High-Throughput Characterization and Optimization of Polyamide Hydrolase Activity Using Open Port Sampling Interface Mass Spectrometry.

Enzymatic biodegradation of polymers, such as polyamides (PA), has the potential to cost-effectively reduce plastic waste, but enhancements in degradation efficiency are needed. Engineering enzymes through directed evolution is one pathway toward identification of critical domains needed for improving activity. However, screening such enzymatic libraries (100s-to-1000s of samples) is time-consuming. Here we demonstrate the use of robotic autosampler (PAL) and immediate drop on demand technology (I.DOT) liquid handling systems coupled with open-port sampling interface-mass spectrometry (OPSI-MS) to screen for PA6 and PA66 hydrolysis by 6-aminohexanoate-oligomer endo-hydrolase (nylon hydrolase, NylC) in a high-throughput (8-20 s/sample) manner. The OPSI-MS technique required minimal sample preparation and was amenable to 96-well plate formats for automated processing. Enzymatic hydrolysis of PA characteristically produced soluble linear oligomer products that could be identified by OPSI-MS. Incubation temperatures and times were optimized for PA6 (65 °C, 24 h) and PA66 (75 °C, 24 h) over 108 experiments. In addition, the I.DOT/OPSI-MS quantified production of PA6 linear dimer (8.3 ± 1.6 µg/mL) and PA66 linear monomer (13.5 ± 1.5 µg/mL) by NylC with a lower limit of detection of 0.029 and 0.032 µg/mL, respectively. For PA6 and PA66, linear oligomer production corresponded to 0.096 ± 0.018% and 0.204 ± 0.028% conversion of dry pellet mass, respectively. The developed methodology is expected to be utilized to assess enzymatic hydrolysis of engineered enzyme libraries, comprising hundreds to thousands of individual samples.

Characterization of a Nitro-Forming Enzyme Involved in Fosfazinomycin Biosynthesis.

N-hydroxylating monooxygenases (NMOs) are a subclass of flavin-dependent enzymes that hydroxylate nitrogen atoms. Recently, unique NMOs that perform multiple reactions on one substrate molecule have been identified. Fosfazinomycin M (FzmM) is one such NMO, forming nitrosuccinate from aspartate (Asp) in the fosfazinomycin biosynthetic pathway in some Streptomyces sp. This work details the biochemical and kinetic analysis of FzmM. Steady-state kinetic investigation shows that FzmM performs a coupled reaction with Asp (kcat, 3.0 ± 0.01 s-1) forming nitrosuccinate, which can be converted to fumarate and nitrite by the action of FzmL. FzmM displays a 70-fold higher kcat/KM value for NADPH compared to NADH and has a narrow optimal pH range (7.5-8.0). Contrary to other NMOs where the kred is rate-limiting, FzmM exhibits a very fast kred (50 ± 0.01 s-1 at 4 °C) with NADPH. NADPH binds at a KD value of ∼400 µM, and hydride transfer occurs with pro-R stereochemistry. Oxidation of FzmM in the absence of Asp exhibits a spectrum with a shoulder at ∼370 nm, consistent with the formation of a C(4a)-hydroperoxyflavin intermediate, which decays into oxidized flavin and hydrogen peroxide at a rate 100-fold slower than the kcat. This reaction is enhanced in the presence of Asp with a slightly faster kox than the kcat, suggesting that flavin dehydration or Asp oxidation is partially rate limiting. Multiple sequence analyses of FzmM to NMOs identified conserved residues involved in flavin binding but not for NADPH. Additional sequence analysis to related monooxygenases suggests that FzmM shares sequence motifs absent in other NMOs.

Structural and Biochemical Characterization of the Flavin-Dependent Siderophore-Interacting Protein from Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic pathogen with a high mortality rate due to multi-drug-resistant strains. The synthesis and uptake of the iron-chelating siderophores acinetobactin (Acb) and preacinetobactin (pre-Acb) have been shown to be essential for virulence. Here, we report the kinetic and structural characterization of BauF, a flavin-dependent siderophore-interacting protein (SIP) required for the reduction of Fe(III) bound to Acb/pre-Acb and release of Fe(II). Stopped-flow spectrophotometric studies of the reductive half-reaction show that BauF forms a stable neutral flavin semiquinone intermediate. Reduction with NAD(P)H is very slow (k obs, 0.001 s-1) and commensurate with the rate of reduction by photobleaching, suggesting that NAD(P)H are not the physiological partners of BauF. The reduced BauF was oxidized by Acb-Fe (k obs, 0.02 s-1) and oxazole pre-Acb-Fe (ox-pre-Acb-Fe) (k obs, 0.08 s-1), a rigid analogue of pre-Acb, at a rate 3-11 times faster than that with molecular oxygen alone. The structure of FAD-bound BauF was solved at 2.85 Å and was found to share a similarity to Shewanella SIPs. The biochemical and structural data presented here validate the role of BauF in A. baumannii iron assimilation and provide information important for drug design.

Biochemical Characterization of the Two-Component Flavin-Dependent Monooxygenase Involved in Valanimycin Biosynthesis.

The flavin reductase (FRED) and isobutylamine N-hydroxylase (IBAH) from Streptomyces viridifaciens constitute a two-component, flavin-dependent monooxygenase system that catalyzes the first step in valanimycin biosynthesis. FRED is an oxidoreductase that provides the reduced flavin to IBAH, which then catalyzes the hydroxylation of isobutylamine (IBA) to isobutylhydroxylamine (IBHA). In this work, we used several complementary methods to investigate FAD binding, steady-state and rapid reaction kinetics, and enzyme-enzyme interactions in the FRED:IBAH system. The affinity of FRED for FADox is higher than its affinity for FADred, consistent with its function as a flavin reductase. Conversely, IBAH binds FADred more tightly than FADox, consistent with its role as a monooxygenase. FRED exhibits a strong preference (28-fold) for NADPH over NADH as the electron source for FAD reduction. Isothermal titration calorimetry was used to study the association of FRED and IBAH. In the presence of FAD, either oxidized or reduced, FRED and IBAH associate with a dissociation constant of 7-8 µM. No interaction was observed in the absence of FAD. These results are consistent with the formation of a protein-protein complex for direct transfer of reduced flavin from the reductase to the monooxygenase in this two-component system.

Structure and function of a flavin-dependent S-monooxygenase from garlic (Allium sativum).

Allicin is a component of the characteristic smell and flavor of garlic (Allium sativum). A flavin-containing monooxygenase (FMO) produced by A. sativum (AsFMO) was previously proposed to oxidize S-allyl-l-cysteine (SAC) to alliin, an allicin precursor. Here, we present a kinetic and structural characterization of AsFMO that suggests a possible contradiction to this proposal. Results of steady-state kinetic analyses revealed that AsFMO exhibited negligible activity with SAC; however, the enzyme was highly active with l-cysteine, N-acetyl-l-cysteine, and allyl mercaptan. We found that allyl mercaptan with NADPH was the preferred substrate-cofactor combination. Rapid-reaction kinetic analyses showed that NADPH binds tightly (KD of ∼2 µm) to AsFMO and that the hydride transfer occurs with pro-R stereospecificity. We detected the formation of a long-wavelength band when AsFMO was reduced by NADPH, probably representing the formation of a charge-transfer complex. In the absence of substrate, the reduced enzyme, in complex with NADP+, reacted with oxygen and formed an intermediate with a spectrum characteristic of C4a-hydroperoxyflavin, which decays several orders of magnitude more slowly than the kcat The presence of substrate enhanced C4a-hydroperoxyflavin formation and, upon hydroxylation, oxidation occurred with a rate constant similar to the kcat The structure of AsFMO complexed with FAD at 2.08-Å resolution features two domains for binding of FAD and NADPH, representative of class B flavin monooxygenases. These biochemical and structural results are consistent with AsFMO being an S-monooxygenase involved in allicin biosynthesis through direct formation of sulfenic acid and not SAC oxidation.

Performing anaerobic stopped-flow spectrophotometry inside of an anaerobic chamber.

The catalytic cycle of most flavin-dependent enzymes can be divided into oxidative and reductive half-reactions. Although some enzymes are oxidized by electron carrier proteins or organic compounds, many use oxygen as the final electron acceptor. In order to properly study the reductive half-reaction of flavin-dependent enzyme that react with oxygen, as in the case of oxidases and monooxygenases, it is necessary to establish anaerobic conditions that will only allow the reduction process to be monitored. The reduced flavoenzyme can be further studied by exposing it to oxygen to monitor the oxidative half-reaction. Anaerobic chambers provide an ideal environment for performing these experiments as they reliably maintain an anaerobic atmosphere inside a large workspace. A common tool used to study flavin-dependent enzymes is the stopped-flow spectrophotometry. This chapter describes methods for performing stopped-flow experiments in an anaerobic chamber. We include information about the chamber components, setting up a stopped-flow spectrophotometer inside of a chamber, preparing anaerobic solutions, and performing experiments to measure the reductive and oxidative half-reactions of flavin-dependent monooxygenases.

Evidence for the Formation of a Radical-Mediated Flavin-N5 Covalent Intermediate.

The redox-neutral reaction catalyzed by 2-haloacrylate hydratase (2-HAH) leads to the conversion of 2-chloroacrylate to pyruvate. Previous mechanistic studies demonstrated the formation of a flavin-iminium ion as an important intermediate in the 2-HAH catalytic cycle. Time-resolved flavin absorbance studies were performed in this study, and the data showed that the enzyme is capable of stabilizing both anionic and neutral flavin semiquinone species. The presence of a radical scavenger decreases the activity in a concentration-dependent manner. These data are consistent with the flavin iminium intermediate occurring by radical recombination.