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Nature's repertoire of bio-halogenase enzymes is intriguing with halogenases from various natural product biosynthetic clusters that carry out site and region-specific halogenation of diverse bioactive precursors and molecules. Currently, we have a comprehensive catalogue of cryptic and non-cryptic halogenases that act on simple to complex aliphatic and aromatic molecular scaffolds. This will open up further synthetic and biosynthetic opportunities for C-H activation, ring formation and functionalization of different molecular structures. In fact, halogenases were exploited over the years for these potential applications, to replace traditional chemical halogenation chemistries toward creating economical and environmentally benign methodologies and also for biosynthetic pathways. This review will discuss our advances in utilizing bio-halogenases to generate both in vivo and in vitro biosynthetic pathways; summarizing all naturals and non-naturals that are synthesized with a direct bio-halogenase incorporation. [ABSTRACT FROM AUTHOR]

Author(s): Mary Ortmayer [1]; Pierre Lafite [1]; Binuraj R. K. Menon [1]; Tewes Tralau [1]; Karl Fisher [1]; Lukas Denkhaus [1]; Nigel S. Scrutton [1]; Stephen E. J. Rigby [1]; [...]

Background: Propane (C3H8) is a volatile hydrocarbon with highly favourable physicochemical properties as a fuel, in addition to existing global markets and infrastructure for storage, distribution and utilization in a wide range of applications. Consequently, propane is an attractive target product in research aimed at developing new renewable alternatives to complement currently used petroleum-derived fuels. This study focuses on the construction and evaluation of alternative microbial biosynthetic pathways for the production of renewable propane. The new pathways utilize CoA intermediates that are derived from clostridial-like fermentative butanol pathways and are therefore distinct from the first microbial propane pathways recently engineered in Escherichia coli. Results: We report the assembly and evaluation of four different synthetic pathways for the production of propane and butanol, designated a) atoB-adhE2 route, b) atoB-TPC7 route, c) nphT7-adhE2 route and d) nphT7-TPC7 route. The highest butanol titres were achieved with the atoB-adhE2 (473 ± 3 mg/L) and atoB-TPC7 (163 ± 2 mg/L) routes. When aldehyde deformylating oxygenase (ADO) was co-expressed with these pathways, the engineered hosts also produced propane. The atoB-TPC7-ADO pathway was the most effective in producing propane (220 ± 3 µg/L). By (i) deleting competing pathways, (ii) including a previously designed ADOA134F variant with an enhanced specificity towards short-chain substrates and (iii) including a ferredoxin-based electron supply system, the propane titre was increased (3.40 ± 0.19 mg/L). Conclusions: This study expands the metabolic toolbox for renewable propane production and provides new insight and understanding for the development of next-generation biofuel platforms. In developing an alternative CoA-dependent fermentative butanol pathway, which includes an engineered ADO variant (ADOA134F), the study addresses known limitations, including the low bio-availability of butyraldehyde precursors and poor activity of ADO with butyraldehyde. [ABSTRACT FROM AUTHOR]

Cobalamin-dependent enzymes enhance the rate of C-Co bond cleavage by up to ~1012-fold to generate cob(II)alamin and a transient adenosyl radical. In the case of the pyridoxal 5'-phosphate (PLP) and cobalamin-dependent enzymes lysine 5,6-aminomutase and ornithine 4,5 aminomutase (OAM), it has been proposed that a large scale domain reorientation of the cobalamin-binding domain is linked to radical catalysis. Here, OAM variants were designed to perturb the interface between the cobalamin-binding domain and the PLP-binding TIM barrel domain. Steady-state and single turnover kinetic studies of these variants, combined with pulsed electron-electron double resonance measurements of spin-labeled OAM were used to provide direct evidence for a dynamic interface between the cobalamin and PLP-binding domains. Our data suggest that following ligand binding-induced cleavage of the Lys629-PLP covalent bond, dynamic motion of the cobalamin-binding domain leads to conformational sampling of the available space. This supports radical catalysis through transient formation of a catalytically competent active state. Crucially, it appears that the formation of the state containing both a substrate/product radical and Co(II) does not restrict cobalamin domain motion. A similar conformational sampling mechanism has been proposed to support rapid electron transfer in a number of dynamic redox systems. [ABSTRACT FROM AUTHOR]

The light-activated enzyme protochiorophyllide oxidoreductase (POR) catalyzes an essential step in the synthesis of the most abundant pigment on Earth, chlorophyll. This unique reaction involves the sequential addition of a hydride and proton across the C17C18 double bond of protochlorophyllide (Pchlide) by dynamically coupled quantum tunneling and is an important model system for studying the mechanism of hydrogen transfer reactions. In the present work, we have combined site-directed mutagenesis studies with a variety of sensitive spectroscopic and kinetic measurements to provide new insights into the mechanistic role of three universally conserved Cys residues in POR. We show that mutation of Cys-226 dramatically alters the catalytic mechanism of the enzyme. In contrast to wild-type POR, the characteristic charge-transfer intermediate, formed upon hydride transfer from NADPH to the C17 position of Pchlide, is absent in C226S variant enzymes. This suggests a concerted hydrogen transfer mechanism where proton transfer only is rate-limiting. Moreover, Pchlide reduction does not require the network of solvent-coupled conformational changes that play a key role in the proton transfer step of wild-type POR. We conclude that this globally important enzyme is finely tuned to facilitate efficient photochemistry, and the removal of a key interaction with Pchlide in the C226S variants significantly affects the local active site structure in POR, resulting in a shorter donoracceptor distance for proton transfer. [ABSTRACT FROM AUTHOR]

The light-activated enzyme NADPH-protochlorophyllide oxidoreductase (POR) catalyzes the trans addition of hydrogen across the C-17-C-18 double bond of protochlorophyllide (Pchlide), a key step in chlotophyll biosynthesis. Similar to other members of the short chain alcohol dehydrogenase/reductase family of enzymes, POR contains a conserved Tyr and Lys residue in the enzyme active site, which are implicated in a proposed reaction mechanism involving proton transfer from the Tyr hydoxyl group to Pchlide. We have analyzed a number of POR variant enzymes altered in these conserved residues using a combination of steady-state turnover, laser photoexcitation studies, and low temperature fluorescence spectroscopy. None of the mutations completely abolished catalytic activity. We demonstrate their importance to catalysis by defining multiple roles in the overall reaction pathway. Mutation of either residue impairs formation of the ground state ternary enzyme-substrate complex, pointing to a key role in substrate binding. By analyzing the most active variant (Y193F), we show that Tyr-193 participates in proton transfer to Pchlide and stabilizes the Pchlide excited state, enabling hydride transfer from NADPH to Pchilde. Thus, in addition to confirming the probable identity of the proton donor in Pchlide reduction, our work defines additional roles for these residues in facilitating hydride transfer through stabilization of the ground and excited states of the ternary enzyme complex. [ABSTRACT FROM AUTHOR]

The light-driven enzyme, protochiorophyllide oxidoreductase (POR), has proven to be an excellent model system for studying the role of protein motions during catalysis. POR catalyzes the trans addition of hydrogen across the C17-C18 double bond of protochlorophyllide (Pchlide), which is a key step in chlorophyll biosynthesis. While we currently have a detailed understanding of the initial photochemical events and the subsequent hydrogen transfer reactions, there remains a lack of information about the slower substrate binding events leading to the formation of the catalytically active ternary complex. As POR is light-activated, it is relatively straightforward to isolate the ternary enzyme-substrate complex in the dark prior to catalysis, which has facilitated the use of a variety of spectroscopic and kinetic probes to study the binding of both substrates. Herein, we provide a detailed kinetic and thermodynamic description of these processes and show that the binding events are complex, involving multiple conformational states en route to the formation of a ternary complex that is primed for photoactivation. The initial binding of NADPH involves three distinct steps, which appear to be necessary for the optimal alignment of the cofactor in the enzyme active site. This is followed by the binding of the Pchlide substrate and subsequent substrate-induced conformational changes within the enzyme that occur prior to the formation of the final "poised" conformational state. These studies, which provide important information on the formation of the reactive conformation, reveal that ternary complex formation is the rate-limiting step in the overall reaction and is controlled by slow conformational changes in the protein. [ABSTRACT FROM AUTHOR]

Protochlorophyllide oxidoreductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide), an essential, regulatory step in chlorophyll biosynthesis. The unique requirement of the enzyme for light has provided the opportunity to investigate how light energy can be harnessed to power biological catalysis and enzyme dynamics. Excited state interactions between the Pchlide molecule and the protein are known to drive the subsequent reaction chemistry. However, the structural features of POR and active site residues that are important for photochemistry and catalysis are currently unknown, because there is no crystal structure for POR. Here, we have used static and time-resolved spectroscopic measurements of a number of active site variants to study the role of a number of residues, which are located in the proposed NADPH/Pchlide binding site based on previous homology models, in the reaction mechanism of POR. Our findings, which are interpreted in the context of a new improved structural model, have identified several residues that are predicted to interact with the coenzyme or substrate. Several of the POR variants have a profound effect on the photochemistry, suggesting that multiple residues are important in stabilizing the excited state required for catalysis. Our work offers insight into how the POR active site geometry is finely tuned by multiple active site residues to support enzyme-mediated photochemistry and reduction of Pchlide, both of which are crucial to the existence of life on Earth., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)