Design, Solution Characterization, and Crystallographic Structure of an Abiological Mn-Porphyrin-Binding Protein Capable of Stabilizing a Mn(V) Species.

protein design offers the opportunity to test our understanding of how metalloproteins perform difficult transformations. Attaining high-resolution structural information is critical to understanding how such designs function. There have been many successes in the design of porphyrin-binding proteins; however, crystallographic characterization has been elusive, limiting what can be learned from such studies as well as the extension to new functions.

The styrene monooxygenase system.

Styrene monooxygenases are soluble two-component flavoproteins that catalyze the NADH and FAD-dependent enantioselective epoxidation of styrene to styrene oxide in the aqueous phase. These enzymes present interesting mechanistic features and potential as catalysts in biotechnological applications ranging from green chemical synthesis to bioremediation. This chapter presents approaches for the expression of the reductase (SMOB, StyB) and epoxidase (SMOA, StyA) components of SMO from pET-vectors as native or N-terminally histidine-tagged proteins in commercial strains of E.

Structure and biochemistry of phenylacetaldehyde dehydrogenase from the Pseudomonas putida S12 styrene catabolic pathway.

Phenylacetaldehyde dehydrogenase catalyzes the NAD-dependent oxidation of phenylactealdehyde to phenylacetic acid in the styrene catabolic and detoxification pathway of Pseudomonas putida (S12). Here we report the structure and mechanistic properties of the N-terminally histidine-tagged enzyme, NPADH. The 2.

Designed metalloprotein stabilizes a semiquinone radical.

Enzymes use binding energy to stabilize their substrates in high-energy states that are otherwise inaccessible at ambient temperature. Here we show that a de novo designed Zn(II) metalloprotein stabilizes a chemically reactive organic radical that is otherwise unstable in aqueous media. The protein binds tightly to and stabilizes the radical semiquinone form of 3,5-di-tert-butylcatechol.

FAD C(4a)-hydroxide stabilized in a naturally fused styrene monooxygenase.

StyA2B represents a new class of styrene monooxygenases that integrates flavin-reductase and styrene-epoxidase activities into a single polypeptide. This naturally-occurring fusion protein offers new avenues for studying and engineering biotechnologically relevant enantioselective biochemical epoxidation reactions. Stopped-flow kinetic studies of StyA2B reported here identify reaction intermediates similar to those reported for the separate reductase and epoxidase components of related two-component systems.

Structure and mechanism of styrene monooxygenase reductase: new insight into the FAD-transfer reaction.

The two-component flavoprotein styrene monooxygenase (SMO) from Pseudomonas putida S12 catalyzes the NADH- and FAD-dependent epoxidation of styrene to styrene oxide. In this study, we investigate the mechanism of flavin reduction and transfer from the reductase (SMOB) to the epoxidase (NSMOA) component and report our findings in light of the 2.2 Å crystal structure of SMOB.

Accelerated hydrolysis of α-halo and α-cyano pyridinium relative to uracil derivatives: a model for ODCase-catalyzed hydrolysis of 6-cyanoUMP.

α-Halo and α-cyano pyridiniums were found to undergo facile hydrolysis, in contrast to the sluggish reactions of corresponding uracils. The greatly enhanced rates found with pyridinium compounds have indicated a possible source of the rate acceleration seen in the hydrolysis of 6-cyanouridine 5'-monophosphate catalyzed by orotidine 5'-monophosphate decarboxylase.

Nature of the reaction intermediates in the flavin adenine dinucleotide-dependent epoxidation mechanism of styrene monooxygenase.

Styrene monooxygenase (SMO) is a two-component flavoenzyme composed of an NADH-specific flavin reductase (SMOB) and FAD-specific styrene epoxidase (NSMOA). NSMOA binds tightly to reduced FAD and catalyzes the stereospecific addition of one atom of molecular oxygen to the vinyl side chain of styrene in the enantioselective synthesis of S-styrene oxide. In this mechanism, molecular oxygen first reacts with NSMOA(FAD(red)) to yield an FAD C(4a)-peroxide intermediate.

Structure and ligand binding properties of the epoxidase component of styrene monooxygenase .

Styrene monooxygenase (SMO) is a two-component flavoprotein monooxygenase that transforms styrene to styrene oxide in the first step of the styrene catabolic and detoxification pathway of Pseudomonas putida S12. The crystal structure of the N-terminally histidine-tagged epoxidase component of this system, NSMOA, determined to 2.3 A resolution, indicates the enzyme exists as a homodimer in which each monomer forms two distinct domains.

Intermolecular electron-transfer reactions in soluble methane monooxygenase: a role for hysteresis in protein function.

Electron transfer from reduced nicotinamide adenine dinucleotide (NADH) to the hydroxylase component (MMOH) of soluble methane monooxygenase (sMMO) primes its non-heme diiron centers for reaction with dioxygen to generate high-valent iron intermediates that convert methane to methanol. This intermolecular electron-transfer step is facilitated by a reductase (MMOR), which contains [2Fe-2S] and flavin adenine dinucleotide (FAD) prosthetic groups. To investigate interprotein electron transfer, chemically reduced MMOR was mixed rapidly with oxidized MMOH in a stopped-flow apparatus, and optical changes associated with reductase oxidation were recorded.

Mechanism of flavin transfer and oxygen activation by the two-component flavoenzyme styrene monooxygenase.

Styrene monooxygenase (SMO) from Pseudomonas putida S12 is a two-component flavoenzyme composed of the NADH-specific flavin reductase, SMOB, and FAD-specific styrene epoxidase, SMOA. Here, we report the cloning, and expression of native and histidine-tagged versions of SMOA and SMOB and studies of the flavin transfer and styrene oxygenation reactions. In the reductive half-reaction, SMOB catalyzes the two-electron reduction of FAD with a turnover number of 3200 s(-1).