Crystal Structures of Physcomitrella patens AOC1 and AOC2: Insights into the Enzyme Mechanism and Differences in Substrate Specificity

In plants, oxylipins regulate developmental processes and defense responses. The first specific step in the biosynthesis of the cyclopentanone class of oxylipins is catalyzed by allene oxide cyclase (AOC) that forms cis(+)-12-oxo-phytodienoic acid. The moss Physcomitrella patens has two AOCs (PpAOC1 and PpAOC2) with different substrate specificities for C₁₈- and C₂₀-derived substrates, respectively. To better understand AOC’s catalytic mechanism and to elucidate the structural properties that explain the differences in substrate specificity, we solved and analyzed the crystal structures of 36 monomers of both apo and ligand complexes of PpAOC1 and PpAOC2. From these data, we propose the following intermediates in AOC catalysis: (1) a resting state of the apo enzyme with a closed conformation, (2) a first shallow binding mode, followed by (3) a tight binding of the substrate accompanied by conformational changes in the binding pocket, and (4) initiation of the catalytic cycle by opening of the epoxide ring. As expected, the substrate dihydro analog cis-12,13S-epoxy-9Z,15Z-octadecadienoic acid did not cyclize in the presence of PpAOC1; however, when bound to the enzyme, it underwent isomerization into the corresponding trans-epoxide. By comparing complex structures of the C₁₈ substrate analog with in silico modeling of the C₂₀ substrate analog bound to the enzyme allowed us to identify three major molecular determinants responsible for the different substrate specificities (i.e. larger active site diameter, an elongated cavity of PpAOC2, and two nonidentical residues at the entrance of the active site).Oxylipins comprise a large family of oxidized fatty acids and metabolites thereof (Acosta and Farmer, 2010). They are abundant in mammals (Funk, 2001) and flowering plants (Creelman and Mulpuri, 2002). In addition, they have been found in fungi (Brodhun and Feussner, 2011) as well as in nonflowering plants like mosses and algae (Andreou et al., 2009). In plants, these lipids serve as signaling molecules regulating developmental processes and mediating defense reactions (Howe and Jander, 2008; Browse, 2009; Acosta and Farmer, 2010). The first committed step in oxylipin biosynthesis is the peroxidation of a polyunsaturated fatty acid containing a 1Z,4Z-pentadiene system by lipoxygenase (LOX) or the peroxidation at the C2 position of a fatty acid by α-dioxygenase. These reactions start the so-called LOX or oxylipin pathway (Feussner and Wasternack, 2002) and are followed by further enzymatic reactions in which the hydroperoxy fatty acid is converted to a set of different secondary products. In the case of LOX-derived hydroperoxy fatty acids, such conversions are mainly catalyzed by members of the cytochrome P450 subfamily Cyp74 (i.e. fatty acid hydroperoxide lyase, divinyl ether synthase, epoxy alcohol synthase, and allene oxide synthase [AOS]; Stumpe and Feussner, 2006; Lee et al., 2008). Additional conversions of the fatty acid hydroperoxide are catalyzed by other proteins, such as LOX or peroxygenase (Mosblech et al., 2009).Jasmonic acid (JA) biosynthesis is one specific branch of the oxylipin pathway. It may start with the release of α-linolenic acid [18:3(n-3)] from membrane lipids by a lipase (Schaller and Stintzi, 2009). This free fatty acid is subsequently oxidized by a 13-LOX to yield 13-hydroperoxy octadecatrienoic acid (13-HPOTE) and converted by the action of AOS into the unstable allene oxide 12,13S-epoxy-9Z,11E,15Z-octadecatrienoic acid (12,13-EOT; Fig. 1). 12,13-EOT is then cyclized by allene oxide cyclase (AOC) to the cyclopentenone derivative cis(+)-12-oxo-phytodienoic acid [cis(+)-OPDA]. In the absence of AOC, the epoxide is hydrolyzed into ketols and racemic 12-oxo-phytodienoic acid (OPDA). cis(+)-OPDA is the first cyclic and biologically active compound in that pathway (Dave and Graham, 2012). While the reactions leading from 18:3(n-3) to cis(+)-OPDA occur in the plastid, all further enzymatic steps resulting in the formation of JA are localized in the peroxisomes (Wasternack, 2007). Here, cis(+)-OPDA is reduced in a NADPH-dependent reaction by cis(+)-OPDA reductase isoform 3 to 3-oxo-2(2′Z-pentenyl)-cyclopentane-1-octanoic acid. This step is followed by activation of the carboxyl group and three steps of β-oxidation and finally leads to the formation of (+)-7-iso-JA (Dave and Graham, 2012).Open in a separate windowFigure 1.Overview of the enzymatic steps in JA biosynthesis with molecular focus (box) on the reaction catalyzed by AOC. JA biosynthesis may start with the release of 18:3(n-3) or roughanic acid from a lipid. Next, the fatty acid is oxidized by a 13-LOX, yielding the 13-hydroperoxy derivative. This serves as a substrate for a subsequent conversion catalyzed by AOS and AOC, yielding the cyclopentenone derivatives cis(+)-OPDA and cis(+)-dinorOPDA, respectively, via an unstable allene oxide. Cyclization of the allene oxide seems to be initiated by one particular Glu residue in the active site of AOC that leads to an opening of the epoxy ring, conformational changes, and a concerted pericyclic ring closure (details are explained in the text). After reduction of the cyclopentenone by cis(+)-OPDA reductase isoform 3 (OPR3), the octanoic or hexanoic side chain is shortened by β-oxidation cycles.The conversion of 13-HPOTE into cis(+)-OPDA was first observed using a flaxseed (Linum usitatissimum) acetone powder preparation and was suggested to take place via a hypothetical epoxide intermediate (Vick et al., 1980). Later studies unequivocally demonstrated that 12,13-EOT (Hamberg, 1987; Brash et al., 1988), an allene oxide formed from 13-HPOTE by AOS (Song and Brash, 1991; Song et al., 1993), serves as substrate for the cyclization reaction catalyzed by AOC (Hamberg and Fahlstadius, 1990). The enzyme was purified (Ziegler et al., 1997), characterized with regard to the substrate specificity (Ziegler et al., 1999), and cloned and recombinantly expressed (Ziegler et al., 2000; Stenzel et al., 2003). In 2006, the crystal structure of an AOC from Arabidopsis (Arabidopsis thaliana; AtAOC2) was solved (Hofmann et al., 2006), and the reaction mechanism as well as the subcellular localization were studied (Schaller et al., 2008). The enzyme crystallized as a homotrimer, with each subunit forming an eight-stranded antiparallel β-barrel harboring a hydrophobic cavity in which the active site of the enzyme is located. While the exterior loops showed a high degree of flexibility, the central part of the enzyme was very rigid, and no induced-fit mechanism could be observed upon binding of a substrate analog (Hofmann et al., 2006). Based on the structure of AtAOC2 in complex with vernolic acid [cis(+/−)-12,13-epoxy-9Z-octadecenoic acid (12,13-EOM)] as an inert substrate analog, the following reaction mechanism has been proposed (Fig. 1, box): the allene oxide substrate binds with its fatty acid backbone deep in the barrel, where it interacts with hydrophobic amino acid residues, while the polar carboxy head group is located on the exterior of the cavity. One particular Glu residue (Glu-23 in AtAOC2) pointing to the Δ^15Z-double bond of the substrate may induce a partial charge separation that leads to a delocalization of the π-electron system, thereby facilitating opening of the epoxide ring. The oxyanion thus formed is stabilized via polar interactions with a catalytic, structurally conserved water molecule that is positioned in the polar cavity of the enzyme formed by two Asn residues (Asn-25 and Asn-53 in AtAOC2, respectively), one Ser (Ser-31 in AtAOC2), and one Pro (Pro-32 in AtAOC2). The ring closure that leads to the formation of the cyclopentenone derivative is achieved by a conformational reorganization of the C10-C11 substrate bond from the trans- to the cis-geometry. Due to steric limitations in the active site, this rotation may be accompanied by a cis/trans-isomerization of the C8-C9 substrate bond. Since the enzyme dictates the stereochemistry of the final ring closure, the released product is exclusively the (+)-enantiomer, cis(+)-OPDA (Schaller et al., 2008). Notably, this reaction competes with the spontaneous decomposition of the allene oxide substrate that leads to the formation of racemic OPDA as well as α-ketols and γ-ketols. This hints toward a low-energy barrier of the cyclization reaction and suggests that AOC does not need much of a catalytic functionality in terms of lowering this barrier (Schaller and Stintzi, 2009). It has been proposed that the enzymatic cyclization reaction is achieved according to the rules of Hoffmann and Woodward (1970) via a concerted pericyclic ring closure while spontaneous cyclization proceeds through a dipolar ring closure (Grechkin et al., 2002). The facts that the allene oxide formed by AOS has a very short half-life in aqueous solution and that natural OPDA is found in its enantiopure cis(+)-configuration suggest that AOS and AOC are coupled. However, no physical interaction of both enzymes may be necessary to form cis(+)-OPDA in vitro (Zerbe et al., 2007).Recently, it was shown that the moss Physcomitrella patens harbors and metabolizes not only C₁₈ but also C₂₀ polyunsaturated fatty acids to form oxylipins (Fig. 2; Stumpe et al., 2010). In particular, it was shown that (12S)-hydroperoxy eicosatetraenoic acid (12-HPETE) is endogenously formed by a bifunctional LOX as the major hydroperoxy fatty acid of arachidonic acid [20:4(n-6)] (Wichard et al., 2004). 12-HPETE serves as a substrate for further conversions either leading to the formation of C₈- and C₉-volatiles (e.g. octenals, octenols, and nonenals) or the cyclopentenone derivative 11-oxo prostatrienoic acid (11-OPTA; Stumpe et al., 2010). Whereas the volatiles are formed by at least two bifunctional LOXs with an additional hydroperoxide lyase activity (Wichard et al., 2004; Senger et al., 2005; Anterola et al., 2009) or by a Cyp74-derived hydroperoxide lyase (Stumpe et al., 2006), 11-OPTA is formed in analogy to the octadecanoids by one particular AOC, PpAOC2, via the allene oxide intermediate formed by PpAOS (Bandara et al., 2009). In contrast, PpAOC1 does not accept the 12-HPETE-derived C₂₀-allene oxide and thus converts only the 13-HPOTE-derived allene oxide.Open in a separate windowFigure 2.AOS/AOC pathways in P. patens. 13-HPOTE is converted by PpAOS to 12,13-EOT, which may either hydrolyze in the absence of PpAOC1 or PpAOC1 to ketols and racemic OPDA or, in the presence of PpAOC1 and PpAOC2, cyclize to cis(+)-OPDA. 12-HPETE is converted by PpAOS to 11,12-EET, which again may either hydrolyze in the absence of PpAOC2 to ketols and racemic OPDA or, in the presence of PpAOC2, cyclize to 11-OPTA.In this study, the crystal structures of PpAOC1 and PpAOC2 were solved. Data were also obtained for mutated forms of PpAOC1 and for PpAOC1 and PpAOC2 in complex with the allene oxide stable analog 12,13-EOD. In this way, detailed information about the allene oxide-to-cyclopentenone conversions promoted by the two AOCs was obtained.     

Crystal and EM Structures of Human Phosphoribosyl Pyrophosphate Synthase I (PRS1) Provide Novel Insights into the Disease-Associated Mutations

Human PRS1, which is indispensable for the biosynthesis of nucleotides, deoxynucleotides and their derivatives, is associated directly with multiple human diseases because of single base mutation. However, a molecular understanding of the effect of these mutations is hampered by the lack of understanding of its catalytic mechanism. Here, we reconstruct the 3D EM structure of the PRS1 apo state. Together with the native stain EM structures of AMPNPP, AMPNPP and R5P, ADP and the apo states with distinct conformations, we suggest the hexamer is the enzymatically active form. Based on crystal structures, sequence analysis, mutagenesis, enzyme kinetics assays, and MD simulations, we reveal the conserved substrates binding motifs and make further analysis of all pathogenic mutants.     

Crystal Structures of Human HMG-CoA Synthase Isoforms Provide Insights into Inherited Ketogenesis Disorders and Inhibitor Design

3-Hydroxy-3-methylglutaryl coenzyme A (CoA) synthase (HMGCS) catalyzes the condensation of acetyl-CoA and acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl CoA. It is ubiquitous across the phylogenetic tree and is broadly classified into three classes. The prokaryotic isoform is essential in Gram-positive bacteria for isoprenoid synthesis via the mevalonate pathway. The eukaryotic cytosolic isoform also participates in the mevalonate pathway but its end product is cholesterol. Mammals also contain a mitochondrial isoform; its deficiency results in an inherited disorder of ketone body formation. Here, we report high-resolution crystal structures of the human cytosolic (hHMGCS1) and mitochondrial (hHMGCS2) isoforms in binary product complexes. Our data represent the first structures solved for human HMGCS and the mitochondrial isoform, allowing for the first time structural comparison among the three isoforms. This serves as a starting point for the development of isoform-specific inhibitors that have potential cholesterol-reducing and antibiotic applications. In addition, missense mutations that cause mitochondrial HMGCS deficiency have been mapped onto the hHMGCS2 structure to rationalize the structural basis for the disease pathology.     

Novel UDP-GalNAc Derivative Structures Provide Insight into the Donor Specificity of Human Blood Group Glycosyltransferase

Two closely related glycosyltransferases are responsible for the final step of the biosynthesis of ABO(H) human blood group A and B antigens. The two enzymes differ by only four amino acid residues, which determine whether the enzymes transfer GalNAc from UDP-GalNAc or Gal from UDP-Gal to the H-antigen acceptor. The enzymes belong to the class of GT-A folded enzymes, grouped as GT6 in the CAZy database, and are characterized by a single domain with a metal dependent retaining reaction mechanism. However, the exact role of the four amino acid residues in the specificity of the enzymes is still unresolved. In this study, we report the first structural information of a dual specificity cis-AB blood group glycosyltransferase in complex with a synthetic UDP-GalNAc derivative. Interestingly, the GalNAc moiety adopts an unusual yet catalytically productive conformation in the binding pocket, which is different from the “tucked under” conformation previously observed for the UDP-Gal donor. In addition, we show that this UDP-GalNAc derivative in complex with the H-antigen acceptor provokes the same unusual binding pocket closure as seen for the corresponding UDP-Gal derivative. Despite this, the two derivatives show vastly different kinetic properties. Our results provide a important structural insight into the donor substrate specificity and utilization in blood group biosynthesis, which can very likely be exploited for the development of new glycosyltransferase inhibitors and probes.     

Insights into Cleavage Specificity from the Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease Complexed with a Peptide Substrate

Picornavirus replication is critically dependent on the correct processing of a polyprotein precursor by 3C protease(s) (3C^pro) at multiple specific sites with related but non-identical sequences. To investigate the structural basis of its cleavage specificity, we performed the first crystallographic structural analysis of non-covalent complexes of a picornavirus 3C^pro with peptide substrates. The X-ray crystal structure of the foot-and-mouth disease virus 3C^pro, mutated to replace the catalytic Cys by Ala and bound to a peptide (APAKQ|LLNFD) corresponding to the P5-P5′ region of the VP1-2A cleavage junction in the viral polyprotein, was determined up to 2.5 Å resolution. Comparison with free enzyme reveals significant conformational changes in 3C^pro on substrate binding that lead to the formation of an extended interface of contact primarily involving the P4-P2′ positions of the peptide. Strikingly, the deep S1′ specificity pocket needed to accommodate P1′-Leu only forms when the peptide binds. Substrate specificity was investigated using peptide cleavage assays to show the impact of amino acid substitutions within the P5-P4′ region of synthetic substrates. The structure of the enzyme-peptide complex explains the marked substrate preferences for particular P4, P2 and P1 residue types, as well as the relative promiscuity at P3 and on the P′ side of the scissile bond. Furthermore, crystallographic analysis of the complex with a modified VP1-2A peptide (APAKE|LLNFD) containing a Gln-to-Glu substitution reveals an identical mode of peptide binding and explains the ability of foot-and-mouth disease virus 3C^pro to cleave sequences containing either P1-Gln or P1-Glu. Structure-based mutagenesis was used to probe interactions within the S1′ specificity pocket and to provide direct evidence of the important contribution made by Asp84 of the Cys-His-Asp catalytic triad to proteolytic activity. Our results provide a new level of detail in our understanding of the structural basis of polyprotein cleavage by 3C^pro.     

Structural Insights into the Substrate Specificity Switch Mechanism of the Type III Protein Export Apparatus

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Crystal Structures of the CERT START Domain with Inhibitors Provide Insights into the Mechanism of Ceramide Transfer

The cytosolic protein CERT transfers ceramide from the endoplasmic reticulum to the Golgi apparatus where ceramide is converted to SM. The C-terminal START (steroidogenic acute regulatory protein-related lipid transfer) domain of CERT binds one ceramide molecule in its central amphiphilic cavity. (1R,3R)-N-(3-Hydroxy-1-hydroxymethyl-3-phenylpropyl)alkanamide (HPA), a synthesized analogue of ceramide, inhibits ceramide transfer by CERT. Here we report crystal structures of the CERT START domain in complex with HPAs of varying acyl chain lengths. In these structures, one HPA molecule is buried in the amphiphilic cavity where the amide and hydroxyl groups of HPA form a hydrogen-bond network with specific amino acid residues. The Ω1 loop, which has been suggested to function as a gate of the cavity, adopts a different conformation when bound to HPA than when bound to ceramide. In the Ω1 loop region, Trp473 shows the largest difference between these two structures. This residue exists inside of the cavity in HPA-bound structures, while it is exposed to the outside of the protein in the apo-form and ceramide-bound complex structures. Surface plasmon resonance experiments confirmed that Trp473 is important for interaction with membranes. These results provide insights into not only the molecular mechanism of inhibition by HPAs but also possible mechanisms by which CERT interacts with ceramide.     

Crystal Structure and Pathophysiological Role of the Pneumococcal Nucleoside-binding Protein PnrA

Nucleotides are important for RNA and DNA synthesis and, despite a de novo synthesis by bacteria, uptake systems are crucial. Streptococcus pneumoniae, a facultative human pathogen, produces a surface-exposed nucleoside-binding protein, PnrA, as part of an ABC transporter system. Here we demonstrate the binding affinity of PnrA to nucleosides adenosine, guanosine, cytidine, thymidine and uridine by microscale thermophoresis and indicate the consumption of adenosine and guanosine by ¹H NMR spectroscopy. In a series of five crystal structures we revealed the PnrA structure and provide insights into how PnrA can bind purine and pyrimidine ribonucleosides but with preference for purine ribonucleosides. Crystal structures of PnrA:nucleoside complexes unveil a clear pattern of interactions in which both the N- and C- domains of PnrA contribute. The ribose moiety is strongly recognized through a conserved network of H-bond interactions, while plasticity in loop 27–36 is essential to bind purine- or pyrimidine-based nucleosides.Further, we deciphered the role of PnrA in pneumococcal fitness in infection experiments. Phagocytosis experiments did not show a clear difference in phagocytosis between PnrA-deficient and wild-type pneumococci. In the acute pneumonia infection model the deficiency of PnrA attenuated moderately virulence of the mutant, which is indicated by a delay in the development of severe lung infections. Importantly, we confirmed the loss of fitness in co-infections, where the wild-type out-competed the pnrA-mutant. In conclusion, we present the PnrA structure in complex with individual nucleosides and show that the consumption of adenosine and guanosine under infection conditions is required for virulence.     

cAMP-dependent protein kinase: crystallographic insights into substrate recognition and phosphotransfer.

The crystal structure of ternary and binary substrate complexes of the catalytic subunit of cAMP-dependent protein kinase has been refined at 2.2 and 2.25 A resolution, respectively. The ternary complex contains ADP and a 20-residue substrate peptide, whereas the binary complex contains the phosphorylated substrate peptide. These 2 structures were refined to crystallographic R-factors of 17.5 and 18.1%, respectively. In the ternary complex, the hydroxyl oxygen OG of the serine at the P-site is 2.7 A from the OD1 atom of Asp 166. This is the first crystallographic evidence showing the direct interaction of this invariant carboxylate with a peptide substrate, and supports the predicted role of Asp 166 as a catalytic base and as an agent to position the serine -OH for nucleophilic attack. A comparison of the substrate and inhibitor ternary complexes places the hydroxyl oxygen of the serine 2.7 A from the gamma-phosphate of ATP and supports a direct in-line mechanism for phosphotransfer. In the binary complex, the phosphate on the Ser interacts directly with the epsilon N of Lys 168, another conserved residue. In the ternary complex containing ATP and the inhibitor peptide, Lys 168 interacts electrostatically with the gamma-phosphate of ATP (Zheng J, Knighton DR, Ten Eyck LF, Karlsson R, Xuong NH, Taylor SS, Sowadski JM, 1993, Biochemistry 32:2154-2161). Thus, Lys 168 remains closely associated with the phosphate in both complexes. A comparison of this binary complex structure with the recently solved structure of the ternary complex containing ATP and inhibitor peptide also reveals that the phosphate atom traverses a distance of about 1.5 A following nucleophilic attack by serine and transfer to the peptide. No major conformational changes of active site residues are seen when the substrate and product complexes are compared, although the binary complex with the phosphopeptide reveals localized changes in conformation in the region corresponding to the glycine-rich loop. The high B-factors for this loop support the conclusion that this structural motif is a highly mobile segment of the protein.     

Structural Insights into the Catalytic Domains of Human Matrix Metalloprotease-2 and Human Matrix Metalloprotease-9: Implications for Substrate Specificities

Structural information for the gelatinases A (MMP-2) and B (MMP-9), two members of the matrix metalloprotease (MMP) family of enzymes, has been elusive. For the first time, computational structures for the catalytic domains of MMP-2 and MMP-9 are reported herein using the program COMPOSER and the reported three-dimensional structures of the fibroblast collagenase (MMP-1), neutrophil collagenase (MMP-8) and stromelysin-1 (MMP-3). The details of the structures of the catalytic domains of gelatinases and interactions with the protein substrate are discussed. The first analysis of the extent of hydrophobicity of surfaces in the active sites of six MMPs (including the two gelatinases reported herein) is presented to provide distinction for substrate specificity among these metalloproteases. The information from the extent of hydrophobicity/hydrophilicity analysis and general topology for these MMPs was utilized in the proposal of a method for categorization of MMPs of known three-dimensional fold. These efforts provide the first information useful to experimentalists working on the biochemical properties of these important members of the MMP family of enzymes, and provide for an opportunity to compare and contrast structures of gelatinases, collagenases and stromelysins.Electronic Supplementary Material available.     

Structural Analysis of a Family 101 Glycoside Hydrolase in Complex with Carbohydrates Reveals Insights into Its Mechanism

O-Linked glycosylation is one of the most abundant post-translational modifications of proteins. Within the secretory pathway of higher eukaryotes, the core of these glycans is frequently an N-acetylgalactosamine residue that is α-linked to serine or threonine residues. Glycoside hydrolases in family 101 are presently the only known enzymes to be able to hydrolyze this glycosidic linkage. Here we determine the high-resolution structures of the catalytic domain comprising a fragment of GH101 from Streptococcus pneumoniae TIGR4, SpGH101, in the absence of carbohydrate, and in complex with reaction products, inhibitor, and substrate analogues. Upon substrate binding, a tryptophan lid (residues 724-WNW-726) closes on the substrate. The closing of this lid fully engages the substrate in the active site with Asp-764 positioned directly beneath C1 of the sugar residue bound within the −1 subsite, consistent with its proposed role as the catalytic nucleophile. In all of the bound forms of the enzyme, however, the proposed catalytic acid/base residue was found to be too distant from the glycosidic oxygen (>4.3 Å) to serve directly as a general catalytic acid/base residue and thereby facilitate cleavage of the glycosidic bond. These same complexes, however, revealed a structurally conserved water molecule positioned between the catalytic acid/base and the glycosidic oxygen. On the basis of these structural observations we propose a new variation of the retaining glycoside hydrolase mechanism wherein the intervening water molecule enables a Grotthuss proton shuttle between Glu-796 and the glycosidic oxygen, permitting this residue to serve as the general acid/base catalytic residue.     

Two Functional States of the CD11b A-Domain: Correlations with Key Features of Two Mn2+-complexed Crystal Structures

In the presence of bound Mn²⁺, the three- dimensional structure of the ligand-binding A-domain from the integrin CR3 (CD11b/CD18) is shown to exist in the “open” conformation previously described only for a crystalline Mg²⁺ complex. The open conformation is distinguished from the “closed” form by the solvent exposure of F302, a direct T209–Mn²⁺ bond, and the presence of a glutamate side chain in the MIDAS site. Approximately 10% of wild-type CD11b A-domain is present in an “active” state (binds to activation-dependent ligands, e.g., iC3b and the mAb 7E3). In the isolated domain and in the holoreceptor, the percentage of the active form can be quantitatively increased or abolished in F302W and T209A mutants, respectively. The iC3b-binding site is located on the MIDAS face and includes conformationally sensitive residues that undergo significant shifts in the open versus closed structures. We suggest that stabilization of the open structure is independent of the nature of the metal ligand and that the open conformation may represent the physiologically active form.     

Crystal Structure of Human Dual-Specificity Tyrosine-Regulated Kinase 3 Reveals New Structural Features and Insights into its Auto-phosphorylation

Dual-specificity tyrosine-regulated kinases (DYRKs) auto-phosphorylate a critical tyrosine residue in their activation loop and phosphorylate their substrate on serine and threonine residues. The auto-phosphorylation occurs intramolecularly and is a one-off event. DYRK3 is selectively expressed at a high level in hematopoietic cells and attenuates erythroblast development, leading to anemia. In the present study, we determined the crystal structure of the mature form of human DYRK3 in complex with harmine, an ATP competitive inhibitor. The crystal structure revealed a phosphorylation site, residue S350, whose phosphorylation increases the stability of DYRK3 and enhances its kinase activity. In addition, our structural and biochemical assays suggest that the N-terminal auto-phosphorylation accessory domain stabilizes the DYRK3 protein, followed by auto-phosphorylation of the tyrosine of the activation loop, which is important for kinase activity. Finally, our docking analysis provides information for the design of novel and potent therapeutics to treat anemia.     

Crystal structure of a chimera of human and Plasmodium falciparum hypoxanthine guanine phosphoribosyltransferases provides insights into oligomerization

The crystal structure of a chimera of Plasmodium falciparum (Pf) and human hypoxanthine guanine phosphoribosyltransferases (HGPRT), which consists of the core of the protein from the human enzyme and the hood region from the Pf enzyme, has been determined as a complex with the product guanosine monophosphate (GMP). The chimera can utilize hypoxanthine, guanine, and xanthine as substrates, similar to the Pf enzyme. It exists as a monomer-dimer mixture in solution, but shifts to a tetramer on addition of phosphoribosyl pyrophosphate (PRPP). The structural studies reveal that the asymmetric unit of the crystal consists of two monomers of the chimeric HGPRT. Surprisingly, the dimer interface of the chimera is the less extensive AC interface of the parent HGPRTs. An analysis of the crystal structures of the various human HGPRTs provides an explanation for the oligomeric characteristics of the chimera. Pro93 and Tyr197 form part of crucial interactions holding together the AB interface in the unliganded or GMP-bound forms of HGPRT, while Pro93 and His26 interact at the interface after binding of PRPP. Replacement of Tyr197 of human HGPRT by Ile207 in the chimera disrupts the interaction at the AB interface in the absence of PRPP. In the presence of PRPP, the interaction between Pro93 and His26 could restore the AB interface, shifting the chimeric enzyme to a tetrameric state. The structure provides valuable insights into the differences in the AB interface between Pf and human HGPRTs, which may be useful for designing selective inhibitors against the parasite enzyme.     

Insights into the fatty acid chain length specificity of the carboxylesterase PA3859 from Pseudomonas aeruginosa: A combined structural,biochemical and computational study

The open reading frame PA3859 of Pseudomonas aeruginosa encodes an intracellular carboxylesterase belonging to a group of microbial enzymes (EC 3.1.1.1) that catalyze the hydrolysis of aliphatic and aromatic esters with a broad substrate specificity. With few exceptions, for this class of enzymes, belonging to the α/β-hydrolase fold superfamily, very little information is available regarding their biochemical activity and in vivo function. The X-ray crystal structure of recombinant PA3859 has been determined for two crystal forms (space groups P2₁ and P2₁2₁2). The kinetic properties of the enzyme were studied using p-nitrophenyl esters as substrates and data fitted to a surface dilution mixed micelle kinetic model. Enzymatic assays and computational docking simulations, pinpointed the enzyme’s preference for esters of palmitic and/or stearic acids and provided insights into the enzyme–substrate favorable binding modes.     

Crystal Structure of Glyceraldehyde-3-Phosphate Dehydrogenase 1 from Methicillin-Resistant Staphylococcus aureus MRSA252 Provides Novel Insights into Substrate Binding and Catalytic Mechanism

The dreaded pathogen Staphylococcus aureus is one of the causes of morbidity and mortality worldwide. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), one of the key glycolytic enzymes, is irreversibly oxidized under oxidative stress and is responsible for sustenance of the pathogen inside the host. With an aim to elucidate the catalytic mechanism and identification of intermediates involved, we describe in this study different crystal structures of GAPDH1 from methicillin-resistant S. aureus MRSA252 (SaGAPDH1) in apo and holo forms of wild type, thioacyl intermediate, and ternary complexes of active-site mutants with physiological substrate d-glyceraldehyde-3-phosphate (G3P) and coenzyme NAD⁺. A new phosphate recognition site, “new P_i” site, similar to that observed in GAPDH from Thermotoga maritima, is reported here, which is 3.40 Å away from the “classical P_i” site. Ternary complexes discussed are representatives of noncovalent Michaelis complexes in the ground state. d-G3P is bound to all the four subunits of C151S.NAD and C151G.NAD in more reactive hydrate (gem-di-ol) form. However, in C151S + H178N.NAD, the substrate is bound to two chains in aldehyde form and in gem-di-ol form to the other two. This work reports binding of d-G3P to the C151G mutant in an inverted manner for the very first time. The structure of the thiaocyl complex presented here is formed after the hydride transfer. The C3 phosphate of d-G3P is positioned at the “P_s” site in the ternary complexes but at the “new P_i” site in the thioacyl complex and C1-O1 bond points opposite to His178 disrupting the alignment between itself and NE2 of His178. A new conformation (Conformation I) of the 209-215 loop has also been identified, where the interaction between phosphate ion at the “new P_i” site and conserved Gly212 is lost. Altogether, inferences drawn from the kinetic analyses and crystal structures suggest the “flip-flop” model proposed for the enzyme mechanism.     

Role of Phe283 in enzymatic reaction of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp.1011: Substrate binding and arrangement of the catalytic site

Cyclodextrin glycosyltransferase (CGTase) belonging to the alpha-amylase family mainly catalyzes transglycosylation and produces cyclodextrins from starch and related alpha-1,4-glucans. The catalytic site of CGTase specifically conserves four aromatic residues, Phe183, Tyr195, Phe259, and Phe283, which are not found in alpha-amylase. To elucidate the structural role of Phe283, we determined the crystal structures of native and acarbose-complexed mutant CGTases in which Phe283 was replaced with leucine (F283L) or tyrosine (F283Y). The temperature factors of the region 259-269 in native F283L increased >10 A(2) compared with the wild type. The complex formation with acarbose not only increased the temperature factors (>10 A(2)) but also changed the structure of the region 257-267. This region is stabilized by interactions of Phe283 with Phe259 and Leu260 and plays an important role in the cyclodextrin binding. The conformation of the side-chains of Glu257, Phe259, His327, and Asp328 in the catalytic site was altered by the mutation of Phe283 with leucine, and this indicates that Phe283 partly arranges the structure of the catalytic site through contacts with Glu257 and Phe259. The replacement of Phe283 with tyrosine decreased the enzymatic activity in the basic pH range. The hydroxyl group of Tyr283 forms hydrogen bonds with the carboxyl group of Glu257, and the pK(a) of Glu257 in F283Y may be lower than that in the wild type.