Betaine-homocysteine methyltransferase: zinc in a distorted barrel

Betaine-homocysteine methyl transferase (BHMT) catalyzes the synthesis of methionine from betaine and homocysteine (Hcy), utilizing a zinc ion to activate Hcy. BHMT is a key liver enzyme that is important for homocysteine homeostasis. X-ray structures of human BHMT in its oxidized (Zn-free) and reduced (Zn-replete) forms, the latter in complex with the bisubstrate analog, S(delta-carboxybutyl)-L-homocysteine, were determined at resolutions of 2.15 A and 2.05 A. BHMT is a (beta/alpha)(8) barrel that is distorted to construct the substrate and metal binding sites. The zinc binding sequences G-V/L-N-C and G-G-C-C are at the C termini of strands beta6 and beta8. Oxidation to the Cys217-Cys299 disulfide and expulsion of Zn are accompanied by local rearrangements. The structures identify Hcy binding fingerprints and provide a prototype for the homocysteine S-methyltransferase family.     

In vitro modification of betaine-homocysteine S-methyltransferase by tissue-type transglutaminase

Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena. To elucidate the physiological roles of transglutaminase at the molecular level, we need to identify its physiological protein substrates and clarify the relationship between transglutaminase modification of protein substrates and biological responses. Here we examined whether betaine-homocysteine S-methyltransferase (BHMT: EC 2.1.1.5) can be a substrate of tissue-type transglutaminase by in vitro experiments using porcine liver BHMT and guinea pig liver transglutarninase. Guinea pig liver transglutaminase incorporated 5-(biotinamido) pentylamine and [3H] histamine into BHMT in a time-dependent manner. Putrescine and spermidine also seemed to be incorporated into BHMT by transglutaminase. In the absence of the primary amines, BHMT subunits were cross-linked intra- and intermolecularly. BHMT activity was decreased significantly through the cross-linking by transglutaminase. Histamine incorporation slightly reduced the BHMT activity. Peptide fragments of BHMT containing the glutamine residues reactive for transglutaminase reaction were isolated through biotin labelling, proteinase digestion, biotin-avidin a affinity separation, and reverse phase HPLC. The results of amino acid sequence analyses of these peptides and sequence homology alignment with other mammalian liver BHMT subunits showed that these reactive glutamine residues were located in the region near the carboxyl terminal of porcine BHMT subunit. These results suggested that the liver BHMT can be modified by tissue-type transglutaminase and its activity is regulated repressively by the modification, especially by the cross-linking. This regulatory reaction might be involved in the regulation of homocysteine metabolism in the liver.     

Betaine homocysteine methyltransferase: gene cloning and expression analysis in rat liver cirrhosis

It has been known for over half a century that homocysteine levels are elevated in liver cirrhosis, but the basis for it is not fully understood. Using differential display, we identified betaine homocysteine methyltransferase (BHMT) as a gene down-regulated in rat liver cirrhosis and most likely involved in this dysregulation. A partial BHMT clone was isolated by screening of a cDNA library with the differential display fragment. The full-length gene was generated by primer extension of cDNA. Expression levels of BHMT in cirrhotic livers of bile duct ligated rats were compared to controls by Northern and Western blotting as well as by enzyme activity measurements. BHMT mRNA levels were reduced to 29+/-23% in established liver cirrhosis induced by bile duct ligation (BDL) as compared to controls. Enzyme assays in crude liver homogenates showed a similar reduction in BHMT activity in bile duct ligated rat livers. By Western blotting, BHMT could be detected in crude liver homogenates of control animals, but was reduced to below the limit of detection in cirrhotic livers. In conclusion, these findings establish a reduced BHMT enzyme activity in cirrhotic rat livers, which may explain the elevated plasma homocysteine levels in cirrhosis.     

Crystal structure of decameric fructose-6-phosphate aldolase from Escherichia coli reveals inter-subunit helix swapping as a structural basis for assembly differences in the transaldolase family

Fructose-6-phosphate aldolase from Escherichia coli is a member of a small enzyme subfamily (MipB/TalC family) that belongs to the class I aldolases. The three-dimensional structure of this enzyme has been determined at 1.93 A resolution by single isomorphous replacement and tenfold non-crystallographic symmetry averaging and refined to an R-factor of 19.9% (R(free) 21.3%). The subunit folds into an alpha/beta barrel, with the catalytic lysine residue on barrel strand beta 4. It is very similar in overall structure to that of bacterial and mammalian transaldolases, although more compact due to extensive deletions of additional secondary structural elements. The enzyme forms a decamer of identical subunits with point group symmetry 52. Five subunits are arranged as a pentamer, and two ring-like pentamers pack like a doughnut to form the decamer. A major interaction within the pentamer is through the C-terminal helix from one monomer, which runs across the active site of the neighbouring subunit. In classical transaldolases, this helix folds back and covers the active site of the same subunit and is involved in dimer formation. The inter-subunit helix swapping appears to be a major determinant for the formation of pentamers rather than dimers while at the same time preserving importing interactions of this helix with the active site of the enzyme. The active site lysine residue is covalently modified, by forming a carbinolamine with glyceraldehyde from the crystallisation mixture. The catalytic machinery is very similar to that of transaldolase, which together with the overall structural similarity suggests that enzymes of the MipB/TALC subfamily are evolutionary related to the transaldolase family.     

Dissecting the catalytic mechanism of betaine-homocysteine S-methyltransferase by use of intrinsic tryptophan fluorescence and site-directed mutagenesis

Betaine-homocysteine S-methyltransferase (BHMT) is a zinc-dependent enzyme that catalyzes the transfer of a methyl group from glycine betaine (Bet) to homocysteine (Hcy) to form dimethylglycine (DMG) and methionine (Met). Previous studies in other laboratories have indicated that catalysis proceeds through the formation of a ternary complex, with a transition state mimicked by the inhibitor S-(delta-carboxybutyl)-l-homocysteine (CBHcy). Using changes in intrinsic tryptophan fluorescence to determine the affinity of human BHMT for substrates, products, or CBHcy, we now demonstrate that the enzyme-substrate complex reaches its transition state through an ordered bi-bi mechanism in which Hcy is the first substrate to bind and Met is the last product released. Hcy, Met, and CBHcy bind to the enzyme to form binary complexes with K(d) values of 7.9, 6.9, and 0.28 microM, respectively. Binary complexes with Bet and DMG cannot be detected with fluorescence as a probe, but Bet and DMG bind tightly to BHMT-Hcy to form ternary complexes with K(d) values of 1.1 and 0.73 microM, respectively. Mutation of each of the seven tryptophan residues in human BHMT provides evidence that the enzyme undergoes two distinct conformational changes that are reflected in the fluorescence of the enzyme. The first is induced when Hcy binds, and the second, when Bet binds. As predicted by the crystal structure of BHMT, the amino acids Trp44 and Tyr160 are involved in binding Bet, and Glu159 in binding Hcy. Replacing these residues by site-directed mutagenesis significantly reduces the catalytic efficiency (V(max)/K(m)) of the enzyme. Replacing Tyr77 with Phe abolishes enzyme activity.     

Corticoadrenal activity regulates in rat betaine-homocysteine <Emphasis Type="Italic">S</Emphasis>-methyltransferase expression with opposites effects in liver and kidney

Betaine-homocysteine S-methyltransferase (BHMT) is an enzyme that converts homocysteine (Hcy) to methionine using betaine as a methyl donor. Betaine also acts as osmolyte in kidney medulla, protecting cells from high extracellular osmolarity. Hepatic BHMT expression is regulated by salt intake. Hormones, particularly corticosteroids, also regulate BHMT expression in rat liver. We investigated to know whether the corticoadrenal activity plays a role in kidney BHMT expression. BHMT activity in rat kidneys is several orders of magnitude lower than in rat livers and only restricted to the renal cortex. This study confirms that corticosteroids stimulate BHMT activity in the liver and, for the first time in an animal model, also up-regulate the BHMT gene expression. Besides, unlike the liver, corticosteroids in rat kidney down-regulate BHMT expression and activity. Given that the classical effect of adrenocortical activity on the kidney is associated with sodium and water re-absorption by the distal tubule leading to volume expansion, by promoting lesser use of betaine as a methyl donor, corticosteroids would preserve betaine for its other role as osmoprotectant against changes in the extracellular osmotic conditions. We conclude that corticosteroids are, at least in part, responsible for the inhibition of BHMT expression and activity in rat kidneys.     

Refined crystal structure of dogfish M4 apo-lactate dehydrogenase

The crystal structure of M4 apo-lactate dehydrogenase from the spiny dogfish (Squalus acanthius) was initially refined by a constrained-restrained, and subsequently restrained, least-squares technique. The final structure contained 286 water molecules and two sulfate ions per subunit and gave an R-factor of 0.202 for difraction data between 8.0 and 2.0 A resolution. The upper limit for the co-ordinate accuracy of the atoms was estimated to be 0.25 A. The elements of secondary structure of the refined protein have not changed from those described previously, except for the appearance of a one-and-a-half turn 3(10) helix immediately after beta J. There is also a short segment of 3(10) helix between beta C and beta D in the part of the chain that connects the two beta alpha beta alpha beta units of the six-stranded parallel sheet (residues Tyr83 to Ala87). Examination of the interactions among the different elements of secondary structure by means of a surface accessibility algorithm supports the four structural clusters in the subunit. The first of the two sulfate ions is in the active site and occupies a cavity near the essential His195. Its nearest protein ligands are Arg171, Asp168 and Asn140. The second sulfate ion is located near the P-axis subunit interface. It is liganded by His188 and Arg173. These two residues are conserved in bacterial lactate dehydrogenase and form part of the fructose 1,6-bisphosphate effector binding site. Two other data sets in which one (collected at pH 7.8) or both (collected at pH 6.0) sulfate ions were replaced by citrate ions were also analyzed. Five cycles of refinement with respect to the pH 6.0 data (25 to 2.8 A resolution) resulted in an R value of 0.191. Only water molecules occupy the subunit boundary anion binding site at pH 7.8. The amino acid sequence was found to be in poor agreement with (2Fobs-Fcalc) electron density maps for the peptide between residues 207 and 211. The original sequence WNALKE was replaced by NVASIK. The essential His195 is hydrogen bonded to Asp168 on one side and Asn140 on the other. The latter residue is part of a turn that contains the only cis peptide bond of the structure at Pro141. The "flexible loop" (residues 97 to 123), which folds down over the active center in ternary complexes of the enzyme with substrate and coenzyme, has a well-defined structure. Analysis of the environment of Tyr237 suggests how its chemical modification inhibits the enzyme.     

Crystal and molecular structures of native and CTP-liganded aspartate carbamoyltransferase from Escherichia coli

Atomic models representing the electron density of two crystalline forms of aspartate carbamoyltransferase from Escherichia coli are reported here. The unliganded form (R32 crystal symmetry) and the CTP-liganded form (P321 crystal symmetry) have been refined independently at resolutions of 3.0 å and 2.8 Å, respectively, each to a crystallographic R-factor of 27%. The molecular models include at least 95% of the theoretical number of atoms for the aspartate Carbamoyltransferase molecule based on chemical sequence information. We provide details of the refinement process for the two structures, and an evaluation of the accuracy of the molecular models.For the most part, the regulatory and catalytic chains of the unliganded enzyme and the CTP-liganded form are in similar conformations. Large conformational differences in the CTP and native forms exist, however, specifically in the region of CTP binding to the regulatory chain. In addition, a segment of ten amino acid residues, which includes Lys83 and Lys84 of the catalytic chain, is disordered in the CTP-liganded form, in contrast to the native structure, where the same residues have refined well into density.Each catalytic monomer of aspartate carbamoyltransferase is in contact with three catalytic chains and two regulatory monomers. Each regulatory monomer borders on one other regulatory chain and two catalytic chains. The catalytic trimera are in contact in the hexamer; residues important to homotropic effects and catalysis (Tyr165 and Tyr232) are integral parts of the interface. We present a thorough survey of interface regions, cataloging polar interactions between sidechains throughout the molecule.We discuss, in context with the present structures, the chemical modifications and mutations of the enzyme. Highlighted specifically are Cys47, Tyr165 and Tyr232, Lys83, Lys84, Trp209 and Trp279 and Gly128, residues of demonstrated importance to the catalytic of regulatory function or aspartate carbamoyltransferase. The spatial arrangement of “active site” residues argues for a catalytic pocket shared between two monomers within catalytic subunit.     

Specific potassium ion interactions facilitate homocysteine binding to betaine‐homocysteine S‐methyltransferase

Betaine‐homocysteine S‐methyltransferase (BHMT) is a zinc‐dependent methyltransferase that uses betaine as the methyl donor for the remethylation of homocysteine to form methionine. This reaction supports S‐adenosylmethionine biosynthesis, which is required for hundreds of methylation reactions in humans. Herein we report that BHMT is activated by potassium ions with an apparent K_M for K⁺ of about 100 µM. The presence of potassium ions lowers the apparent K_M of the enzyme for homocysteine, but it does not affect the apparent K_M for betaine or the apparent k_cat for either substrate. We employed molecular dynamics (MD) simulations to theoretically predict and protein crystallography to experimentally localize the binding site(s) for potassium ion(s). Simulations predicted that K⁺ ion would interact with residues Asp26 and/or Glu159. Our crystal structure of BHMT bound to homocysteine confirms these sites of interaction and reveals further contacts between K⁺ ion and BHMT residues Gly27, Gln72, Gln247, and Gly298. The potassium binding residues in BHMT partially overlap with the previously identified DGG (Asp26‐Gly27‐Gly28) fingerprint in the Pfam 02574 group of methyltransferases. Subsequent biochemical characterization of several site‐specific BHMT mutants confirmed the results obtained by the MD simulations and crystallographic data. Together, the data herein indicate that the role of potassium ions in BHMT is structural and that potassium ion facilitates the specific binding of homocysteine to the active site of the enzyme. Proteins 2014; 82:2552–2564. © 2014 Wiley Periodicals, Inc.     

Effects of hyperhomocysteinemia and betaine–homocysteine S-methyltransferase inhibition on hepatocyte metabolites and the proteome

Both cardiovascular disease and liver injury are major public health issues. Hyperhomocysteinemia has been linked to cardiovascular diseases, and defects in methyl group metabolism, often resulting in hyperhomocysteinemia, are among the key molecular events postulated to play a role in liver injury. We employed proteomics and metabolomics analyses of human hepatocytes in primary cell culture to explore the spectrum of proteins and associated metabolites affected by the disruption of methyl group metabolism. We treated the hepatocytes with homocysteine (Hcy, 0.1 mM and 2 mM) to follow the impact of hyperhomocysteinemia, and in parallel, we used a specific inhibitor of betaine–homocysteine S-methyltransferase (BHMT) to extend our understanding of the physiological functions of the enzyme. The major effect of BHMT inhibition was a 50% decrease in S-adenosylmethionine levels. The treatments with Hcy resulted in multiple changes in the metabolite levels depending on the treatment modality. The BHMT inhibition and 0.1 mM Hcy treatment induced only moderate changes in the hepatocyte proteome and secretome, while the changes induced by the 2 mM Hcy treatment were extensive. Phosphatidylethanolamine carboxykinase and ornithine aminotransferase were up-regulated about two fold indicating an intervention into metabolism. Cellular proliferation was suspended, secretome composition was changed and signs of apoptosis were discernible. We have detected fibrinogen gamma dimers, which might have a role as a potentially new biomarker of early liver injury. Finally, we have demonstrated the failed maturation of apolipoprotein A1, which might be a new mechanism of disruption of cholesterol efflux from tissues.     

Crystal structures of creatininase reveal the substrate binding site and provide an insight into the catalytic mechanism

Creatininase from Pseudomonas putida is a member of the urease-related amidohydrolase superfamily. The crystal structure of the Mn-activated enzyme has been solved by the single isomorphous replacement method at 1.8A resolution. The structures of the native creatininase and the Mn-activated creatininase-creatine complex have been determined by a difference Fourier method at 1.85 A and 1.6 A resolution, respectively. We found the disc-shaped hexamer to be roughly 100 A in diameter and 50 A in thickness and arranged as a trimer of dimers with 32 (D3) point group symmetry. The enzyme is a typical Zn2+ enzyme with a binuclear metal center (metal1 and metal2). Atomic absorption spectrometry and X-ray crystallography revealed that Zn2+ at metal1 (Zn1) was easily replaced with Mn2+ (Mn1). In the case of the Mn-activated enzyme, metal1 (Mn1) has a square-pyramidal geometry bound to three protein ligands of Glu34, Asp45, and His120 and two water molecules. Metal2 (Zn2) has a well-ordered tetrahedral geometry bound to the three protein ligands of His36, Asp45, and Glu183 and a water molecule. The crystal structure of the Mn-activated creatininase-creatine complex, which is the first structure as the enzyme-substrate/inhibitor complex of creatininase, reveals that significant conformation changes occur at the flap (between the alpha5 helix and the alpha6 helix) of the active site and the creatine is accommodated in a hydrophobic pocket consisting of Trp174, Trp154, Tyr121, Phe182, Tyr153, and Gly119. The high-resolution crystal structure of the creatininase-creatine complex enables us to identify two water molecules (Wat1 and Wat2) that are possibly essential for the catalytic mechanism of the enzyme. The structure and proposed catalytic mechanism of the creatininase are different from those of urease-related amidohydrolase superfamily enzymes. We propose a new two-step catalytic mechanism possibly common to creatininases in which the Wat1 acts as the attacking nucleophile in the water-adding step and the Wat2 acts as the catalytic acid in the ring-opening step.     

Betaine:homocysteine methyltransferase from rat liver: purification and inhibition by a boronic acid substrate analog.

Betaine:homocysteine methyltransferase (BHMT) from rat liver has been highly purified by an efficient procedure requiring only two chromatographic steps: Sephadex G-100 chromatography and fast protein liquid chromatography chromatofocusing. A 170-fold purification and 7.5% overall yield were achieved. Chromatofocusing yielded three active forms of BHMT with pI values near 8.0, 7.6, and 7.0. The subunit molecular weight of each active form is 45,000 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the native enzyme has a molecular weight of 270,000 as determined by exclusion chromatography. The stability of the purified enzyme was found to be potentiated by the presence of 1 mM dimethylglycine and 1 mM homocysteine. Boronate analogs of betaine (pinanyl N,N,N-trimethylaminomethaneboronate) (4) and dimethylglycine (pinanyl N,N-dimethylaminomethaneboronate) were synthesized from pinanyl iodomethaneboronate (3) and trimethylamine or dimethylamine, respectively. The free acid of the betaine analog (5) was reversibly generated from (4). The inhibition of BHMT by (5) appears competitive with a Ki = 45 microM. Since the Km for betaine measured with the purified enzyme is near 0.1 mM, the boronic acid analog of betaine appears to function effectively as a substrate analog inhibitor of BHMT. The analog does not appear to act as a methyl donor to homocysteine when (5) is substituted for betaine in the enzyme reaction. In addition, an enzyme assay based upon C3-cyano reverse phase HPLC detection of the o-phthalaldehyde derivative of methionine was developed as an alternative to the standard radiochemical assay. Betaine:homocysteine methyltransferase in the picomole range can be quantitated using this assay as indicated by a linear response of enzyme activity to protein concentration.     

Activation and induction of glycine N-methyltransferase by retinoids are tissue- and gender-specific

Betaine-homocysteine S-methyltransferase (BHMT; EC2.1.1.5) is a zinc metalloenzyme that catalyzes the transfer of a methyl group from betaine to homocysteine to produce dimethylglycine and Met, respectively. This enzyme is a member of a family of zinc-dependent methyltransferases that use thiols or selenols as methyl acceptors and which contain the following motif: G[ILV]NCX(20, 100)[ALV]X(2)[ILV]GGCCX(3)PX(2)I. We recently reported that the three cysteine residues within this motif function as ligands to zinc in BHMT because changing any of them to alanine abolished zinc-binding and enzyme activity (A. P. Breksa, III, and T. A. Garrow, 1999, Biochemistry 38, 13991-13998). To determine if other amino acid residues in this motif were critical for enzyme function, the two regions defined by the motif in human BHMT, GVNCH(218) and VRYIGGCCGFEPYHI(307), were subjected to semirandom and random site-directed mutagenesis. Mutant enzymes were classified as either active or inactive based on their ability to complement the Met auxotrophy of Escherichia coli strain J5-3. The Gly residue at position 214 was found to be absolutely essential for complementation. The positions occupied by Gly297, Gly298, and Gly301 favored substitutions of small amino acids like Ala and Ser. We hypothesize that these Gly residues provide the necessary flexibility to the Zn-binding region to permit coordination of the metal.     

Molecular structure of flavocytochrome b2 at 2.4 A resolution

The crystal structure of flavocytochrome b2 has been solved at 3.0 A resolution by the method of multiple isomorphous replacement with anomalous scattering. Area detector data from native and two heavy-atom derivative crystals were used. The phases were refined by the B.C. Wang phase-filtering procedure utilizing the 67% (v/v) solvent content of the crystals. A molecular model was built first on a minimap and then on computer graphics from a combination of maps both averaged and not averaged about the molecular symmetry axis. The structure was extended to 2.4 A resolution using film data recorded at a synchrotron and refined by the Hendrickson-Konnert procedure. The molecule, a tetramer of Mr 230,000, is located on a crystallographic 2-fold axis and possesses local 4-fold symmetry. Each subunit is composed of two domains, one binding a heme and the other an FMN prosthetic group. In subunit 1, both the cystochrome and the flavin-binding domain are visible in the electron density map. In subunit 2 the cytochrome domain is disordered. However, in the latter, a molecule of pyruvate, the product of the enzymatic reaction, is bound at the active site. The cytochrome domain consists of residues 1 to 99 and is folded in a fashion similar to the homologous soluble fragment of cytochrome b5. The flavin binding domain contains a parallel beta 8 alpha 8 barrel structure and is composed of residues 100 to 486. The remaining 25 residues form a tail that wraps around the molecular 4-fold axis and is in contact with each remaining subunit. The FMN moiety, which is located at the C-terminal end of the central beta-barrel, is mostly sequestered from solvent; it forms hydrogen bond interactions with main- and side-chain atoms from six of the eight beta-strands. The interaction of Lys349 with atoms N-1 and O-2 of the flavin ring is probably responsible for stabilization of the anionic form of the flavin semiquinone and hydroquinone and enhancing the reactivity of atom N-5 toward sulfite. The binding of pyruvate at the active site in subunit 2 is stabilized by interaction of its carboxylate group with the side-chain atoms of Arg376 and Tyr143. Residues His373 and Tyr254 interact with the keto-oxygen atom and are involved in catalysis. In contrast, four water molecules occupy the substrate-binding site in subunit 1 and Tyr143 forms a hydrogen bond to the ordered heme propionate group. Otherwise the two flavin-binding domains are identical within experimental error.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tyrosine cross-linking reveals interfacial dynamics in adeno-associated viral capsids during infection

Viral capsid dynamics are often observed during infectious events such as cell surface attachment, entry and genome release. Structural analysis of adeno-associated virus (AAV), a helper-dependent parvovirus, revealed a cluster of surface-exposed tyrosine residues at the icosahedral two-fold symmetry axis. We exploited the latter observation to carry out selective oxidation of Tyr residues, which yielded cross-linked viral protein (VP) subunit dimers, effectively "stitching" together the AAV capsid two-fold interface. Characterization of different Tyr-to-Phe mutants confirmed that the formation of cross-linked VP dimers is mediated by dityrosine adducts and requires the Tyr704 residue, which crosses over from one neighboring VP subunit to the other. When compared to unmodified capsids, Tyr-cross-linked AAV displayed decreased transduction efficiency in cell culture. Surprisingly, further biochemical and quantitative microscopy studies revealed that restraining the two-fold interface hinders externalization of buried VP N-termini, which contain a phospholipase A2 domain and nuclear localization sequences critical for infection. These adverse effects caused by tyrosine oxidation support the notion that interfacial dynamics at the AAV capsid two-fold symmetry axis play a role in externalization of VP N-termini during infection.     

Structural analysis of the 2.8 A model of Xylose isomerase from Actinoplanes missouriensis

The structure of Xylose isomerase (X.I.) from Actinoplanes missouriensis has been solved to 2.8 Angstroms resolution. Phases were determined from a single Eu3+ derivative and from the noncrystallographic 222 symmetry of the tetrameric molecule. An atomic model was built and subjected to restrained crystallographic refinement. The resulting model is shown to be closely similar to the recently reported X.I.'s structures from three other bacterial sources. Each monomer is found to be composed of an eight-stranded alpha/beta "T.I.M." barrel forming an N-terminal domain of 328 residues followed by a large loop of 66 residues embracing an adjacent subunit. Analysis of intersubunit packing shows that the X.I. tetramer is an assembly of two tight dimers. The beta barrel fits a simple hyperboloid model as other T.I.M. barrels do. The active site, identified as the binding site for the inhibitor xylitol, is located at the carboxyl end of the beta strands in the barrel next to a pair of binding sites for Eu3+ ions, which are assumed to be sites for the divalent ions involved in catalysis. Active sites in the tetramer are oriented towards the interface between dimers. It is suggested that subunit interfaces might stabilize the active site region and this might explain the oligomeric nature of other alpha/beta barrel enzymes.     

Crystal structure of Rab geranylgeranyltransferase at 2.0 A resolution

BACKGROUND: Rab geranylgeranyltransferase (RabGGT) catalyzes the addition of two geranylgeranyl groups to the C-terminal cysteine residues of Rab proteins, which is crucial for membrane association and function of these proteins in intracellular vesicular trafficking. Unlike protein farnesyltransferase (FT) and type I geranylgeranyltransferase, which both prenylate monomeric small G proteins or short peptides, RabGGT can prenylate Rab only when Rab is in a complex with Rab escort protein (REP). RESULTS: The crystal structure of rat RabGGT at 2.0 A resolution reveals an assembly of four distinct structural modules. The beta subunit forms an alpha-alpha barrel that contains most of the residues in the active site. The alpha subunit consists of a helical domain, an immunoglobulin (Ig)-like domain, and a leucine-rich repeat (LRR) domain. The N-terminal region of the alpha subunit binds to the active site in the beta subunit; residue His2alpha directly coordinates a zinc ion. The prenyl-binding pocket of RabGGT is deeper than that in FT. CONCLUSIONS: LRR and Ig domains are often involved in protein-protein interactions; in RabGGT they might participate in the recognition and binding of REP. The binding of the N-terminal peptide of the alpha subunit to the active site suggests an autoinhibition mechanism that might contribute to the inability of RabGGT to recognize short peptides or Rab alone as its substrate. Replacement of residues Trp102beta and Tyr154beta in FT by Ser48beta and Leu99beta, respectively, in RabGGT largely determine the different lipid-binding specificities of the two enzymes.