism of action and potential uses in bioremediation and enzymatic synthesis. These enzyme complexes use a small molecular weight effector protein to modulate the function of the hydroxylase. However, the origin of these functional changes is poorly understood. Here, we report the structures of the biologically relevant effector protein-hydroxylase complex of toluene 4-monooxygenase in 2 redox states. The structures reveal a number of coordinated changes that occur up to 25 Å from the active site and poise the diiron center for catalysis. The results provide a structural basis for the changes observed in a number of the measurable properties associated with effector protein binding. This description provides insight into the functional role of effector protein binding in all carboxylate-bridged diiron hydroxylases.crystal structure ͉ iron enzyme ͉ mechanism ͉ oxygenase C arboxylate-bridged diiron enzymes provide essential biological functions such as O 2 transport, iron sequestration, deoxyribonucleotide synthesis, fatty acid desaturation, and hydrocarbon hydroxylation (1). All diiron hydroxylase complexes include a multisubunit hydroxylase, electron transfer proteins, and a cofactorless effector protein that is unique to the diiron hydroxylase family (2). Effector proteins are required for catalysis, and their presence is associated with improved coupling (3), shifts in redox potential (4), increased rate of catalysis (3), more efficient activation of O 2 (5), and changes in regiospecificity (3, 6). Correlation of how the effector protein may induce these phenomena ultimately requires examination of high-resolution structures of the stoichiometric protein-protein complexes in multiple redox states. Previously available structures with smallmolecule analogs (7,8) or partial occupancy of an effector protein binding site (9) have provided some insight into regions of the hydroxylase that might be perturbed by these interactions. Surprisingly, however, changes in the active site were not observed, although these might reasonably be anticipated based on numerous spectroscopic studies of this enzyme family (10). Consequently, further information is needed to better understand the role of effector protein binding in catalysis by diiron hydroxylases.Toluene 4-monooxygenase (T4moH*; 200 kDa) is composed of TmoA, TmoE, and TmoB polypeptides and has an (␣␤␥) 2 quaternary structure (11). This enzyme hydroxylates toluene with high regiospecificity in the presence of its effector protein, T4moD (3, 12). Here, we report X-ray structures of resting T4moH, the stoichiometric complex of resting T4moH with T4moD, and the sodium dithionite-reduced complex to resolutions of 1.9, 1.9, and 1.7 Å respectively [see supporting information (SI) Table S1 for refinement statistics]. Comparison of these structures revealed changes within the active site and ...

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Insulin degrading enzyme (IDE) plays key roles in degrading peptides vital in type two diabetes, Alzheimer's, inflammation, and other human diseases. However, the process through which IDE recognizes peptides that tend to form amyloid fibrils remained unsolved. We used cryoEM to understand both the apo- and insulin-bound dimeric IDE states, revealing that IDE displays a large opening between the homologous ~55 kDa N- and C-terminal halves to allow selective substrate capture based on size and charge complementarity. We also used cryoEM, X-ray crystallography, SAXS, and HDX-MS to elucidate the molecular basis of how amyloidogenic peptides stabilize the disordered IDE catalytic cleft, thereby inducing selective degradation by substrate-assisted catalysis. Furthermore, our insulin-bound IDE structures explain how IDE processively degrades insulin by stochastically cutting either chain without breaking disulfide bonds. Together, our studies provide a mechanism for how IDE selectively degrades amyloidogenic peptides and offers structural insights for developing IDE-based therapies.

Antibody Fab fragments have been exploited with significant success to facilitate the structure determination of challenging macromolecules as crystallization chaperones and as molecular fiducial marks for single particle cryo-electron microscopy approaches. However, the inherent flexibility of the "elbow" regions, which link the constant and variable domains of the Fab, can introduce disorder and thus diminish their effectiveness. We have developed a phage display engineering strategy to generate synthetic Fab variants that significantly reduces elbow flexibility, while maintaining their high affinity and stability. This strategy was validated using previously recalcitrant Fab-antigen complexes where introduction of an engineered elbow region enhanced crystallization and diffraction resolution. Furthermore, incorporation of the mutations appears to be generally portable to other synthetic antibodies and may serve as a universal strategy to enhance the success rates of Fabs as structure determination chaperones.

A diiron hydroxylase reaction typically begins by combination of O2 with a diferrous center to form reactive intermediates capable of hydrocarbon hydroxylation. In this natural cycle, reducing equivalents are provided by specific interactions with electron transfer proteins. The biological process can be bypassed by combining H2O2 with a diferric center, i.e., peroxide-shunt catalysis. Here we show that toluene 4-monooxygenase has a peroxide-shunt reaction that is approximately 600-fold slower than catalysis driven by biological electron transfer. However, the toluene 4-monooxygenase hydroxylase-effector protein complex was stable in the presence of 300 mM H2O2, suggesting overall benign effects of the exogenous oxidant on active site structure and function. The X-ray structure of the toluene 4-monooxygenase hydroxylase-effector protein complex determined from crystals soaked in H2O2 revealed a bound diatomic molecule, assigned to a cis-mu-1,2-peroxo bridge. This peroxo species resides in an active site position adjacent to the hydrogen-bonding substructure established by effector protein binding and faces into the distal cavity where substrate must bind during regiospecific aromatic ring hydroxylation catalysis. These results provide a new structural benchmark for how activated intermediates may be formed and dispatched during diiron hydroxylase catalysis.

Immunoglobulin binding proteins (IBPs) are broadly used as reagents for the purification and detection of antibodies. Among the IBPs, the most widely used are Protein-A and Protein-G. The C2 domain of Protein-G from Streptococcus is a multi-specific protein domain; it possesses a high affinity (KD ~ 10 nM) for the Fc region of the IgG, but a much lower affinity (KD ~ low εM) for the constant domain of the antibody fragment (Fab), which limits some of its applications. Here, we describe the engineering of the Protein-G interface using phage display to create Protein-G-A1, a variant with 8 point mutations and an approximately 100-fold improved affinity over the parent domain for the 4D5 Fab scaffold. Protein-G-A1 is capable of robust binding to Fab fragments for numerous applications. Furthermore, we isolated a variant with pH-dependent affinity, demonstrating a 10,000-fold change in affinity from pH 7 to 4. Additional rational mutagenesis endowed Protein-G with significantly enhanced stability in basic conditions relative to the parent domain while maintaining high affinity to the Fab. This property is particularly useful to regenerate Protein-G affinity columns. Lastly, the affinity-matured Protein-G-A1 variant was tethered together to produce dimers capable of providing multivalent affinity enhancement to a low affinity antibody fragment-antigen interaction. Engineered Protein-G variants should find widespread application in the use of Fab-based affinity reagents.