Assuntos
Proteínas AAA/química , Proteínas AAA/genética , Proteínas AAA/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Desenvolvimento de Medicamentos , Humanos , Simulação de Dinâmica Molecular , Mutação , Conformação ProteicaRESUMO
Mitochondrial protein quality control requires the action of proteases to remove damaged or unnecessary proteins and perform key regulatory cleavage events. Important components of the quality control network are the mitochondrial AAA proteases, which capture energy from ATP hydrolysis to destabilize and degrade protein substrates on both sides of the inner membrane. Dysfunction of these proteases leads to the breakdown of mitochondrial proteostasis and is linked to the development of severe human diseases. In this review, we will describe recent insights into the structure and motions of the mitochondrial AAA proteases and related enzymes. Together, these studies have revealed the mechanics of ATP-driven protein destruction and significantly advanced our understanding of how these proteases maintain mitochondrial health.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Deficiências na Proteostase/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Humanos , Membranas Mitocondriais/patologia , Proteínas Mitocondriais/genética , Deficiências na Proteostase/genética , Deficiências na Proteostase/patologiaRESUMO
Mitochondrial AAA+ quality-control proteases regulate diverse aspects of mitochondrial biology through specialized protein degradation, but the underlying mechanisms of these enzymes remain poorly defined. The mitochondrial AAA+ protease AFG3L2 is of particular interest, as genetic mutations localized throughout AFG3L2 are linked to diverse neurodegenerative disorders. However, a lack of structural data has limited our understanding of how mutations impact enzymatic function. Here, we used cryoelectron microscopy (cryo-EM) to determine a substrate-bound structure of the catalytic core of human AFG3L2. This structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins. Many disease-relevant mutations localize to these unique structural features of AFG3L2 and distinctly influence its activity and stability. Our results provide a molecular basis for neurological phenotypes associated with different AFG3L2 mutations and establish a structural framework to understand how different members of the AAA+ superfamily achieve specialized biological functions.
Assuntos
Proteases Dependentes de ATP/química , ATPases Associadas a Diversas Atividades Celulares/química , Proteínas Mitocondriais/química , Mutação , Proteases Dependentes de ATP/genética , Proteases Dependentes de ATP/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Microscopia Crioeletrônica , Células HEK293 , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Transtornos Heredodegenerativos do Sistema Nervoso/metabolismo , Humanos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Domínios ProteicosRESUMO
The increasing use of medical marijuana highlights the importance of developing a better understanding of cannabinoid metabolism. Phytocannabinoids, including ∆9-tetrahydrocannabinol (THC), are metabolized and inactivated by cytochrome P450 enzymes primarily within the liver. The lipophilic nature of cannabinoids necessitates mechanism(s) to facilitate their intracellular transport to metabolic enzymes. Here, we test the central hypothesis that liver-type fatty acid binding protein (FABP1) mediates phytocannabinoid transport and subsequent inactivation. Using X-ray crystallography, molecular modeling, and in vitro binding approaches we demonstrate that FABP1 accommodates one molecule of THC within its ligand binding pocket. Consistent with its role as a THC carrier, biotransformation of THC was reduced in primary hepatocytes obtained from FABP1-knockout (FABP1-KO) mice. Compared to their wild-type littermates, administration of THC to male and female FABP1-KO mice potentiated the physiological and behavioral effects of THC. The stark pharmacodynamic differences were confirmed upon pharmacokinetic analyses which revealed that FABP1-KO mice exhibit reduced rates of THC biotransformation. Collectively, these data position FABP1 as a hepatic THC transport protein and a critical mediator of cannabinoid inactivation. Since commonly used medications bind to FABP1 with comparable affinities to THC, our results further suggest that FABP1 could serve a previously unrecognized site of drug-drug interactions.
Assuntos
Dronabinol/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Animais , Sítios de Ligação , Biotransformação , Células Cultivadas , Cristalografia por Raios X , Dronabinol/administração & dosagem , Proteínas de Ligação a Ácido Graxo/química , Feminino , Hepatócitos/metabolismo , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Modelos MolecularesRESUMO
Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. Identifying how AFG3L2 selects substrates from the diverse complement of matrix-localized proteins is essential for understanding mitochondrial protein biogenesis and quality control. Here, we create solubilized forms of AFG3L2 to examine the enzyme's substrate specificity mechanisms. We show that conserved residues within the presequence of the mitochondrial ribosomal protein, MrpL32, target the subunit to the protease for processing into a mature form. Moreover, these residues can act as a degron, delivering diverse model proteins to AFG3L2 for degradation. By determining the sequence of degradation products from multiple substrates using mass spectrometry, we construct a peptidase specificity profile that displays constrained product lengths and is dominated by the identity of the residue at the P1' position, with a strong preference for hydrophobic and small polar residues. This specificity profile is validated by examining the cleavage of both fluorogenic reporter peptides and full polypeptide substrates bearing different P1' residues. Together, these results demonstrate that AFG3L2 contains multiple modes of specificity, discriminating between potential substrates by recognizing accessible degron sequences and performing peptide bond cleavage at preferred patterns of residues within the compartmental chamber.
Assuntos
Proteases Dependentes de ATP/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Ribossômicas/metabolismo , Proteases Dependentes de ATP/química , ATPases Associadas a Diversas Atividades Celulares/química , Sequência de Aminoácidos , Humanos , Proteínas Mitocondriais/química , Proteólise , Proteínas Ribossômicas/química , Solubilidade , Especificidade por SubstratoRESUMO
We present an atomic model of a substrate-bound inner mitochondrial membrane AAA+ quality control protease in yeast, YME1. Our ~3.4-angstrom cryo-electron microscopy structure reveals how the adenosine triphosphatases (ATPases) form a closed spiral staircase encircling an unfolded substrate, directing it toward the flat, symmetric protease ring. Three coexisting nucleotide states allosterically induce distinct positioning of tyrosines in the central channel, resulting in substrate engagement and translocation to the negatively charged proteolytic chamber. This tight coordination by a network of conserved residues defines a sequential, around-the-ring adenosine triphosphate hydrolysis cycle that results in stepwise substrate translocation. A hingelike linker accommodates the large-scale nucleotide-driven motions of the ATPase spiral relative to the planar proteolytic base. The translocation mechanism is likely conserved for other AAA+ ATPases.
Assuntos
Proteases Dependentes de ATP/química , Membranas Mitocondriais/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Proteases Dependentes de ATP/ultraestrutura , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Hidrólise , Modelos Moleculares , Domínios Proteicos , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Especificidade por SubstratoRESUMO
Mitochondria perform numerous functions necessary for the survival of eukaryotic cells. These activities are coordinated by a diverse complement of proteins encoded in both the nuclear and mitochondrial genomes that must be properly organized and maintained. Misregulation of mitochondrial proteostasis impairs organellar function and can result in the development of severe human diseases. ATP-driven AAA+ proteins play crucial roles in preserving mitochondrial activity by removing and remodeling protein molecules in accordance with the needs of the cell. Two mitochondrial AAA proteases, i-AAA and m-AAA, are anchored to either face of the mitochondrial inner membrane, where they engage and process an array of substrates to impact protein biogenesis, quality control, and the regulation of key metabolic pathways. The functionality of these proteases is extended through multiple substrate-dependent modes of action, including complete degradation, partial processing, or dislocation from the membrane without proteolysis. This review discusses recent advances made toward elucidating the mechanisms of substrate recognition, handling, and degradation that allow these versatile proteases to control diverse activities in this multifunctional organelle.
RESUMO
The HslUV proteolytic machine consists of HslV, a double-ring self-compartmentalized peptidase, and one or two AAA+ HslU ring hexamers that hydrolyze ATP to power the unfolding of protein substrates and their translocation into the proteolytic chamber of HslV. Here, we use genetic tethering and disulfide bonding strategies to construct HslU pseudohexamers containing mixtures of ATPase active and inactive subunits at defined positions in the hexameric ring. Genetic tethering impairs HslV binding and degradation, even for pseudohexamers with six active subunits, but disulfide-linked pseudohexamers do not have these defects, indicating that the peptide tether interferes with HslV interactions. Importantly, pseudohexamers containing different patterns of hydrolytically active and inactive subunits retain the ability to unfold protein substrates and/or collaborate with HslV in their degradation, supporting a model in which ATP hydrolysis and linked mechanical function in the HslU ring operate by a probabilistic mechanism.
Assuntos
Trifosfato de Adenosina/química , Endopeptidase Clp/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Desdobramento de Proteína , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Domínio Catalítico , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
The i-AAA protease is a component of the mitochondrial quality control machinery that regulates respiration, mitochondrial dynamics, and protein import. The protease is required to select specific substrates for degradation from among the diverse complement of proteins present in mitochondria, yet the rules that govern this selection are unclear. Here, we reconstruct the yeast i-AAA protease, Yme1p, to examine the in vitro degradation of two intermembrane space chaperone subunits, Tim9 and Tim10. Yme1p degrades Tim10 more rapidly than Tim9 despite high sequence and structural similarity, and loss of Tim10 is accelerated by the disruption of conserved disulfide bonds within the substrate. An unstructured N-terminal region of Tim10 is necessary and sufficient to target the substrate to the protease through recognition of a short phenylalanine-rich motif, and the presence of similar motifs in other small Tim proteins predicts robust degradation by the protease. Together, these results identify the first specific degron sequence within a native i-AAA protease substrate.
Assuntos
Proteases Dependentes de ATP/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Dissulfetos , Cinética , Proteínas do Complexo de Importação de Proteína Precursora MitocondrialRESUMO
The human YME1L protease is a membrane-anchored AAA+ enzyme that controls proteostasis at the inner membrane and intermembrane space of mitochondria. Understanding how YME1L recognizes substrates and catalyses ATP-dependent degradation has been hampered by the presence of an insoluble transmembrane anchor that drives hexamerization of the catalytic domains to form the ATPase active sites. Here, we overcome this limitation by replacing the transmembrane domain with a soluble hexameric coiled coil to produce active YME1L hexamers that can be studied in vitro. We use these engineered proteases to reveal principles of substrate processing by YME1L. Degradation by YME1L requires substrates to present an accessible signal sequence and is not initiated simply by substrate unfolding. The protease is also capable of processively unfolding substrate proteins with substantial thermodynamic stabilities. Lastly, we show that YME1L discriminates between degradation signals by amino acid composition, implying the use of sequence-specific signals in mitochondrial proteostasis.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Metaloendopeptidases/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/genética , Sequência de Aminoácidos , Biocatálise , Humanos , Metaloendopeptidases/química , Metaloendopeptidases/genética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Engenharia de Proteínas/métodos , Multimerização Proteica , Desdobramento de Proteína , Proteólise , Proteostase , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
BACKGROUND: Fatty acid amide hydrolase 2 (FAAH2) is a hydrolase that mediates the degradation of endocannabinoids in man. Alterations in the endocannabinoid system are associated with a wide variety of neurologic and psychiatric conditions, but the phenotype and biochemical characterization of patients with genetic defects of FAAH2 activity have not previously been described. We report a male with autistic features with an onset before the age of 2 years who subsequently developed additional features including anxiety, pseudoseizures, ataxia, supranuclear gaze palsy, and isolated learning disabilities but was otherwise cognitively intact as an adult. METHODS AND RESULTS: Whole exome sequencing identified a rare missense mutation in FAAH2, hg19: g.57475100G > T (c.1372G > T) resulting in an amino acid change (p.Ala458Ser), which was Sanger confirmed as maternally inherited and absent in his healthy brother. Alterations in lipid metabolism with abnormalities of the whole blood acyl carnitine profile were found. Biochemical and molecular modeling studies confirmed that the p.Ala458Ser mutation results in partial inactivation of FAAH2. Studies in patient derived fibroblasts confirmed a defect in FAAH2 activity resulting in altered levels of endocannabinoid metabolites. CONCLUSIONS: We propose that genetic alterations in FAAH2 activity contribute to neurologic and psychiatric disorders in humans.
Assuntos
Amidoidrolases/metabolismo , Ansiedade/patologia , Doenças do Sistema Nervoso Central/patologia , Depressão/patologia , Adulto , Amidoidrolases/genética , Ansiedade/genética , Doenças do Sistema Nervoso Central/genética , Clonagem Molecular , Depressão/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Masculino , Modelos Moleculares , Mutação de Sentido Incorreto , Conformação ProteicaRESUMO
ClpX, a AAA+ ring homohexamer, uses the energy of ATP binding and hydrolysis to power conformational changes that unfold and translocate target proteins into the ClpP peptidase for degradation. In multiple crystal structures, some ClpX subunits adopt nucleotide-loadable conformations, others adopt unloadable conformations, and each conformational class exhibits substantial variability. Using mutagenesis of individual subunits in covalently tethered hexamers together with fluorescence methods to assay the conformations and nucleotide-binding properties of these subunits, we demonstrate that dynamic interconversion between loadable and unloadable conformations is required to couple ATP hydrolysis by ClpX to mechanical work. ATP binding to different classes of subunits initially drives staged allosteric changes, which set the conformation of the ring to allow hydrolysis and linked mechanical steps. Subunit switching between loadable and unloadable conformations subsequently isomerizes or resets the configuration of the nucleotide-loaded ring and is required for mechanical function.
Assuntos
Adenosina Trifosfatases/química , Trifosfato de Adenosina/metabolismo , Endopeptidase Clp/química , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Chaperonas Moleculares/química , Nucleotídeos/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Conformação Proteica , Dobramento de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , ProteóliseRESUMO
In the Escherichia coli ClpXP protease, a hexameric ClpX ring couples ATP binding and hydrolysis to mechanical protein unfolding and translocation into the ClpP degradation chamber. Rigid-body packing between the small AAA+ domain of each ClpX subunit and the large AAA+ domain of its neighbor stabilizes the hexamer. By connecting the parts of each rigid-body unit with disulfide bonds or linkers, we created covalently closed rings that retained robust activity. A single-residue insertion in the hinge that connects the large and small AAA+ domains and forms part of the nucleotide-binding site uncoupled ATP hydrolysis from productive unfolding. We propose that ATP hydrolysis drives changes in the conformation of one hinge and its flanking domains and that the changes are propagated around the AAA+ ring through the topologically constrained set of rigid-body units and hinges to produce coupled ring motions that power substrate unfolding.
Assuntos
Adenosina Trifosfatases/metabolismo , Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Subunidades Proteicas/metabolismo , Desdobramento de Proteína , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Endopeptidase Clp/química , Endopeptidase Clp/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Hidrólise , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Mutação , Multimerização Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , ProteóliseRESUMO
ClpX is a AAA+ machine that uses the energy of ATP binding and hydrolysis to unfold native proteins and translocate unfolded polypeptides into the ClpP peptidase. The crystal structures presented here reveal striking asymmetry in ring hexamers of nucleotide-free and nucleotide-bound ClpX. Asymmetry arises from large changes in rotation between the large and small AAA+ domains of individual subunits. These differences prevent nucleotide binding to two subunits, generate a staggered arrangement of ClpX subunits and pore loops around the hexameric ring, and provide a mechanism for coupling conformational changes caused by ATP binding or hydrolysis in one subunit to flexing motions of the entire ring. Our structures explain numerous solution studies of ClpX function, predict mechanisms for pore elasticity during translocation of irregular polypeptides, and suggest how repetitive conformational changes might be coupled to mechanical work during the ATPase cycle of ClpX and related molecular machines.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Endopeptidase Clp/química , Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Cristalografia por Raios X , Modelos Moleculares , Nucleotídeos/metabolismo , Conformação Proteica , Dobramento de ProteínaRESUMO
The structure of A. thaliana imidazoleglycerol-phosphate dehydratase, an enzyme of histidine biosynthesis and a target for the triazole phosphonate herbicides, has been determined to 3.0 A resolution. The structure is composed of 24 identical subunits arranged in 432 symmetry and shows how the formation of a novel dimanganese cluster is crucial to the assembly of the active 24-mer from an inactive trimeric precursor and to the formation of the active site of the enzyme. Molecular modeling suggests that the substrate is bound to the manganese cluster as an imidazolate moiety that subsequently collapses to yield a diazafulvene intermediate. The mode of imidazolate recognition exploits pseudosymmetry at the active site arising from a combination of the assembly of the particle and the pseudosymmetry present in each subunit as a result of gene duplication. This provides an intriguing example of the role of evolution in the design of Nature's catalysts.
Assuntos
Arabidopsis/enzimologia , Hidroliases/química , Modelos Moleculares , Sequência de Aminoácidos , Sítios de Ligação/genética , Catálise , Hidroliases/genética , Manganês/química , Dados de Sequência Molecular , Conformação Proteica , Subunidades ProteicasRESUMO
Imidazoleglycerol-phosphate dehydratase catalyses the sixth step of the histidine-biosynthesis pathway in plants and microorganisms and has been identified as a possible target for the development of novel herbicides. Arabidopsis thaliana IGPD has been cloned and overexpressed in Escherichia coli, purified and subsequently crystallized in the presence of manganese. Under these conditions, the inactive trimeric form of the metal-free enzyme is assembled into a fully active species consisting of a 24-mer exhibiting 432 symmetry. X-ray diffraction data have been collected to 3.0 A resolution from a single crystal at 293 K. The crystal belongs to space group R3, with approximate unit-cell parameters a = b = 157.9, c = 480.0 A, alpha = beta = 90, gamma = 120 degrees and with either 16 or 24 subunits in the asymmetric unit. A full structure determination is under way in order to provide insights into the mode of subunit assembly and to initiate a programme of rational herbicide design.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Hidroliases/química , Hidroliases/metabolismo , Cristalização , Cristalografia por Raios XRESUMO
Galactokinase (GalK) catalyses the first step of the Leloir pathway of galactose metabolism, the ATP-dependent phosphorylation of galactose to galactose-1-phosphate. In man, defects in galactose metabolism can result in disorders with severe clinical consequences, and deficiencies in galactokinase have been linked with the development of cataracts within the first few months of life. The crystal structure of GalK from Pyrococcus furiosus in complex with MgADP and galactose has been determined to 2.9 A resolution to provide insights into the substrate specificity and catalytic mechanism of the enzyme. The structure consists of two domains with the active site in a cleft at the domain interface. Inspection of the substrate binding pocket identifies the amino acid residues involved in galactose and nucleotide binding and points to both structural and mechanistic similarities with other enzymes of the GHMP kinase superfamily to which GalK belongs. Comparison of the sequence of the Gal3p inducer protein, which is related to GalK and which forms part of the transcriptional activation of the GAL gene cluster in the yeast Saccharomyces cerevisiae, has led to an understanding of the molecular basis of galactose and nucleotide recognition. Finally, the structure has enabled us to further our understanding on the functional consequences of mutations in human GalK which cause galactosemia.
Assuntos
Galactoquinase/química , Galactoquinase/metabolismo , Pyrococcus furiosus/enzimologia , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Galactoquinase/genética , Galactose/metabolismo , Galactosemias/enzimologia , Galactosemias/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação Proteica , Dobramento de Proteína , Pyrococcus furiosus/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Especificidade por Substrato , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Galactokinase catalyses the conversion of galactose to galactose-1-phosphate as the first step in the Leloir pathway, a metabolic route that eventually enables the degradation of galactose via the glycolytic pathway. Galactokinases have been isolated from a wide range of prokaryotic and eukaryotic organisms and the enzyme has been identified as a member of the GHMP kinase (galactokinase, homoserine kinase, mevalonate kinase and phosphomevalonate kinase) superfamily. Pyrococcus furiosus galactokinase was cloned, expressed in Escherichia coli, purified and crystallized using the hanging-drop method of vapour diffusion with ammonium sulfate as the precipitant. The crystals belong to the space group C222(1), with more than eight subunits in the asymmetric unit and with approximate unit-cell parameters a = 211.7, b = 355.4, c = 165.5 A, alpha = beta = gamma = 90 degrees. The crystals diffract X-rays to 2.9 A resolution on a synchrotron-radiation source. Determination of the structure will provide insights into the molecular basis of substrate recognition and catalysis of this enzyme, for which no structures are currently available.