Directed evolution circumvents our profound ignorance of how a protein's sequence encodes its function by using iterative rounds of random mutation and artificial selection to discover new and useful proteins. Proteins can be tuned to adapt to new functions or environments by simple adaptive walks involving small numbers of mutations. Directed evolution studies have shown how rapidly some proteins can evolve under strong selection pressures and, because the entire 'fossil record' of evolutionary intermediates is available for detailed study, they have provided new insight into the relationship between sequence and function. Directed evolution has also shown how mutations that are functionally neutral can set the stage for further adaptation.

/pdf/exploring-protein-fitness-landscapes-by-directed-evolution-4wy4f2twej.pdf

Exploring protein fitness landscapes by directed evolution

In chemical terms, the regio- and stereoselective hydroxylation of hydrocarbon C-H bonds is a very difficult transformation. Nevertheless, these reactions are deftly catalyzed by a variety of metalloenzymes, among which the most diverse are the many members of the cytochrome P450 family. Cytochrome P450 enzymes are found in most classes of organisms, including bacteria, fungi, plants, insects, and mammals. Thousands of such proteins are now known (http://drnelson.utmem.edu/cytochromeP450.html), including 57 in the human genome (1), 20 in Mycobacterium tuberculosis (2), 272 in Arabidopsis (3), and the amazing number of 457 in rice (4). The nomenclature for these enzymes is based on their sequence similarity when appropriately aligned, a somewhat arbitrary similarity cutoff (approximately >40% identity) being used to define members of a family and a higher cutoff (approximately >55% identity) members of a subfamily (5). Thus CYP3A4 corresponds to the fourth enzyme in family 3, subfamily A. This nomenclature allows the naming of enzymes without regard to their origin or specific properties.

The mammalian, plant, and fungal proteins are commonly membrane bound and are relatively difficult to manipulate, but the bacterial proteins are usually soluble, monomeric proteins. For that reason, much of the early research on mechanisms of cytochrome P450 enzymes was carried out with bacterial enzymes, particularly with the prototypical enzyme CYP101 (P450cam) from Pseudomonas putida (6, 7). From a chemist's point of view, there is a particular interest in the thermophilic enzymes, which currently include CYP119 (8-10), P450st (11), CYP174A1 (12), and CYP231A2 (13). The thermal stability of these enzymes makes them attractive starting points for the development of industrially useful catalysts. In this context, particular attention has also focused on CYP102 (P450BM3), a self-sufficient enzyme from Bacillus megaterium in which the flavoprotein protein required for transfer of electrons from NADPH is fused to the hemoprotein (14). The resulting simplicity and high catalytic rate have led to extensive efforts to engineer this protein for practical catalytic purposes (15-19). Although these proteins have properties that make them particularly attractive for engineering purposes, the large reservoir of P450 enzymes that collectively catalyze an astounding diversity of reactions suggests that P450 catalysis will develop into a highly useful technology.

The cytochrome P450 enzymes are defined by the presence in the proteins of a heme (iron protoporphyrin IX) prosthetic group coordinated on the proximal side by a thiolate ion (20, 21). This feature gives rise to the spectroscopic signature that defines these enzymes, as the thiolate-ligated ferrous-CO complex is characterized by a Soret absorption maximum at ∼450 nm (21). A thiolate-coordinated heme group is present in all P450 enzymes, although not all proteins with such coordination are members of this superfamily. One obvious exception, for example, is chloroperoxidase, which has a thiolate-coordinated heme group but normally catalyzes a very different reaction than the P450 enzymes (21-23).

Although there are unusual P450 enzymes, such as the thromboxane and prostacyclin synthases (24), or CYP152 from Sphingomonas paucimobilis or Bacillus subtilis (25, 26), that normally utilize peroxides as substrates, the defining reaction for P450 enzymes is the reductive activation of molecular oxygen. In this reaction, one of the oxygen atoms of molecular oxygen is inserted into the substrate and the other oxygen atom is reduced to a molecule of water. With one exception to date (27, 28), the electrons required for this reduction of molecular oxygen derive from reduced pyridine nucleotides (NADH or NADPH). The overall equation for the reaction thus adheres to the formula: RH + NAD(P)H + O2 + H+ -> ROH + NAD(P)+ + H2O, where RH stands for a substrate with a hydroxylatable site. P450 enzymes therefore belong to the monooxygenase class of enzymes that only insert one of the oxygen atoms of molecular oxygen into their substrates. However, under appropriate circumstances or with specific substrates, other P450-catalyzed reactions can be observed, including desaturation, carbon-carbon bond scission, and carbon-carbon bond formation (29, 30). This review specifically focuses on P450-catalyzed hydrocarbon hydroxylation, the reaction that is most characteristic of P450 enzymes and that has been most extensively investigated. However, the principles that apply in these reactions also apply to other hydroxylation reactions, including those that occur on carbons adjacent to nitrogen, sulfur, or oxygen.

Hydrocarbon hydroxylation by cytochrome P450 enzymes.

Synthetic polymers containing metal centres are emerging as an interesting and broad class of easily processable materials with properties and functions that complement those of state-of-the-art organic macromolecular materials. A diverse range of different metal centres can be harnessed to tune macromolecular properties, from transition- and main-group metals to lanthanides. Moreover, the linkages that bind the metal centres can vary almost continuously from strong, essentially covalent bonds that lead to irreversible or 'static' binding of the metal to weak and labile, non-covalent coordination interactions that allow for reversible, 'dynamic' or 'metallosupramolecular', binding. Here we review recent advances and challenges in the field and illustrate developments towards applications as emissive and photovoltaic materials; as optical limiters; in nanoelectronics, information storage, nanopatterning and sensing; as macromolecular catalysts and artificial enzymes; and as stimuli-responsive materials. We focus on materials in which the metal centres provide function; although they can also play a structural role, systems where this is solely their purpose have not been discussed.

Functional soft materials from metallopolymers and metallosupramolecular polymers

Enzymes are increasingly being used as biocatalysts in the generation of products that have until now been derived using traditional chemical processes. Such products range from pharmaceutical and agrochemical building blocks to fine and bulk chemicals and, more recently, components of biofuels. For a biocatalyst to be effective in an industrial process, it must be subjected to improvement and optimization, and in this respect the directed evolution of enzymes has emerged as a powerful enabling technology. Directed evolution involves repeated rounds of (i) random gene library generation, (ii) expression of genes in a suitable host and (iii) screening of libraries of variant enzymes for the property of interest. Both in vitro screening–based methods and in vivo selection–based methods have been applied to the evolution of enzyme function and properties. Significant developments have occurred recently, particularly with respect to library design, screening methodology, applications in synthetic transformations and strategies for the generation of new enzyme function.

Directed evolution drives the next generation of biocatalysts

Metalloproteins catalyse some of the most complex and important processes in nature, such as photosynthesis and water oxidation An ultimate test of our knowledge of how metalloproteins work is to design new metalloproteins Doing so not only can reveal hidden structural features that may be missing from studies of native metalloproteins and their variants, but also can result in new metalloenzymes for biotechnological and pharmaceutical applications Although it is much more challenging to design metalloproteins than non-metalloproteins, much progress has been made in this area, particularly in functional design, owing to recent advances in areas such as computational and structural biology

Design of functional metalloproteins

Cytochrome P450 enzymes (P450s) are exceptional oxygenating catalysts with enormous potential in drug discovery, chemical synthesis, bioremediation, and biotechnology. Compared to their natural counterparts, however, engineered P450s often exhibit poor catalytic and cofactor coupling efficiencies. Obtaining native-like catalytic proficiencies is a mandatory first step towards utilizing the power of these versatile oxygenases in chemical synthesis. Cytochrome P450BM3 (119 kDa, B. megaterium) catalyzes the subterminal hydroxylation of long-chain (C12–C20) fatty acids. Its high activity and catalytic self-sufficiency (heme and diflavin reductase domains are fused in a single polypeptide chain) 5] make P450BM3 an excellent platform for biocatalysis. However, despite numerous reports of the heme domain being engineered to accept nonnative substrates, including short-chain fatty acids, aromatic compounds, alkanes, and alkenes, reports of preparative-scale applications of P450BM3 remain scarce. [9] P450BM3 function is finely regulated through conformational rearrangements in the heme and reductase domains and possibly also through hinged domain motions. 10] Hydroxylation of fatty acids occurs almost fully coupled to cofactor (NADPH) utilization (93–96% depending on the substrate). In the presence of nonnative substrates or when amino acid substitutions are introduced, the mechanisms controlling efficient catalysis in P450s are disrupted, leading to the formation of reactive oxygen species and rapid enzyme inactivation. High coupling efficiencies on substrates whose physicochemical properties are substantially different from the native substrates have not been achieved, and coupling efficiencies ranging from less than 1% to 30– 40% are typical. 8] Strategies for addressing this “coupling problem” are needed in order to take engineered P450s to larger-scale applications. Selective hydroxylation of short alkanes is a long-standing problem, for which no practical catalysts are available. In an effort to produce P450BM3-based biocatalysts for selective hydroxylation of small alkanes, we previously engineered this enzyme to accept propane and ethane (35E11 variant). Despite greater than 5000 total turnover (TTN) supported in vitro, the utility of this catalyst remained limited because of its poor in vivo performance (see below), which was mostly due to the low efficiencies for coupling the product formation to cofactor consumption (17.4% for propane and 0.01% for ethane oxidation). Our goal was to engineer a P450BM3 variant with nativelike activity and coupling efficiency towards a structurally challenging, nonnative substrate (propane) and evaluate the impact of these features on performance in preparative-scale biotransformations. To this end, we used a domain-based protein-engineering strategy, in which the heme, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) domains of the 35E11 variant were evolved separately in the context of the holoenzyme, and beneficial mutations were recombined in a final step (Figure 1). Previous work suggested that mutations in the reductase and linker regions can affect catalytic properties. However, no systematic engineering efforts had been undertaken to engineer the complete 1048 amino acid holoenzyme. Holoenzyme libraries outlined in Figure 1 were created using random, saturation, and site-directed mutagenesis and

Engineered Alkane‐Hydroxylating Cytochrome P450BM3 Exhibiting Nativelike Catalytic Properties

Engineered alkane-hydroxylating cytochrome P450(BM3) exhibiting nativelike catalytic properties.

Enzymes of the AlkB and CYP153 families catalyze the first step in the catabolism of medium-chain-length alkanes, selective oxidation of the alkane to the 1-alkanol, and enable their host organisms to utilize alkanes as carbon sources. Small, gaseous alkanes, however, are converted to alkanols by evolutionarily unrelated methane monooxygenases. Propane and butane can be oxidized by CYP enzymes engineered in the laboratory, but these produce predominantly the 2-alkanols. Here we report the in vivo-directed evolution of two medium-chain-length terminal alkane hydroxylases, the integral membrane di-iron enzyme AlkB from Pseudomonas putida GPo1 and the class II-type soluble CYP153A6 from Mycobacterium sp. strain HXN-1500, to enhance their activity on small alkanes. We established a P. putida evolution system that enables selection for terminal alkane hydroxylase activity and used it to select propane- and butane-oxidizing enzymes based on enhanced growth complementation of an adapted P. putida GPo12(pGEc47ΔB) strain. The resulting enzymes exhibited higher rates of 1-butanol production from butane and maintained their preference for terminal hydroxylation. This in vivo evolution system could be useful for directed evolution of enzymes that function efficiently to hydroxylate small alkanes in engineered hosts.

/pdf/in-vivo-evolution-of-butane-oxidation-by-terminal-alkane-10iy3frid9.pdf

In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6

Polysaccharides comprise an extremely important class of biopolymers that play critical roles in a wide range of biological processes, but the synthesis of these compounds is challenging because of their complex structures We have developed a chemoenzymatic method for regioselective deprotection of monosaccharide substrates using engineered Bacillus megaterium cytochrome P450 (P450BM3) demethylases that provides a highly efficient means to access valuable intermediates, which can be converted to a wide range of substituted monosaccharides and polysaccharides Demethylases displaying high levels of regioselectivity toward a number of protected monosaccharides were identified using a combination of protein and substrate engineering, suggesting that this approach ultimately could be used in the synthesis of a wide range of substituted mono- and polysaccharides for studies in chemistry, biology, and medicine

https://www.pnas.org/content/pnas/106/39/16550.full.pdf

Mike M. Chen

Papers

Engineered Alkane‐Hydroxylating Cytochrome P450BM3 Exhibiting Nativelike Catalytic Properties

Engineered alkane-hydroxylating cytochrome P450(BM3) exhibiting nativelike catalytic properties.

In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6

Chemoenzymatic elaboration of monosaccharides using engineered cytochrome P450BM3 demethylases