Chlorophyllase from Reveals an Emerging Model for Controlling Chlorophyll Hydrolysis.

Chlorophyll (Chl) is one of Nature's most complex pigments to biosynthesize and derivatize. This pigment is vital for survival and also paradoxically toxic if overproduced or released from a protective protein scaffold. Therefore, along with the mass production of Chl, organisms also invest in mechanisms to control its degradation and recycling.

Purification and characterization of a Rieske oxygenase and its NADH-regenerating partner proteins.

The Rieske non-heme iron oxygenases (Rieske oxygenases) comprise a class of metalloenzymes that are involved in the biosynthesis of complex natural products and the biodegradation of aromatic pollutants. Despite this desirable catalytic repertoire, industrial implementation of Rieske oxygenases has been hindered by the multicomponent nature of these enzymes and their requirement for expensive reducing equivalents in the form of a reduced nicotinamide adenine dinucleotide cosubstrate (NAD(P)H). Fortunately, however, some Rieske oxygenases co-occur with accessory proteins, that through a downstream reaction, recycle the needed NAD(P)H for catalysis.

Structure-driven development of a biomimetic rare earth artificial metalloprotein.

The 2011 discovery of the first rare earth-dependent enzyme in methylotrophic AM1 prompted intensive research toward understanding the unique chemistry at play in these systems. This enzyme, an alcohol dehydrogenase (ADH), features a La ion closely associated with redox-active coenzyme pyrroloquinoline quinone (PQQ) and is structurally homologous to the Ca-dependent ADH from the same organism. AM1 also produces a periplasmic PQQ-binding protein, PqqT, which we have now structurally characterized to 1.

Custom tuning of Rieske oxygenase reactivity.

Rieske oxygenases use a Rieske-type [2Fe-2S] cluster and a mononuclear iron center to initiate a range of chemical transformations. However, few details exist regarding how this catalytic scaffold can be predictively tuned to catalyze divergent reactions. Therefore, in this work, using a combination of structural analyses, as well as substrate and rational protein-based engineering campaigns, we elucidate the architectural trends that govern catalytic outcome in the Rieske monooxygenase TsaM.

The NADH recycling enzymes TsaC and TsaD regenerate reducing equivalents for Rieske oxygenase chemistry.

Many microorganisms use both biological and nonbiological molecules as sources of carbon and energy. This resourcefulness means that some microorganisms have mechanisms to assimilate pollutants found in the environment. One such organism is Comamonas testosteroni, which metabolizes 4-methylbenzenesulfonate and 4-methylbenzoate using the TsaMBCD pathway.

Leveraging a Structural Blueprint to Rationally Engineer the Rieske Oxygenase TsaM.

Rieske nonheme iron oxygenases use two metallocenters, a Rieske-type [2Fe-2S] cluster and a mononuclear iron center, to catalyze oxidation reactions on a broad range of substrates. These enzymes are widely used by microorganisms to degrade environmental pollutants and to build complexity in a myriad of biosynthetic pathways that are industrially interesting. However, despite the value of this chemistry, there is a dearth of understanding regarding the structure-function relationships in this enzyme class, which limits our ability to rationally redesign, optimize, and ultimately exploit the chemistry of these enzymes.

A structure-function analysis of chlorophyllase reveals a mechanism for activity regulation dependent on disulfide bonds.

Chlorophyll pigments are used by photosynthetic organisms to facilitate light capture and mediate the conversion of sunlight into chemical energy. Due to the indispensable nature of this pigment and its propensity to form reactive oxygen species, organisms heavily invest in its biosynthesis, recycling, and degradation. One key enzyme implicated in these processes is chlorophyllase, an α/β hydrolase that hydrolyzes the phytol tail of chlorophyll pigments to produce chlorophyllide molecules.

Engineering Rieske oxygenase activity one piece at a time.

Enzyme engineering plays a central role in the development of biocatalysts for biotechnology, chemical and pharmaceutical manufacturing, and environmental remediation. Rational design of proteins has historically relied on targeting active site residues to confer a protein with desirable catalytic properties. However, additional "hotspots" are also known to exist beyond the active site.

Engineering a Conformationally Switchable Artificial Metalloprotein.

Many naturally occurring metalloenzymes are gated by rate-limiting conformational changes, and there exists a critical interplay between macroscopic structural rearrangements of the protein and subatomic changes affecting the electronic structure of embedded metallocofactors. Despite this connection, most artificial metalloproteins (ArMs) are prepared in structurally rigid protein hosts. To better model the natural mechanisms of metalloprotein reactivity, we have developed conformationally switchable ArMs (swArMs) that undergo a large-scale structural rearrangement upon allosteric effector binding.

Rieske Oxygenase Catalyzed C-H Bond Functionalization Reactions in Chlorophyll Biosynthesis.

Rieske oxygenases perform precise C-H bond functionalization reactions in anabolic and catabolic pathways. These reactions are typically characterized as monooxygenation or dioxygenation reactions, but other divergent reactions are also catalyzed by Rieske oxygenases. Chlorophyll(ide) oxygenase (CAO), for example is proposed to catalyze two monooxygenation reactions to transform a methyl-group into the formyl-group of Chlorophyll .

Cobalamin-Dependent Radical -Adenosylmethionine Enzymes: Capitalizing on Old Motifs for New Functions.

The members of the radical -adenosylmethionine (SAM) enzyme superfamily are responsible for catalyzing a diverse set of reactions in a multitude of biosynthetic pathways. Many members of this superfamily accomplish their transformations using the catalytic power of a 5'-deoxyadenosyl radical (5'-dAdo•), but there are also enzymes within this superfamily that bind auxiliary cofactors and extend the catalytic repertoire of SAM. In particular, the cobalamin (Cbl)-dependent class synergistically uses Cbl to facilitate challenging methylation and radical rearrangement reactions.

Purification and structural elucidation of a cobalamin-dependent radical SAM enzyme.

The cobalamin (Cbl)-dependent radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster, SAM, and Cbl to carry out remarkable catalytic feats in a large number of biosynthetic pathways. However, despite the abundance of annotated Cbl-dependent radical SAM enzymes, relatively few molecular details exist regarding how these enzymes function. Traditionally, challenges associated with purifying and reconstituting Cbl-dependent radical SAM enzymes have hindered biochemical studies aimed at elucidating the structures and mechanisms of these enzymes.

Structural and mechanistic basis for redox sensing by the cyanobacterial transcription regulator RexT.

Organisms have a myriad of strategies for sensing, responding to, and combating reactive oxygen species, which are unavoidable consequences of aerobic life. In the heterocystous cyanobacterium Nostoc sp. PCC 7120, one such strategy is the use of an ArsR-SmtB transcriptional regulator RexT that senses HO and upregulates expression of thioredoxin to maintain cellular redox homeostasis.

Design principles for site-selective hydroxylation by a Rieske oxygenase.

Rieske oxygenases exploit the reactivity of iron to perform chemically challenging C-H bond functionalization reactions. Thus far, only a handful of Rieske oxygenases have been structurally characterized and remarkably little information exists regarding how these enzymes use a common architecture and set of metallocenters to facilitate a diverse range of reactions. Herein, we detail how two Rieske oxygenases SxtT and GxtA use different protein regions to influence the site-selectivity of their catalyzed monohydroxylation reactions.

Structural basis for divergent C-H hydroxylation selectivity in two Rieske oxygenases.

Biocatalysts that perform C-H hydroxylation exhibit exceptional substrate specificity and site-selectivity, often through the use of high valent oxidants to activate these inert bonds. Rieske oxygenases are examples of enzymes with the ability to perform precise mono- or dioxygenation reactions on a variety of substrates. Understanding the structural features of Rieske oxygenases responsible for control over selectivity is essential to enable the development of this class of enzymes for biocatalytic applications.

Functional Annotation of ABHD14B, an Orphan Serine Hydrolase Enzyme.

The metabolic serine hydrolase family is, arguably, one of the largest functional enzyme classes in mammals, including humans, comprising 1-2% of the total proteome. This enzyme family uses a conserved nucleophilic serine residue in the active site to perform diverse hydrolytic reactions and consists of proteases, lipases, esterases, amidases, and transacylases, which are prototypical members of this family. In humans, this enzyme family consists of >250, of which approximately 40% members remain unannotated, in terms of both their endogenous substrates and the biological pathways that they regulate.

Mechanism of frataxin "bypass" in human iron-sulfur cluster biosynthesis with implications for Friedreich's ataxia.

In humans, mitochondrial iron-sulfur cluster biosynthesis is an essential biochemical process mediated by the assembly complex consisting of cysteine desulfurase (NFS1), LYR protein (ISD11), acyl-carrier protein (ACP), and the iron-sulfur cluster assembly scaffold protein (ISCU2). The protein frataxin (FXN) is an allosteric activator that binds the assembly complex and stimulates the cysteine desulfurase and iron-sulfur cluster assembly activities. FXN depletion causes loss of activity of iron-sulfur-dependent enzymes and the development of the neurodegenerative disease Friedreich's ataxia.

Aerobic Enzymes and Their Radical SAM Enzyme Counterparts in Tetrapyrrole Pathways.

Microorganisms have lifestyles and metabolism adapted to environmental niches, which can be very broad or highly restricted. Molecular oxygen (O) is currently variably present in microenvironments and has driven adaptation and microbial differentiation over the course of evolution on Earth. Obligate anaerobes use enzymes and cofactors susceptible to low levels of O and are restricted to O-free environments, whereas aerobes typically take advantage of O as a reactant in many biochemical pathways and may require O for essential biochemical reactions.

A Rich Man, Poor Man Story of S-Adenosylmethionine and Cobalamin Revisited.

S-adenosylmethionine (AdoMet) has been referred to as both "a poor man's adenosylcobalamin (AdoCbl)" and "a rich man's AdoCbl," but today, with the ever-increasing number of functions attributed to each cofactor, both appear equally rich and surprising. The recent characterization of an organometallic species in an AdoMet radical enzyme suggests that the line that differentiates them in nature will be constantly challenged. Here, we compare and contrast AdoMet and cobalamin (Cbl) and consider why Cbl-dependent AdoMet radical enzymes require two cofactors that are so similar in their reactivity.

A B-dependent radical SAM enzyme involved in oxetanocin A biosynthesis.

Oxetanocin A (OXT-A) is a potent antitumour, antiviral and antibacterial compound. Biosynthesis of OXT-A has been linked to a plasmid-borne Bacillus megaterium gene cluster that contains four genes: oxsA, oxsB, oxrA and oxrB. Here we show that both the oxsA and oxsB genes are required for the production of OXT-A.

Vitamin B in the spotlight again.

The ability of cobalamin to coordinate different upper axial ligands gives rise to a diversity of reactivity. Traditionally, adenosylcobalamin is associated with radical-based rearrangements, and methylcobalamin with methyl cation transfers. Recently, however, a new role for adenosylcobalamin has been discovered as a light sensor, and a methylcobalamin-dependent enzyme has been identified that is suggested to transfer a methyl anion.

An HD domain phosphohydrolase active site tailored for oxetanocin-A biosynthesis.

HD domain phosphohydrolase enzymes are characterized by a conserved set of histidine and aspartate residues that coordinate an active site metallocenter. Despite the important roles these enzymes play in nucleotide metabolism and signal transduction, few have been both biochemically and structurally characterized. Here, we present X-ray crystal structures and biochemical characterization of the Bacillus megaterium HD domain phosphohydrolase OxsA, involved in the biosynthesis of the antitumor, antiviral, and antibacterial compound oxetanocin-A.