High resolution crystal structures of human kynurenine aminotransferase‐I bound to PLP cofactor,and in complex with aminooxyacetate

In this study, we report two high‐resolution structures of the pyridoxal 5′ phosphate (PLP)‐dependent enzyme kynurenine aminotransferase‐I (KAT‐I). One is the native structure with the cofactor in the PLP form bound to Lys247 with the highest resolution yet available for KAT‐I at 1.28 Å resolution, and the other with the general PLP‐dependent aminotransferase inhibitor, aminooxyacetate (AOAA) covalently bound to the cofactor at 1.54 Å. Only small conformational differences are observed in the vicinity of the aldimine (oxime) linkage with which the PLP forms the Schiff base with Lys247 in the 1.28 Å resolution native structure, in comparison to other native PLP‐bound structures. We also report the inhibition of KAT‐1 by AOAA and aminooxy‐phenylpropionic acid (AOPP), with IC50s of 13.1 and 5.7 μM, respectively. The crystal structure of the enzyme in complex with the inhibitor AOAA revealed that the cofactor is the PLP form with the external aldimine linkage. The location of this oxime with the PLP, which forms in place of the native internal aldimine linkage of PLP of the native KAT‐I, is away from the position of the native internal aldimine, with the free Lys247 substantially retaining the orientation of the native structure. Tyr101, at the active site, was observed in two conformations in both structures.     

Structure of the external aldimine form of PglE,an aminotransferase required for N,N'‐diacetylbacillosamine biosynthesis

N,N'‐diacetylbacillosamine is a novel sugar that plays a key role in bacterial glycosylation. Three enzymes are required for its biosynthesis in Campylobacter jejuni starting from UDP‐GlcNAc. The focus of this investigation, PglE, catalyzes the second step in the pathway. It is a PLP‐dependent aminotransferase that converts UDP‐2‐acetamido‐4‐keto‐2,4,6‐trideoxy‐d ‐glucose to UDP‐2‐acetamido‐4‐amino‐2,4,6‐trideoxy‐d ‐glucose. For this investigation, the structure of PglE in complex with an external aldimine was determined to a nominal resolution of 2.0 Å. A comparison of its structure with those of other sugar aminotransferases reveals a remarkable difference in the manner by which PglE accommodates its nucleotide‐linked sugar substrate.     

Crystal structure of New Delhi metallo-β-lactamase reveals molecular basis for antibiotic resistance

β‐Lactams are the most commonly prescribed class of antibiotics and have had an enormous impact on human health. Thus, it is disquieting that an enzyme called New Delhi metallo‐β‐lactamase‐1 (NDM‐1) can confer Enterobacteriaceae with nearly complete resistance to all β‐lactam antibiotics including the carbapenams. We have determined the crystal structure of Klebsiella pneumoniae apo‐NDM‐1 to 2.1‐Å resolution. From the structure, we see that NDM‐1 has an expansive active site with a unique electrostatic profile, which we propose leads to a broader substrate specificity. In addition, NDM‐1 undergoes important conformational changes upon substrate binding. These changes have not been previously observed in metallo‐β‐lactamase enzymes and may have a direct influence on substrate recognition and catalysis.     

Crystal structure of the S187F variant of human liver alanine: Aminotransferase associated with primary hyperoxaluria type I and its functional implications

The substitution of Ser187, a residue located far from the active site of human liver peroxisomal alanine:glyoxylate aminotransferase (AGT), by Phe gives rise to a variant associated with primary hyperoxaluria type I. Unexpectedly, previous studies revealed that the recombinant form of S187F exhibits a remarkable loss of catalytic activity, an increased pyridoxal 5′‐phosphate (PLP) binding affinity and a different coenzyme binding mode compared with normal AGT. To shed light on the structural elements responsible for these defects, we solved the crystal structure of the variant to a resolution of 2.9 Å. Although the overall conformation of the variant is similar to that of normal AGT, we noticed: (i) a displacement of the PLP‐binding Lys209 and Val185, located on the re and si side of PLP, respectively, and (ii) slight conformational changes of other active site residues, in particular Trp108, the base stacking residue with the pyridine cofactor moiety. This active site perturbation results in a mispositioning of the AGT‐pyridoxamine 5′‐phosphate (PMP) complex and of the external aldimine, as predicted by molecular modeling studies. Taken together, both predicted and observed movements caused by the S187F mutation are consistent with the following functional properties of the variant: (i) a 300‐ to 500‐fold decrease in both the rate constant of L‐alanine half‐transamination and the k_cat of the overall transamination, (ii) a different PMP binding mode and affinity, and (iii) a different microenvironment of the external aldimine. Proposals for the treatment of patients bearing S187F mutation are discussed on the basis of these results. Proteins 2013; 81:1457–1465. © 2013 Wiley Periodicals, Inc.     

Structural investigation on WlaRG from Campylobacter jejuni: A sugar aminotransferase

Campylobacter jejuni is a Gram‐negative bacterium that represents a leading cause of human gastroenteritis worldwide. Of particular concern is the link between C. jejuni infections and the subsequent development of Guillain‐Barré syndrome, an acquired autoimmune disorder leading to paralysis. All Gram‐negative bacteria contain complex glycoconjugates anchored to their outer membranes, but in most strains of C. jejuni, this lipoglycan lacks the O‐antigen repeating units. Recent mass spectrometry analyses indicate that the C. jejuni 81116 (Penner serotype HS:6) lipoglycan contains two dideoxyhexosamine residues, and enzymological assay data show that this bacterial strain can synthesize both dTDP‐3‐acetamido‐3,6‐dideoxy‐d ‐glucose and dTDP‐3‐acetamido‐3,6‐dideoxy‐d ‐galactose. The focus of this investigation is on WlaRG from C. jejuni, which plays a key role in the production of these unusual sugars by functioning as a pyridoxal 5′‐phosphate dependent aminotransferase. Here, we describe the first three‐dimensional structures of the enzyme in various complexes determined to resolutions of 1.7 Å or higher. Of particular significance are the external aldimine structures of WlaRG solved in the presence of either dTDP‐3‐amino‐3,6‐dideoxy‐d ‐galactose or dTDP‐3‐amino‐3,6‐dideoxy‐d ‐glucose. These models highlight the manner in which WlaRG can accommodate sugars with differing stereochemistries about their C‐4′ carbon positions. In addition, we present a corrected structure of WbpE, a related sugar aminotransferase from Pseudomonas aeruginosa, solved to 1.3 Å resolution.     

Structure of the bacterial type II NADH dehydrogenase: a monotopic membrane protein with an essential role in energy generation

Non‐proton pumping type II NADH dehydrogenase (NDH‐2) plays a central role in the respiratory metabolism of bacteria, and in the mitochondria of fungi, plants and protists. The lack of NDH‐2 in mammalian mitochondria and its essentiality in important bacterial pathogens suggests these enzymes may represent a potential new drug target to combat microbial pathogens. Here, we report the first crystal structure of a bacterial NDH‐2 enzyme at 2.5 Å resolution from Caldalkalibacillus thermarum. The NDH‐2 structure reveals a homodimeric organization that has a unique dimer interface. NDH‐2 is localized to the cytoplasmic membrane by two separated C‐terminal membrane‐anchoring regions that are essential for membrane localization and FAD binding, but not NDH‐2 dimerization. Comparison of bacterial NDH‐2 with the yeast NADH dehydrogenase (Ndi1) structure revealed non‐overlapping binding sites for quinone and NADH in the bacterial enzyme. The bacterial NDH‐2 structure establishes a framework for the structure‐based design of small‐molecule inhibitors.     

Structure of the full‐length Serratia marcescens acetyltransferase AAC(3)‐Ia in complex with coenzyme A

Acyl‐coenzyme A‐dependent N‐acetyltransferases (AACs) catalyze the modification of aminoglycosides rendering the bacteria carrying such enzymes resistant to this class of antibiotics. Here we present the crystal structure of AAC(3)‐Ia enzyme from Serratia marcescens in complex with coenzyme A determined to 1.8 Å resolution. This enzyme served as an architype for the AAC enzymes targeting the amino group at Position 3 of aminoglycoside main aminocyclitol ring. The structure of this enzyme has been previously determined only in truncated form and was interpreted as distinct from subsequently characterized AACs. The reason for the unusual arrangement of secondary structure elements of AAC(3)‐Ia was not further investigated. By determining the full‐length structure of AAC(3)‐Ia we establish that this enzyme adopts the canonical AAC fold conserved across this family and it does not undergo through significant rearrangement of secondary structure elements upon ligand binding as was proposed previously. In addition, our results suggest that the C‐terminal tail in AAC(3)‐Ia monomer forms intramolecular hydrogen bonds that contributes to formation of stable dimer, representing the predominant oligomeric state for this enzyme.     

The macro domain as fusion tag for carrier‐driven crystallization

Obtaining well‐ordered crystals remains a significant challenge in protein X‐ray crystallography. Carrier‐driven crystallization can facilitate crystal formation and structure solution of difficult target proteins. We obtained crystals of the small and highly flexible SPX domain from the yeast vacuolar transporter chaperone 4 (Vtc4) when fused to a C‐terminal, non‐cleavable macro tag derived from human histone macroH2A1.1. Initial crystals diffracted to 3.3 Å resolution. Reductive protein methylation of the fusion protein yielded a new crystal form diffracting to 2.1 Å. The structures were solved by molecular replacement, using isolated macro domain structures as search models. Our findings suggest that macro domain tags can be employed in recombinant protein expression in E. coli, and in carrier‐driven crystallization.     

Structure and function of an acetyl xylan esterase (Est2A) from the rumen bacterium Butyrivibrio proteoclasticus

Butyrivibrio proteoclasticus is a significant component of the microbial population of the rumen of dairy cattle. It is a xylan‐degrading organism whose genome encodes a large number of open reading frames annotated as fiber‐degrading enzymes. We have determined the three‐dimensional structure of Est2A, an acetyl xylan esterase from B. proteoclasticus, at 2.1 Å resolution, along with the structure of an inactive mutant (H351A) at 2.0 Å resolution. The structure reveals two domains—a C‐terminal SGNH domain and an N‐terminal jelly‐roll domain typical of CE2 family structures. The structures are accompanied by experimentally determined enzymatic parameters against two model substrates, para‐nitrophenyl acetate and para‐nitrophenyl butyrate. The suite of fiber‐degrading enzymes produced by B. proteoclasticus provides a rich source of new enzymes of potential use in industrial settings. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Human α‐amino‐β‐carboxymuconate‐ε‐semialdehyde decarboxylase (ACMSD): A structural and mechanistic unveiling

Human α‐amino‐β‐carboxymuconate‐ε‐semialdehyde decarboxylase determines the fate of tryptophan metabolites in the kynurenine pathway by controlling the quinolinate levels for de novo nicotinamide adenine dinucleotide biosynthesis. The unstable nature of its substrate has made gaining insight into its reaction mechanism difficult. Our electron paramagnetic resonance (EPR) spectroscopic study on the Cu‐substituted human enzyme suggests that the native substrate does not directly ligate to the metal ion. Substrate binding did not result in a change of either the hyperfine structure or the super‐hyperfine structure of the EPR spectrum. We also determined the crystal structure of the human enzyme in its native catalytically active state (at 1.99 Å resolution), a substrate analogue‐bound form (2.50 Å resolution), and a selected active site mutant form with one of the putative substrate binding residues altered (2.32 Å resolution). These structures illustrate that each asymmetric unit contains three pairs of dimers. Consistent with the EPR findings, the ligand‐bound complex structure shows that the substrate analogue does not directly coordinate to the metal ion but is bound to the active site by two arginine residues through noncovalent interactions. Proteins 2015; 83:178–187. © 2014 Wiley Periodicals, Inc.     

Structure determination of feline panleukopenia virus empty particles

Various crystal forms of the single-stranded DNA, feline panleukopenia virus (FPV), a parvovirus, have been grown of both full virions and empty particles. The structure of empty particles crystallized in an orthorhombic space group P2₁2₁2₁, with unit cell dimensions a = 380.1 Å, b = 379.3 Å, and c = 350.9 Å, has been determined to 3.3 Å resolution. The data were collected using oscillation photography with synchrotron radiation. The orientations of the empty capsids in the unit cell were determined using a self-rotation function and their positions were obtained with an R-factor search using canine parvovirus (CPV) as a model. Phases were then calculated, based on the CPV model, to 6.0 Å resolution and gradually extended to 3.3 Å resolution by molecular replacement electron density averaging. The resultant electron density was readily interpreted in terms of the known amino acid sequence. The structure is contrasted to that of CPV in terms of host range, neutralization by antibodies, hemagglutination properties, and binding of genomic DNA. © Wiley-Liss, Inc.     

Structural insights into catalysis by βC-S lyase from Streptococcus anginosus

Hydrogen sulfide (H₂S) is a causative agent of oral malodor and may play an important role in the pathogenicity of oral bacteria such as Streptococcus anginosus. In this microorganism, H₂S production is associated with βC‐S lyase (Lcd) encoded by lcd gene, which is a pyridoxal 5′‐phosphate (PLP)‐dependent enzyme that catalyzes the α,β‐elimination of sulfur‐containing amino acids. When Lcd acts on L ‐cysteine, H₂S is produced along with pyruvate and ammonia. To understand the H₂S‐producing mechanism of Lcd in detail, we determined the crystal structures of substrate‐free Lcd (internal aldimine form) and two reaction intermediate complexes (external aldimine and α‐aminoacrylate forms). The formation of intermediates induced little changes in the overall structure of the enzyme and in the active site residues, with the exception of Lys234, a PLP‐binding residue. Structural and mutational analyses highlighted the importance of the active site residues Tyr60, Tyr119, and Arg365. In particular, Tyr119 forms a hydrogen bond with the side chain oxygen atom of L ‐serine, a substrate analog, in the external aldimine form suggesting its role in the recognition of the sulfur atom of the true substrate (L ‐cysteine). Tyr119 also plays a role in fixing the PLP cofactor at the proper position during catalysis through binding with its side chain. Finally, we partly modified the catalytic mechanism known for cystalysin, a βC‐S lyase from Treponema denticola, and proposed an improved mechanism, which seems to be common to the βC‐S lyases from oral bacteria. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Three‐dimensional structure of a sugar N‐formyltransferase from Francisella tularensis

N‐formylated sugars have been observed on the O‐antigens of such pathogenic Gram‐negative bacteria as Campylobacter jejuni and Francisella tularensis. Until recently, however, little was known regarding the overall molecular architectures of the N‐formyltransferases that are required for the biosynthesis of these unusual sugars. Here we demonstrate that the protein encoded by the wbtj gene from F. tularensis is an N‐formyltransferase that functions on dTDP‐4‐amino‐4,6‐dideoxy‐d ‐glucose as its substrate. The enzyme, hereafter referred to as WbtJ, demonstrates a strict requirement for N¹⁰‐formyltetrahydrofolate as its carbon source. In addition to the kinetic analysis, the three‐dimensional structure of the enzyme was solved in the presence of dTDP‐sugar ligands to a nominal resolution of 2.1 Å. Each subunit of the dimeric enzyme is dominated by a “core” domain defined by Met 1 to Ser 185. This core motif harbors the active site residues. Following the core domain, the last 56 residues fold into two α‐helices and a β‐hairpin motif. The hairpin motif is responsible primarily for the subunit:subunit interface, which is characterized by a rather hydrophobic pocket. From the study presented here, it is now known that WbtJ functions on C‐4′ amino sugars. Another enzyme recently investigated in the laboratory, WlaRD, formylates only C‐3′ amino sugars. Strikingly, the quaternary structures of WbtJ and WlaRD are remarkably different. In addition, there are several significant variations in the side chains that line their active site pockets, which may be important for substrate specificity. Details concerning the kinetic and structural properties of WbtJ are presented.     

High resolution crystal structure of Clostridium propionicum β‐alanyl‐CoA:ammonia lyase,a new member of the “hot dog fold” protein superfamily

Clostridium propionicum is the only organism known to ferment β‐alanine, a constituent of coenzyme A (CoA) and the phosphopantetheinyl prosthetic group of holo‐acyl carrier protein. The first step in the fermentation is a CoA‐transfer to β‐alanine. Subsequently, the resulting β‐alanyl‐CoA is deaminated by the enzyme β‐alanyl‐CoA:ammonia lyase (Acl) to reversibly form ammonia and acrylyl‐CoA. We have determined the crystal structure of Acl in its apo‐form at a resolution of 0.97 Å as well as in complex with CoA at a resolution of 1.59 Å. The structures reveal that the enyzme belongs to a superfamily of proteins exhibiting a so called “hot dog fold” which is characterized by a five‐stranded antiparallel β‐sheet with a long α‐helix packed against it. The functional unit of all “hot dog fold” proteins is a homodimer containing two equivalent substrate binding sites which are established by the dimer interface. In the case of Acl, three functional dimers combine to a homohexamer strongly resembling the homohexamer formed by YciA‐like acyl‐CoA thioesterases. Here, we propose an enzymatic mechanism based on the crystal structure of the Acl·CoA complex and molecular docking. Proteins 2014; 82:2041–2053. © 2014 Wiley Periodicals, Inc.     

Purification,crystallization, and preliminary X-ray studies of 10-formyltetrahydrofolate synthetase from Clostridia acidici-urici

The monofunctional enzyme 10-formyltetrahydrofolate synthetase (THFS), which is responsible for the recruitment of single carbon units from the formate pool into a variety of folate-dependent biosynthetic pathways, has been subcloned, purified, and crystallized. The crystals belong to space group P2₁, with unit cell dimensions a= 102.4 Å b= 116.5 Å c= 115.8 Å and β = 103.5 Å. The crystal unit cell and diffraction is consistent with an asymmetric unit consisting of the enzyme tetramer, and a specific volume of the unit cell of 2.7 Å₃/Da. The crystals diffract to at least 2.3 Å resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector. © 1997 Wiley-Liss, Inc.     

Purification,crystallization, and preliminary X-ray studies of a bifunctional 5,10-methenyl/methylene tetrahydrofolate cyclohydrolase/dehydrogenase from Escherichia coli

A bifunctional enzyme that catalyzes the conversion of formyltetrahydrofolate to methylene-tetrahydrofolate (5,10-methenyltetrahydrofolate cyclohydrolase and 5,10-methylene tetrahydrofolate dehydrogenease), has been subcloned from a cDNA library, purified to homogeneity, and crystallized. The crystals belong to space group I222, with unit cell dimensions of a= 64.5 Å b= 84.9 Å c= 146.1 Å. The crystal unit cell and diffraction is consistent with an asymmetric unit consisting of the enzyme monomer, and a specific volume of the unit cell of 3.2 ų/Da. The crystals diffract to at least 2.8 Å resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector. A 2.56 Å resolution native data set has been collected at beamline X12-C at the NSLS. © 1997 Wiley-Liss, Inc.     

Unique coenzyme binding mode of hyperthermophilic archaeal sn‐glycerol‐1‐phosphate dehydrogenase from Pyrobaculum calidifontis

A gene encoding an sn‐glycerol‐1‐phosphate dehydrogenase (G1PDH) was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. In contrast to conventional G1PDHs, the expressed enzyme showed strong preference for NADH: the reaction rate (V_max) with NADPH was only 2.4% of that with NADH. The crystal structure of the enzyme was determined at a resolution of 2.45 Å. The asymmetric unit consisted of one homohexamer. Refinement of the structure and HPLC analysis showed the presence of the bound cofactor NADPH in subunits D, E, and F, even though it was not added in the crystallization procedure. The phosphate group at C2’ of the adenine ribose of NADPH is tightly held through the five biased hydrogen bonds with Ser40 and Thr42. In comparison with the known G1PDH structure, the NADPH molecule was observed to be pushed away from the normal coenzyme binding site. Interestingly, the S40A/T42A double mutant enzyme acquired much higher reactivity than the wild‐type enzyme with NADPH, which suggests that the biased interactions around the C2’‐phosphate group make NADPH binding insufficient for catalysis. Our results provide a unique structural basis for coenzyme preference in NAD(P)‐dependent dehydrogenases. Proteins 2016; 84:1786–1796. © 2016 Wiley Periodicals, Inc.     

Crystallization and preliminary X-ray diffraction studies of leishmanolysin,the major surface metalloproteinase from Leishmania major

The membrane-bound GPI-anchored zinc metalloproteinase leishmanolysin purified from Leishmania major promastigotes has been crystallized in its mature form. Two crystal forms of leishmanolysin have been grown by the vapor diffusion method using 2-methyl-2,4-pentanediol as the precipitant. Macroseeding techniques were employed to produce large single crystals. Protein microhet-erogeneity in molecular size and charge was incorporated into both crystal forms. The tetragonal crystal form belongs to the space group P4₁2₁2 or the enantiomorph P4₃2₁2, has unit cell parameters of a = b = 63.6 Å, c = 251.4 Å, and contains one molecule per asymmetric unit. The second crystal form is monoclinic, space group C2, with unit cell dimensions a = 107.2 Å, b = 90.6 Å, c = 70.6 Å, β = 110.6°, and also contains one molecule per asymmetric unit. Both crystal forms diffract X-rays beyond 2.6 Å resolution and are suitable for X-ray analysis. Native diffraction data sets have been collected and the structure determination of leishmanolysin using a combination of the isomorphous replacement and the molecular replacement methods is in progress. © 1995 Wiley-Liss, Inc.     

Mechanism of 8-amino-7-oxononanoate synthase: spectroscopic, kinetic, and crystallographic studies

8-Amino-7-oxononanoate synthase (also known as 7-keto-8-aminopelargonate synthase, EC 2.3.1.47) is a pyridoxal 5'-phosphate-dependent enzyme which catalyzes the decarboxylative condensation of L-alanine with pimeloyl-CoA in a stereospecific manner to form 8(S)-amino-7-oxononanoate. This is the first committed step in biotin biosynthesis. The mechanism of Escherichia coli AONS has been investigated by spectroscopic, kinetic, and crystallographic techniques. The X-ray structure of the holoenzyme has been refined at a resolution of 1.7 A (R = 18.6%, R(free) = 21. 2%) and shows that the plane of the imine bond of the internal aldimine deviates from the pyridine plane. The structure of the enzyme-product external aldimine complex has been refined at a resolution of 2.0 A (R = 21.2%, R(free) = 27.8%) and shows a rotation of the pyridine ring with respect to that in the internal aldimine, together with a significant conformational change of the C-terminal domain and subtle rearrangement of the active site hydrogen bonding. The first step in the reaction, L-alanine external aldimine formation, is rapid (k(1) = 2 x 10(4) M(-)(1) s(-)(1)). Formation of an external aldimine with D-alanine, which is not a substrate, is significantly slower (k(1) = 125 M(-)(1) s(-)(1)). Binding of D-alanine to AONS is enhanced approximately 2-fold in the presence of pimeloyl-CoA. Significant substrate quinonoid formation only occurs upon addition of pimeloyl-CoA to the preformed L-alanine external aldimine complex and is preceded by a distinct lag phase ( approximately 30 ms) which suggests that binding of the pimeloyl-CoA causes a conformational transition of the enzyme external aldimine complex. This transition, which is inferred by modeling to require a rotation around the Calpha-N bond of the external aldimine complex, promotes abstraction of the Calpha proton by Lys236. These results have been combined to form a detailed mechanistic pathway for AONS catalysis which may be applied to the other members of the alpha-oxoamine synthase subfamily.     

Crystallization of the NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae

Saccharomyces cerevisiae possesses three isozymes of 5,10-methylenetetrahydrofolate dehydrogenase (MTD). The NAD-dependent enzyme is the first monofunctional form found in eukaryotes. Here we report its crystallization in a form suitable for high-resolution structure. The space group is P4₂2₁2 with cell constants a = b = 75.9, c = 160.0 Å, and there is one 36 kDa molecule in the asymmetric unit. Crystals diffract to 2.9 Å resolution. Proteins 26:481–482 © 1996 Wiley-Liss, Inc.