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Matthew H Sazinsky

Publications and source records attributed to Matthew H Sazinsky.

13 recordsLinked to original sources
PubMed2006

X-ray structure of a hydroxylase-regulatory protein complex from a hydrocarbon-oxidizing multicomponent monooxygenase, Pseudomonas sp. OX1 phenol hydroxylase.

Phenol hydroxylase (PH) belongs to a family of bacterial multicomponent monooxygenases (BMMs) with carboxylate-bridged diiron active sites. Included are toluene/o-xylene (ToMO) and soluble methane (sMMO) monooxygenase. PH hydroxylates aromatic compounds, but unlike sMMO, it cannot oxidize alkanes despite having a similar dinuclear iron active site. Important for activity is formation of a complex between the hydroxylase and a regulatory protein component. To address how structural features of BMM hydroxylases and their component complexes may facilitate the catalytic mechanism and choice of substrate, we determined X-ray structures of native and SeMet forms of the PH hydroxylase (PHH) in complex with its regulatory protein (PHM) to 2.3 A resolution. PHM binds in a canyon on one side of the (alphabetagamma)2 PHH dimer, contacting alpha-subunit helices A, E, and F approximately 12 A above the diiron core. The structure of the dinuclear iron center in PHH resembles that of mixed-valent MMOH, suggesting an Fe(II)Fe(III) oxidation state. Helix E, which comprises part of the iron-coordinating four-helix bundle, has more pi-helical character than analogous E helices in MMOH and ToMOH lacking a bound regulatory protein. Consequently, conserved active site Thr and Asn residues translocate to the protein surface, and an approximately 6 A pore opens through the four-helix bundle. Of likely functional significance is a specific hydrogen bond formed between this Asn residue and a conserved Ser side chain on PHM. The PHM protein covers a putative docking site on PHH for the PH reductase, which transfers electrons to the PHH diiron center prior to O2 activation, suggesting that the regulatory component may function to block undesired reduction of oxygenated intermediates during the catalytic cycle. A series of hydrophobic cavities through the PHH alpha-subunit, analogous to those in MMOH, may facilitate movement of the substrate to and/or product from the active site pocket. Comparisons between the ToMOH and PHH structures provide insights into their substrate regiospecificities.

Binding Sites↗
PubMed2006

X-ray crystal structures of manganese(II)-reconstituted and native toluene/o-xylene monooxygenase hydroxylase reveal rotamer shifts in conserved residues and an enhanced view of the protein interior.

We report the X-ray crystal structures of native and manganese(II)-reconstituted toluene/o-xylene monooxygenase hydroxylase (ToMOH) from Pseudomonas stutzeri OX1 to 1.85 and 2.20 A resolution, respectively. The structures reveal that reduction of the dimetallic active site is accompanied by a carboxylate shift and alteration of the coordination environment for dioxygen binding and activation. A rotamer shift in a strategically placed asparagine 202 accompanies dimetallic center reduction and is proposed to influence protein component interactions. This rotamer shift is conserved between ToMOH and the corresponding residue in methane monooxygenase hydroxylase (MMOH). Previously unidentified hydrophobic pockets similar to those present in MMOH are assigned.

Crystallography, X-Ray↗
PubMed2006

Structural insights into dioxygen-activating copper enzymes.

Copper-containing enzymes that react with O(2) play a key role in many biological processes. Mononuclear, dinuclear and trinuclear copper centers function in O(2) binding, activation and subsequent substrate oxidation. Recent advances in the structural biology of O(2)-activating copper enzymes range from the identification of novel copper centers, such as that of particulate methane monooxygenase, to the elucidation of the details of O(2) binding and reactivity in peptidylglycine alpha-hydroxylating monooxygenase. Structures of phenoxazinone synthase and Fet3 contribute to our understanding of multicopper oxidases. Additionally, details of the tyrosinase structure provide new insight into how dicopper sites confer substrate specificity. A common theme for each of these enzymes is that the protein scaffold plays a major role in dictating the overall function.

Catalytic Domain↗
PubMed2006

Structure of the actuator domain from the Archaeoglobus fulgidus Cu(+)-ATPase.

Copper homeostasis is maintained in part by membrane-bound P(1B)-type ATPases that are found in all organisms and drive the transport of this essential, yet toxic, metal ion across cellular membranes. CopA from Archaeoglobus fulgidus is a hyperthermophilic member of this ATPase subfamily and is homologous to the human Wilson and Menkes disease ATPases. To gain insight into Cu(+)-ATPase function, the structure of the CopA actuator domain (A-domain) was determined to 1.65 A resolution. The CopA A-domain functions to couple ATP hydrolysis in the ATP binding domain (ATPBD) with structural rearrangements of critical transmembrane segments. Its fold is quite similar to that of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1) A-domain, with the exception of an external loop region. On the basis of sequence and structural comparisons, specific residues that probably interact with the CopA ATPBD have been identified. Comparisons to the Wilson and Menkes disease A-domains reveal the presence of an additional loop that may be associated with regulatory functions in eukaryotic Cu(+)-ATPases. Finally, several mutations in the Wilson and Menkes disease ATPases occur in the A-domain, and their likely effects on function can be inferred from the CopA A-domain structure.

Adenosine Triphosphatases↗
PubMed2006

Structure of the ATP binding domain from the Archaeoglobus fulgidus Cu+-ATPase.

The P-type ATPases translocate cations across membranes using the energy provided by ATP hydrolysis. CopA from Archaeoglobus fulgidus is a hyperthermophilic ATPase responsible for the cellular export of Cu+ and is a member of the heavy metal P1B-type ATPase subfamily, which includes the related Wilson and Menkes diseases proteins. The Cu+-ATPases are distinct from their P-type counter-parts in ion binding sequences, membrane topology, and the presence of cytoplasmic metal binding domains, suggesting that they employ alternate forms of regulation and novel mechanisms of ion transport. To gain insight into Cu+-ATPase function, the structure of the CopA ATP binding domain (ATPBD) was determined to 2.3 A resolution. Similar to other P-type ATPases, the ATPBD includes nucleotide binding (N-domain) and phosphorylation (P-domain) domains. The ATPBD adopts a closed conformation similar to the nucleotide-bound forms of the Ca2+-ATPase. The CopA ATPBD is much smaller and more compact, however, revealing the minimal elements required for ATP binding, hydrolysis, and enzyme phosphorylation. Structural comparisons to the AMP-PMP-bound form of the Escherichia coli K+-transporting Kdp-ATPase and to the Wilson disease protein N-domain indicate that the five conserved N-domain residues found in P1B-type ATPases, but not in the other families, most likely participate in ATP binding. By contrast, the P-domain includes several residues conserved among all P-type ATPases. Finally, the CopA ATPBD structure provides a basis for understanding the likely structural and functional effects of various mutations that lead to Wilson and Menkes diseases.

Adenosine Triphosphatases↗
PubMed2006

Correlating structure with function in bacterial multicomponent monooxygenases and related diiron proteins.

Bacterial multicomponent monooxygenases (BMMs) catalyze the O2-dependent hydroxylation of hydrocarbons at a carboxylate-bridged diiron center similar to those that occur in a variety of dimetallic oxygen-utilizing enzymes. BMMs have found numerous biodegradation and biocatalytic applications. Recent investigations have begun to reveal how BMMs perform their C-H bond activation chemistry and why these enzymes may be mechanistically different from other related diiron proteins. The structures of the BMM component proteins and of complexes between them provide insights into the tuning of the dinuclear iron center and the enzyme mechanism. Selected findings are compared and contrasted with the properties of other carboxylate-bridged diiron proteins, revealing common structural and functional themes.

Bacteria↗
PubMed2005

X-ray absorption spectroscopic study of the reduced hydroxylases of methane monooxygenase and toluene/o-xylene monooxygenase: differences in active site structure and effects of the coupling proteins MMOB and ToMOD.

The diiron active sites of the reduced hydroxylases from methane monooxygenase (MMOH(red)) and toluene/o-xylene monooxygenase (ToMOH(red)) have been investigated by X-ray absorption spectroscopy (XAS). Results of Fe K-edge and extended X-ray absorption fine structure analysis reveal subtle differences between the hydroxylases that may be correlated to access of the active site. XAS data were also recorded for each hydroxylase in the presence of its respective coupling protein. MMOB affects the outer-shell scattering contributions in the diiron site of MMOH(red), whereas ToMOD exerts its main effect on the first-shell ligation of ToMOH(red); it also causes a slight decrease in the Fe-Fe separation. These results provide an initial step toward delineating the differences in structure and reactivity in bacterial multicomponent monooxygenase proteins.

Bacteria↗
PubMed2005

Product bound structures of the soluble methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath): protein motion in the alpha-subunit.

The soluble methane monooxygenase hydroxylase (MMOH) alpha-subunit contains a series of cavities that delineate the route of substrate entrance to and product egress from the buried carboxylate-bridged diiron center. The presence of discrete cavities is a major structural difference between MMOH, which can hydroxylate methane, and toluene/o-xylene monooxygenase hydroxylase (ToMOH), which cannot. To understand better the functions of the cavities and to investigate how an enzyme designed for methane hydroxylation can also accommodate larger substrates such as octane, methylcubane, and trans-1-methyl-2-phenylcyclopropane, MMOH crystals were soaked with an assortment of different alcohols and their X-ray structures were solved to 1.8-2.4 A resolution. The product analogues localize to cavities 1-3 and delineate a path of product exit and/or substrate entrance from the active site to the surface of the protein. The binding of the alcohols to a position bridging the two iron atoms in cavity 1 extends and validates previous crystallographic, spectroscopic, and computational work indicating this site to be where substrates are hydroxylated and products form. The presence of these alcohols induces perturbations in the amino acid side-chain gates linking pairs of cavities, allowing for the formation of a channel similar to one observed in ToMOH. Upon binding of 6-bromohexan-1-ol, the pi helix formed by residues 202-211 in helix E of the alpha-subunit is extended through residue 216, changing the orientations of several amino acid residues in the active site cavity. This remarkable secondary structure rearrangement in the four-helix bundle has several mechanistic implications for substrate accommodation and the function of the effector protein, MMOB.

Alcohols↗
PubMed2005

Fourier transform infrared characterization of the azido complex of methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath).

The azido complex formed in oxidized methane monooxygenase from Methylococcus capsulatus (Bath) was investigated with resonance Raman and FTIR techniques. These experiments show the presence of a nuas(NNN) at approximately 2077 cm-1 which splits to two components at 2059 and 2073 cm-1 with 15N14N2. The vibrational data are assigned to an azido complex bound terminally to one iron(III) at the diiron center. When the azido complex is illuminated at 15 K, a new nuas(NNN) is observed at 2136 cm-1 which is assigned to a photodissociated HN3 within the substrate pocket. We propose a model where an aqua ligand engages a hydrogen bond interaction with the 1N atom of the azido group and acts as at a proton donor during the photolysis process.

Azides↗
PubMed2004

Preparation and X-ray structures of metal-free, dicobalt and dimanganese forms of soluble methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath).

A three-component soluble methane monooxygenase (sMMO) enzyme system catalyzes the hydroxylation of methane to methanol at a carboxylate-bridged diiron center housed in the alpha-subunit of the hydroxylase (MMOH). Catalysis is facilitated by the presence of a regulatory protein (MMOB) and inhibited by MMOD, a protein of unknown function encoded in the sMMO operon. Both MMOB and MMOD are presumed to bind to the same region of the MMOH alpha-subunit. A colorimetric method for monitoring removal of Fe(II) from MMOH was developed using 1,10-phenanthroline and yields apo MMOH with <0.1 Fe/homodimer. With the use of this method, it was possible to investigate the X-ray structure of the apoenzyme and to perform metal reconstitution studies. Using MMOH from Methylococccus capsulatus (Bath), the effects of MMOB and MMOD on metal binding were studied and structural perturbations relevant to the function of this enzyme were identified. X-ray crystal structures of the apo, Mn(II)-soaked, and Co(II)-grown MMOH, determined to 2.3 A or greater resolution, reveal that the presence of metal ions is essential for the proper folding of helices E, F, and H of the alpha-subunit. The active sites of Mn(II)-soaked and Co(II)-grown MMOH are similar to that of reduced, native MMOH with notable differences in the metal-metal distances and ligand coordination sphere that may reflect how this dinuclear metal center might change in the presence of MMOB. MMOB and MMOD decrease the rate of removal of Fe(II) from the enzyme by 22- and 16-fold, respectively. On the basis of previous studies, it is hypothesized that MMOB, and perhaps MMOD, function to block solvent access to the MMOH active site. Finally, ITC studies and the observed disorder in helices E, F, and H in the apo and Mn(II)-soaked structures suggest that these regions of MMOH are critical for MMOB and MMOD binding.

Apoproteins↗
PubMed2004

Determination by X-ray absorption spectroscopy of the Fe-Fe separation in the oxidized form of the hydroxylase of methane monooxygenase alone and in the presence of MMOD.

The diiron active site in the hydroxylase of Methylococcus capsulatus (Bath) methane monooxygenase (MMOH) has been studied in the oxidized form by X-ray absorption spectroscopy (XAS). Previous investigations by XAS and X-ray crystallography have identified two different distances (3.0 and 3.4 angstroms) between the two Fe atoms in the dinuclear site. The present study has employed a systematic extended X-ray absorption fine structure (EXAFS) fitting methodology, utilizing known and simulated active site and relevant model structures, to determine unambiguously the Fe-Fe separation in the oxidized form of MMOH. Consistent and unique fits were only possible for an Fe-Fe distance of 3.0 angstroms. This methodology was then applied to study potential changes in the active site local structure in the presence of MMOD, a protein of unknown function in multicomponent MMO. Fe K-edge and EXAFS analyses revealed negligible changes in the diiron site electronic and geometric structure upon addition of MMOD to oxidized MMOH.

Binding Sites↗
PubMed2004

Crystal structure of the toluene/o-xylene monooxygenase hydroxylase from Pseudomonas stutzeri OX1. Insight into the substrate specificity, substrate channeling, and active site tuning of multicomponent monooxygenases.

The four-component toluene/o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 is capable of oxidizing arenes, alkenes, and haloalkanes at a carboxylate-bridged diiron center similar to that of soluble methane monooxygenase (sMMO). The remarkable variety of substrates accommodated by ToMO invites applications ranging from bioremediation to the regio- and enantiospecific oxidation of hydrocarbons on an industrial scale. We report here the crystal structures of the ToMO hydroxylase (ToMOH), azido ToMOH, and ToMOH containing the product analogue 4-bromophenol to 2.3 A or greater resolution. The catalytic diiron(III) core resembles that of the sMMO hydroxylase, but aspects of the alpha2beta2gamma2 tertiary structure are notably different. Of particular interest is a 6-10 A-wide channel of approximately 35 A in length extending from the active site to the protein surface. The presence of three bromophenol molecules in this space confirms this route as a pathway for substrate entrance and product egress. An analysis of the ToMOH active site cavity offers insights into the different substrate specificities of multicomponent monooxygenases and explains the behavior of mutant forms of homologous enzymes described in the literature.

Azides↗