Structure and function of aerotolerant, multiple-turnover THI4 thiazole synthases

Plant and fungal THI4 thiazole synthases produce the thiamin thiazole moiety in aerobic conditions via a single-turnover suicide reaction that uses an active-site Cys residue as sulfur donor. Multiple-turnover (i.e. catalytic) THI4s lacking an active-site Cys (non-Cys THI4s) that use sulfide as sulfur donor have been biochemically characterized – but only from archaeal methanogens that are anaer­obic, O2-sensitive hyperthermophiles from sulfide-rich habitats. These THI4s prefer iron as cofactor. A survey of prokaryote genomes uncovered non-Cys THI4s in aerobic mesophiles from sulfide-poor habitats, suggesting that multiple-turnover THI4 operation is possible in aerobic, mild, low-sulfide conditions. This was confirmed by testing 23 representative non-Cys THI4s for complementation of an Escherichia coli ΔthiG thiazole auxotroph in aerobic conditions. Sixteen were clearly active, and more so when intracellular sulfide level was raised by supplying Cys, demonstrating catalytic function in the presence of O2 at mild temperatures and indicating use of sulfide or a sulfide metabolite as sulfur donor. Comparative genomic evidence linked non-Cys THI4s with proteins from families that bind, transport, or metabolize cobalt or other heavy metals. The crystal structure of the aerotolerant bacterial Thermovibrio ammonificans THI4 was determined to probe the molecular basis of aerotolerance. The structure suggested no large deviations compared to the structures of THI4s from O2-sensitive methanogens, but is consistent with an alternative catalytic metal. Together with complementation data, use of cobalt rather than iron was supported. We conclude that catalytic THI4s can indeed operate aerobically and that the metal cofactor inserted is a likely natural determinant of aerotolerance.

Structure and function of aerotolerant, multiple-turnover THI4 thiazole synthases

Plant and fungal THI4 thiazole synthases produce the thiamin thiazole moiety in aerobic conditions via a single–turnover suicide reaction that uses an active–site Cys residue as sulfur donor. Multiple turnover (i.e. catalytic) THI4s lacking an active–site Cys (non–Cys THI4s) that use sulfide as sulfur donor have been characterized—but only from archaeal methanogens that are anaerobic, O2–sensitivehyperthermophiles from sulfide–rich habitats. These THI4s prefer iron as cofactor. A survey of prokaryote genomes uncovered non–Cys THI4s in aerobic mesophiles from sulfide–poor habitats, suggesting that multiple–turnover THI4 operation is possible in aerobic, mild, low–sulfide conditions. This was confirmed by testing 23 representative non–Cys THI4s for complementation of an Escherichia coli ΔthiG thiazole auxotroph in aerobic conditions. Sixteen were active, and more so when intracellular sulfidelevel was raised by supplying Cys, demonstrating that they function in the presence of O2 at mild temperatures and indicating they use sulfide or a sulfide metabolite as sulfur donor. Comparative genomic evidence linked non–Cys THI4s with proteins from families that bind, transport, or metabolize cobalt or other heavy metals. The crystal structure of the aerotolerant bacterial Thermovibrio ammonificans THI4 was determined to probe the molecular basis of aerotolerance. The structure suggested no large deviations compared to the structures of THI4s from O2–sensitive methanogens but is consistent with an alternative catalytic metal. Together with complementation data, the use of cobalt rather than iron was supported. We conclude that catalytic THI4s can indeed operate aerobically and that the metal cofactor inserted is a likely natural determinant of aerotolerance.

Cage effects control the mechanism of methane hydroxylation in zeolites

Catalytic conversion of methane to methanol remains an economically tantalizing but fundamentally challenging goal. Current technologies based on zeolites deactivate too rapidly for practical application. We found that similar active sites hosted in different zeolite lattices can exhibit markedly different reactivity with methane, depending on the size of the zeolite pore apertures. Whereas zeolite with large pore apertures deactivates completely after a single turnover, 40% of active sites in zeolite with small pore apertures are regenerated, enabling a catalytic cycle. Detailed spectroscopic characterization of reaction intermediates and density functional theory calculations show that hindered diffusion through small pore apertures disfavors premature release of CH3 radicals from the active site after C-H activation, thereby promoting radical recombination to form methanol rather than deactivated Fe-OCH3 centers elsewhere in the lattice.

Single-Turnover Variable Chlorophyll Fluorescence as a Tool for Assessing Phytoplankton Photosynthesis and Primary Productivity: Opportunities, Caveats and Recommendations

Phytoplankton photosynthetic physiology can be investigated through single-turnover variable chlorophyll fluorescence (ST-ChlF) approaches, which carry unique potential to autonomously collect data at high spatial and temporal resolution. Over the past decades, significant progress has been made in the development and application of ST-ChlF methods in aquatic ecosystems, and in the interpretation of the resulting observations. At the same time, however, an increasing number of sensor types, sampling protocols, and data processing algorithms have created confusion and uncertainty among potential users, with a growing divergence of practice among different research groups. In this review, we assist the existing and upcoming user community by providing an overview of current approaches and consensus recommendations for the use of ST-ChlF measurements to examine in-situ phytoplankton productivity and photo-physiology. We argue that a consistency of practice and adherence to basic operational and quality control standards is critical to ensuring data inter-comparability. Large datasets of inter-comparable and globally coherent ST-ChlF observations hold the potential to reveal large-scale patterns and trends in phytoplankton photo-physiology, photosynthetic rates and bottom-up controls on primary productivity. As such, they hold great potential to provide invaluable physiological observations on the scales relevant for the development and validation of ecosystem models and remote sensing algorithms.

Phytoplankton Photophysiology Utilities: A Python Toolbox for the Standardization of Processing Active Chlorophyll-a Fluorescence Data

The uptake and application of single turnover chlorophyll fluorometers to the study of phytoplankton ecosystem status and microbial functions has grown considerably in the last two decades. However, standardization of measurement protocols, processing of fluorescence transients and quality control of derived photosynthetic parameters is still lacking and makes community goals of large global databases of high-quality data unrealistic. We introduce the Python package Phytoplankton Photophysiology Utilities (PPU), an adaptable and open-source interface between Fast Repetition Rate and Fluorescence Induction and Relaxation instruments and python. The PPU package includes a variety of functions for the loading, processing and quality control of single turnover fluorescence transients from many commercially available instruments. PPU provides the user with greater flexibility in the application of the Kolber-Prasil-Falkowski model; tools for plotting, quality control, correcting instrument biases and high-throughput processing with ease; and a greater appreciation for the uncertainties in derived photosynthetic parameters. Using data from three research cruises across different biogeochemical regimes, we provide example applications of PPU to fit raw active chlorophyll-a fluorescence data from three commercial instruments and demonstrate tools which help to reduce uncertainties in the final fitted parameters.

A Dynamic Substrate is Required for MhuD-catalyzed Degradation of Heme to Mycobilin

The non-canoncial heme oxygenase MhuD from <i>Mycobacterium tuberculosis</i> binds a heme substrate that adopts a dynamic equilibrium between planar and out-of-plane ruffled conformations. MhuD degrades this substrate to an unusual mycobilin product via successive monooxygenation and dioxygenation reactions. This article establishes a causal relationship between heme substrate dynamics and MhuD-catalyzed heme degradation resulting in a refined enzymatic mechanism. UV/Vis absorption (Abs) and electrospray ionization mass spectrometry (ESI-MS) data demonstrated that a second-sphere substitution favoring population of the ruffled heme conformation changed the rate-limiting step of the reaction resulting in a measurable build-up of the monooxygenated meso-hydroxyheme intermediate. In addition, UV/Vis Abs and ESI-MS data for a second-sphere variant that favored the planar substrate conformation showed that this change altered the enzymatic mechanism resulting in an alpha-biliverdin product. Single-turnover kinetic analyses for three MhuD variants revealed that the rate of heme monooxygenation depends upon the population of the ruffled substrate conformation. These kinetic analyses also revealed that the rate of meso-hydroxyheme dioxygenation by MhuD depends upon the population of the planar substrate conformation. Thus, the ruffled haem conformation supports rapid heme monooxygenation by MhuD, but further oxygenation to the mycobilin product is inhibited. In contrast, the planar substrate conformation exhibits altered heme monooxygenation regiospecificity followed by rapid oxygenation of meso-hydroxyheme. Altogether, these data yielded a refined enzymatic mechanism for MhuD where access to both substrate conformations is needed for rapid incorporation of three oxygen atoms into heme yielding mycobilin.<br>

Functional and structural characterization of a flavoprotein monooxygenase essential for biogenesis of tryptophylquinone cofactor

Bioconversion of peptidyl amino acids into enzyme cofactors is an important post-translational modification. Here, we report a flavoprotein, essential for biosynthesis of a protein-derived quinone cofactor, cysteine tryptophylquinone, contained in a widely distributed bacterial enzyme, quinohemoprotein amine dehydrogenase. The purified flavoprotein catalyzes the single-turnover dihydroxylation of the tryptophylquinone-precursor, tryptophan, in the protein substrate containing triple intra-peptidyl crosslinks that are pre-formed by a radical S-adenosylmethionine enzyme within the ternary complex of these proteins. Crystal structure of the peptidyl tryptophan dihydroxylase reveals a large pocket that may dock the protein substrate with the bound flavin adenine dinucleotide situated close to the precursor tryptophan. Based on the enzyme-protein substrate docking model, we propose a chemical reaction mechanism of peptidyl tryptophan dihydroxylation catalyzed by the flavoprotein monooxygenase. The diversity of the tryptophylquinone-generating systems suggests convergent evolution of the peptidyl tryptophan-derived cofactors in different proteins.

A Dynamic Substrate is Required for MhuD-catalyzed Degradation of Heme to Mycobilin

The non-canoncial heme oxygenase MhuD from <i>Mycobacterium tuberculosis</i> binds a heme substrate that adopts a dynamic equilibrium between planar and out-of-plane ruffled conformations. MhuD degrades this substrate to an unusual mycobilin product via successive monooxygenation and dioxygenation reactions. This article establishes a causal relationship between heme substrate dynamics and MhuD-catalyzed heme degradation resulting in a refined enzymatic mechanism. UV/Vis absorption (Abs) and electrospray ionization mass spectrometry (ESI-MS) data demonstrated that a second-sphere substitution favoring population of the ruffled heme conformation changed the rate-limiting step of the reaction resulting in a measurable build-up of the monooxygenated meso-hydroxyheme intermediate. In addition, UV/Vis Abs and ESI-MS data for a second-sphere variant that favored the planar substrate conformation showed that this change altered the enzymatic mechanism resulting in an alpha-biliverdin product. Single-turnover kinetic analyses for three MhuD variants revealed that the rate of heme monooxygenation depends upon the population of the ruffled substrate conformation. These kinetic analyses also revealed that the rate of meso-hydroxyheme dioxygenation by MhuD depends upon the population of the planar substrate conformation. Thus, the ruffled haem conformation supports rapid heme monooxygenation by MhuD, but further oxygenation to the mycobilin product is inhibited. In contrast, the planar substrate conformation exhibits altered heme monooxygenation regiospecificity followed by rapid oxygenation of meso-hydroxyheme. Altogether, these data yielded a refined enzymatic mechanism for MhuD where access to both substrate conformations is needed for rapid incorporation of three oxygen atoms into heme yielding mycobilin.<br>

A Dynamic Substrate is Required for MhuD-catalyzed Degradation of Heme to Mycobilin

The non-canoncial heme oxygenase MhuD from <i>Mycobacterium tuberculosis</i> binds a heme substrate that adopts a dynamic equilibrium between planar and out-of-plane ruffled conformations. MhuD degrades this substrate to an unusual mycobilin product via successive monooxygenation and dioxygenation reactions. This article establishes a causal relationship between heme substrate dynamics and MhuD-catalyzed heme degradation resulting in a revised enzymatic mechanism. UV/Vis absorption (Abs) and electrospray ionization mass spectrometry (ESI-MS) data demonstrated that a second-sphere substitution favoring population of the ruffled heme conformation changed the rate-limiting step of the reaction resulting in a measurable build-up of the monooxygenated meso-hydroxyheme intermediate. In addition, UV/Vis Abs and ESI-MS data for a second-sphere variant that favored the planar substrate conformation showed that this change altered the enzymatic mechanism resulting in an alpha-biliverdin product. Single-turnover kinetic analyses for three MhuD variants revealed that the rate of heme monooxygenation depends upon the population of the ruffled substrate conformation. These kinetic analyses also revealed that the rate of meso-hydroxyheme dioxygenation by MhuD depends upon the population of the planar substrate conformation. Thus, the ruffled haem conformation supports rapid heme monooxygenation by MhuD, but further oxygenation to the mycobilin product is inhibited. In contrast, the planar substrate conformation exhibits altered heme monooxygenation regiospecificity followed by rapid oxygenation of meso-hydroxyheme. Altogether, these data yielded a revised enzymatic mechanism for MhuD where access to both substrate conformations is needed for rapid incorporation of three oxygen atoms into heme yielding mycobilin.<br>