Structure and mechanistic implications of a uroporphyrinogen III synthase-product complex

Uroporphyrinogen III synthase (U3S) catalyzes the asymmetrical cyclization of a linear tetrapyrrole to form the physiologically relevant uroporphyrinogen III (uro'gen III) isomer during heme biosynthesis. Here, we report four apoenzyme and one product complex crystal structures of the Thermus thermophilus (HB27) U3S protein. The overlay of eight crystallographically unique U3S molecules reveals a huge range of conformational flexibility, including a "closed" product complex. The product, uro'gen III, binds between the two domains and is held in place by a network of hydrogen bonds between the product's side chain carboxylates and the protein's main chain amides. Interactions of the product A and B ring carboxylate side chains with both structural domains of U3S appear to dictate the relative orientation of the domains in the closed enzyme conformation and likely remain intact during catalysis. The product C and D rings are less constrained in the structure, consistent with the conformational changes required for the catalytic cyclization with inversion of D ring orientation. A conserved tyrosine residue is potentially positioned to facilitate loss of a hydroxyl from the substrate to initiate the catalytic reaction.     

Crystal structure of uroporphyrinogen III synthase from Pseudomonas syringae pv. tomato DC3000

Uroporphyrinogen III synthase (U3S) is one of the key enzymes in the biosynthesis of tetrapyrrole compounds. It catalyzes the cyclization of the linear hydroxymethylbilane (HMB) to uroporphyrinogen III (uro’gen III). We have determined the crystal structure of U3S from Pseudomonas syringae pv. tomato DC3000 (psU3S) at 2.5 Å resolution by the single wavelength anomalous dispersion (SAD) method. Each psU3S molecule consists of two domains interlinked by a two-stranded antiparallel β-sheet. The conformation of psU3S is different from its homologous proteins because of the flexibility of the linker between the two domains, which might be related to this enzyme’s catalytic properties. Based on mutation and activity analysis, a key residue, Arg219, was found to be important for the catalytic activity of psU3S. Mutation of Arg219 to Ala caused a decrease in enzymatic activity to about 25% that of the wild type enzyme. Our results provide the structural basis and biochemical evidence to further elucidate the catalytic mechanism of U3S.     

A HECT domain E3 enzyme assembles novel polyubiquitin chains

Although polyubiquitin chains linked through Lys(29) of ubiquitin have been implicated in the targeting of certain substrates to proteasomes, the signaling properties of these chains are poorly understood. We previously described a ubiquitin-protein isopeptide ligase (E3) from erythroid cells that assembles polyubiquitin chains through either Lys(29) or Lys(48) of ubiquitin (Mastrandrea, L. D., You, J., Niles, E. G., and Pickart, C. M. (1999) J. Biol. Chem. 274, 27299-27306). Here we describe the purification of this E3 based on its affinity for a linear fusion of ubiquitin to the ubiquitin-conjugating enzyme UbcH5A. Among five major polypeptides in the affinity column eluate, the activity of interest was assigned to the product of a previously cloned human cDNA known as KIAA10 (Nomura, N., Miyajima, N., Sazuka, T., Tanaka, A., Kawarabayasi, Y., Sato, S., Nagase, T., Seki, N., Ishikawa, K., and Tabata, S. (1994) DNA Res. 1, 27-35). The KIAA10 protein is a member of the HECT (homologous to E6-AP carboxyl terminus) domain family of E3s. These E3s share a conserved C-terminal (HECT) domain that functions in the catalysis of ubiquitination, while their divergent N-terminal domains function in cognate substrate binding (Huibregtse, J. M., Scheffner, M., Beaudenon, S., and Howley, P. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2563-2567). Recombinant KIAA10 catalyzed the assembly of both Lys(29)- and Lys(48)-linked polyubiquitin chains. Surprisingly, the C-terminal 428 residues of KIAA10 were both necessary and sufficient for this activity, suggesting that the ability to assemble polyubiquitin chains may be a general property of HECT domains. The N-terminal domain of KIAA10 interacted in vitro with purified 26 S proteasomes and with the isolated S2/Rpn1 subunit of the proteasome's 19 S regulatory complex, suggesting that the N-terminal domains of HECT E3s may function in proteasome binding as well as substrate binding.     

Enzyme catalysis: removing chemically 'essential' residues by site-directed mutagenesis

Enzymatic catalysis relies on the action of the amino acid side chains arrayed in the enzyme active sites. Usually, only two or three 'essential' residues are directly involved in the bond making and breaking steps leading to product formation. For the past 20 years, enzymologists have been addressing the role of such residues by changing them into chemically inert side chains. Removal of an 'essential' group often does not abolish activity, but can significantly alter the catalytic mechanism. Such results underscore the sophistication of enzyme catalysis and the functional plasticity of enzyme active sites.     

The structure of aconitase

The crystal structure of the 80,000 Da Fe-S enzyme aconitase has been solved and refined at 2.1 A resolution. The protein contains four domains; the first three from the N-terminus are closely associated around the [3Fe-4S] cluster with all three cysteine ligands to the cluster being provided by the third domain. Association of the larger C-terminal domain with the first three domains creates an extensive cleft leading to the Fe-S cluster. Residues from all four domains contribute to the active site region, which is defined by the Fe-S cluster and a bound SO4(2-) ion. This region of the structure contains 4 Arg, 3 His, 3 Ser, 2 Asp, 1 Glu, 3 Asn, and 1 Gln residues, as well as several bound water molecules. Three of these side chains reside on a three-turn 3(10) helix in the first domain. The SO4(2-) ion is bound 9.3 A from the center of the [3Fe-4S] cluster by the side chains of 2 Arg and 1 Gln residues. Each of 3 His side chains in the putative active site is paired with Asp or Glu side chains.     

Site-directed mutagenesis of the cysteinyl residues and the active-site serine residue of bacterial D-amino acid transaminase

Each of the three cysteinyl residues per subunit in D-amino acid transaminase from a thermophilic species of Bacillus has been changed to a glycine residue (C142G, C164G, and C212G) by site-directed mutagenesis. The mutant enzymes were detected by Western blots and a stain for activity. After purification to homogeneity, each mutant protein had the same activity as the wild-type enzyme. Thus, none of the Cys residues are essential for catalysis. Each protein when denatured showed the expected titer of two SH groups per subunit. In the native state, each of the three mutant proteins exhibited nearly the same slow rate of titration of SH groups as the wild-type protein with about one SH group titratable over a period of 4 h. Conversion of Ser-146, adjacent to Lys-145 to which the coenzyme pyridoxal phosphate is bound, to an alanine residue (S146A) does not alter the catalytic activity but has a significant effect on the SH titration behavior. Thus, three to four of the six SH groups of S146A are titratable by DTNB. The rapid SH titration of S146A is prevented by the presence of D-alanine. This finding suggests that the change of Ser-146 to Ala at the active site promotes the exposure and rapid titration of a Cys residue in that region. The rapid SH titration of S146A by DTNB is accompanied by a loss of enzyme activity. Two of the mutant enzymes, C142G and S146A, lose activity at 4 degrees C and also upon freezing and thawing. The mutant enzymes C164G and C212G show the same degree of thermostability as the wild-type enzyme.     

Role of hydrogen bonding in the active site of human manganese superoxide dismutase

The side chain of Gln143, a conserved residue in manganese superoxide dismutase (MnSOD), forms a hydrogen bond with the manganese-bound solvent and is critical in maintaining catalytic activity. The side chains of Tyr34 and Trp123 form hydrogen bonds with the carboxamide of Gln143. We have replaced Tyr34 and Trp123 with Phe in single and double mutants of human MnSOD and measured their catalytic activity by stopped-flow spectrophotometry and pulse radiolysis. The replacements of these side chains inhibited steps in the catalysis as much as 50-fold; in addition, they altered the gating between catalysis and formation of a peroxide complex to yield a more product-inhibited enzyme. The replacement of both Tyr34 and Trp123 in a double mutant showed that these two residues interact cooperatively in maintaining catalytic activity. The crystal structure of Y34F/W123F human MnSOD at 1.95 A resolution suggests that this effect is not related to a conformational change in the side chain of Gln143, which does not change orientation in Y34F/W123F, but rather to more subtle electronic effects due to the loss of hydrogen bonding to the carboxamide side chain of Gln143. Wild-type MnSOD containing Trp123 and Tyr34 has approximately the same thermal stability compared with mutants containing Phe at these positions, suggesting the hydrogen bonds formed by these residues have functional rather than structural roles.     

Modulation of selenium-dependent glutathione peroxidase (Se-GSH-Px) activity in mice

Selenium-dependent glutathione peroxidase activity was assessed in the liver, kidney, lung and blood of mice from seven strains (129/ReJ, BALB/c, C3H/HeSnJ, C3H/S, C57BL/6J, Csb, and S.W.) at five ages (newborn, 21, 70, 175 and +500 days old). Activity was highest in the liver (0.25 U/mg protein) followed by blood hemolysate (0.16 U/mg protein) with kidney and lung displaying similar, comparatively lower levels of activity (0.14 and 0.12 U/mg protein respectively). Although activity was shown by statistical analysis to be not significantly different among the strains (p = 0.05), age-associated, strain-specific changes in enzyme activity were noted to be highly significant (p = 0.001). Also, ethanol administered in drinking water resulted in a marked reduction in selenium-dependent glutathione peroxidase activity during both short- (1-2 weeks) and long- (5-6 weeks) term treatment periods. Changes in this enzyme due to aging and after exposure to xenobiotics such as ethanol may have serious ramifications given the importance of this enzyme in the detoxification of reactive oxygen metabolites.     

A computational method for design of connected catalytic networks in proteins

Computational design of new active sites has generally proceeded by geometrically defining interactions between the reaction transition state(s) and surrounding side‐chain functional groups which maximize transition‐state stabilization, and then searching for sites in protein scaffolds where the specified side‐chain–transition‐state interactions can be realized. A limitation of this approach is that the interactions between the side chains themselves are not constrained. An extensive connected hydrogen bond network involving the catalytic residues was observed in a designed retroaldolase following directed evolution. Such connected networks could increase catalytic activity by preorganizing active site residues in catalytically competent orientations, and enabling concerted interactions between side chains during catalysis, for example, proton shuffling. We developed a method for designing active sites in which the catalytic side chains, in addition to making interactions with the transition state, are also involved in extensive hydrogen bond networks. Because of the added constraint of hydrogen‐bond connectivity between the catalytic side chains, to find solutions, a wider range of interactions between these side chains and the transition state must be considered. Our new method starts from a ChemDraw‐like two‐dimensional representation of the transition state with hydrogen‐bond donors, acceptors, and covalent interaction sites indicated, and all placements of side‐chain functional groups that make the indicated interactions with the transition state, and are fully connected in a single hydrogen‐bond network are systematically enumerated. The RosettaMatch method can then be used to identify realizations of these fully‐connected active sites in protein scaffolds. The method generates many fully‐connected active site solutions for a set of model reactions that are promising starting points for the design of fully‐preorganized enzyme catalysts.     

Conformational exchange of aromatic side chains characterized by L-optimized TROSY-selected 13C CPMG relaxation dispersion

Protein dynamics on the millisecond time scale commonly reflect conformational transitions between distinct functional states. NMR relaxation dispersion experiments have provided important insights into biologically relevant dynamics with site-specific resolution, primarily targeting the protein backbone and methyl-bearing side chains. Aromatic side chains represent attractive probes of protein dynamics because they are over-represented in protein binding interfaces, play critical roles in enzyme catalysis, and form an important part of the core. Here we introduce a method to characterize millisecond conformational exchange of aromatic side chains in selectively (13)C labeled proteins by means of longitudinal- and transverse-relaxation optimized CPMG relaxation dispersion. By monitoring (13)C relaxation in a spin-state selective manner, significant sensitivity enhancement can be achieved in terms of both signal intensity and the relative exchange contribution to transverse relaxation. Further signal enhancement results from optimizing the longitudinal relaxation recovery of the covalently attached (1)H spins. We validated the L-TROSY-CPMG experiment by measuring fast folding-unfolding kinetics of the small protein CspB under native conditions. The determined unfolding rate matches perfectly with previous results from stopped-flow kinetics. The CPMG-derived chemical shift differences between the folded and unfolded states are in excellent agreement with those obtained by urea-dependent chemical shift analysis. The present method enables characterization of conformational exchange involving aromatic side chains and should serve as a valuable complement to methods developed for other types of protein side chains.     

Functional role of residue 193 (chymotrypsin numbering) in serine proteases: influence of side chain length and beta-branching on the catalytic activity of blood coagulation factor XIa

In serine proteases, Gly193 (chymotrypsin numbering) is conserved with rare exception. Mutants of blood coagulation proteases have been reported with Glu, Ala, Arg or Val substitutions for Gly193. To further understand the role of Gly193 in protease activity, we replaced it with Ala or Val in coagulation factor XIa (FXIa). For comparison to the reported FXIa Glu193 mutant, we prepared FXIa with Asp (short side chain) or Lys (opposite charge) substitutions. Binding of p-aminobenzamidine (pAB) and diisopropylfluorphosphate (DFP) were impaired 1.6-36-fold and 35-478-fold, respectively, indicating distortion of, or altered accessibility to, the S1 and oxyanion-binding sites. Val or Asp substitutions caused the most impairment. Salt bridge formation between the amino terminus of the mature protease moiety at Ile16 and Asp194, essential for catalysis, was impaired 1.4-4-fold. Mutations reduced catalytic efficiency of tripeptide substrate hydrolysis 6-280-fold, with Val or Asp causing the most impairment. Further studies were directed toward macromolecular interactions with the FXIa mutants. kcat for factor IX activation was reduced 8-fold for Ala and 400-1100-fold for other mutants, while binding of the inhibitors antithrombin and amyloid beta-precursor protein Kunitz domain (APPI) was impaired 13-2300-fold and 22-27000-fold, respectively. The data indicate that beta-branching of the side chain of residue 193 is deleterious for interactions with pAB, DFP and amidolytic substrates, situations where no S2'-P2' interactions are involved. When an S2'-P2' interaction is involved (factor IX, antithrombin, APPI), beta-branching and increased side chain length are detrimental. Molecular models indicate that the mutants have impaired S2' binding sites and that beta-branching causes steric conflicts with the FXIa 140-loop, which could perturb the local tertiary structure of the protease domain. In conclusion, enzyme activity is impaired in FXIa when Gly193 is replaced by a non-Gly residue, and residues with side chains that branch at the beta-carbon have the greatest effect on catalysis and binding of substrates.     

Infrared studies reveal unique vibrations associated with the PGK-ATP-3-PG ternary complex

Phosphoglycerate kinase (PGK) catalyzes a reversible phospho-transfer reaction between ATP and 3-phosphoglycerate (3-PG) that is thought to require a hinge-bending motion in the protein that brings two separate substrate-binding domains together. We have used difference infrared spectroscopy to better understand the conformational changes that are unique to the PGK-ATP-3-PG complex. Caged nucleotides (caged-ADP and caged-ATP) were used to initiate nucleotide binding to PGK or PGK-3-PG complexes. The difference spectra include those of PGK-ATP minus PGK, PGK-3-PG-ATP minus PGK-3-PG, PGK-3-PG-ADP minus PGK-3-PG, and PGK-ADP minus PGK. The resulting spectra were compared in attempts to identify bands associated with each PGK complex. In addition, complementary activity assays were performed in the presence of caged-nucleotides. While PGK activity decreased in the presence of caged-ADP, the activity was not influenced by the addition of caged-ATP. The activity assay results suggest that the caged-ADP may interact with PGK substrate binding site(s) and inhibit phospho-transfer. Therefore, additional difference infrared nucleotide exchange experiments were used to isolate the differences between ADP and ATP binding to PGK. Difference FTIR spectra obtained on PGK-nucleotide-3-PG complexes show distinct bands that may result from amino acid side chains as well as structural changes in the hinge region and/or increased interactions such as salt bridges forming between the two domains. The infrared data obtained on the active ternary complexes show evidence of changes in alpha-helix and beta-structures as well as signals consistent with Arg, Asn, His, Lys, Asp, Glu, and additional side chains that are uniquely perturbed in the active ternary complex as compared to other PGK complexes.     

Criteria to design green enzymatic processes in ionic liquid/supercritical carbon dioxide systems

Five different ionic liquids (ILs) based on quaternary ammonium cations, with functional side chains ((3-hydroxypropyl)-trimethyl-, (3-cyanopropyl)-trimethyl-, butyl-trimethyl-, (5-cyanopentyl)-trimethyl- and hexyl-trimethyl-) associated with the same anion (bis(trifluoromethane)sulfonyl amide)), were synthesized, and their suitability for Candida antarctica lipase B (CALB)-catalyzed ester synthesis in IL/supercritical carbon dioxide (scCO(2)) biphasic systems was assayed. Catalytic efficiency of the system has been analyzed as a function of both enzyme properties and mass-transfer phenomena criteria. First, the suitability of these ILs as enzymic reaction media was tested for the kinetic resolution of rac-phenylethanol. All ILs were found to be suitable media for enzyme catalysis, the best catalytic parameter (5.3 U/mg specific activity, 94.9% selectivity) being obtained for the (5-cyanopentyl)-trimethylammonium. Second, enzyme stability in all of the ILs was studied at 50 degrees C over a period of 50 days, and data were analyzed by a two-step kinetic deactivation model. All of the ILs were shown to act as stabilizing agents with respect to hexane, producing an increase in the free energy of deactivation (to 25 kJ/mol protein) and an improvement in the half-life time of the enzyme (2000-fold), which agrees with the observed increased hydrophobicity of the cation alkyl side chain (measured by Hansen's solubility parameter, delta). By using two different CALB-IL systems with different hydrophobicity in the cation, continuous processes to synthesize six different short chain alkyl esters (butyl acetate, butyl propionate, butyl butyrate, hexyl propionate, hexyl butyrate, and octyl propionate) in scCO(2) at 10 MPa and 50 degrees C were carried out. Both rate-limiting parameters (synthetic activity and scCO(2)-ILs mass-transfer phenomena) were related with the delta-parameter of the ILs-alkyl chain and reagents.     

Role of an interdomain Gly-Gly sequence at the regulatory-substrate domain interface in the regulation of Escherichia coli. D-3-phosphoglycerate dehydrogenase

The regulatory and substrate binding domains of D-3-phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) from Escherichia coli are connected by a single polypeptide strand that contains a Gly-Gly sequence approximately midway between the domains. The potential flexibility of this sequence and its strategic location between major domain structures suggests that it may function in the conformational change leading from effector binding to inhibition of the active site. Site-directed mutagenesis of this region (Gly-336-Gly-337) supports this hypothesis. When bulky side chains were substituted for the glycines at these positions, substantial changes in the ability of serine to inhibit the enzyme were seen with little effect on the activity of the enzyme. The effect of these substitutions could be alleviated by placing a new glycine residue at position 335, immediately flanking the original glycine pair. On the other hand, substituting a glycine at position 338 revealed a critical role for the side chain of Arg-338. This residue may function in stabilizing the conformation about the Gly-Gly turn, resulting in a specific orientation of the adjacent domains relative to each other. Rotation about the phi or psi bonds of either Gly-336 or Gly-337 would have a profound effect on this orientation. The data are consistent with this as a role for the Gly-Gly sequence between the regulatory and substrate binding domains of PGDH.     

Role of syndecan-4 side chains in turkey satellite cell apoptosis and focal adhesion formation

S4 (syndecan-4) is a cell membrane heparan sulfate proteoglycan that functions in muscle growth and development. It is composed of a central core protein and two types of side chains: GAGs (glycosaminoglycans) and N-glycosylated (N-linked glycosylated) chains. The N-glycosylated chains and GAG chains are required for S4 to regulate turkey myogenic satellite cell proliferation. The objective of the current study was to determine whether the S4 side chains regulate cell proliferation through muscle cell focal adhesion formation and apoptosis. S4 mutants with only one or without any N-glycosylated chains attached to the core protein with or without GAG chains were generated to study the function of N-glycosylated chains and the interaction between N-glycosylated chains and GAG chains. The wild-type S4 and all of the S4 side chain mutants were transfected into turkey myogenic satellite cells. Cell apoptosis and focal adhesion formation were measured, and PKCα (protein kinase Cα) cell membrane localization was investigated. S4 increased FAK (focal adhesion kinase) activity and the deletion of the side chains decreased this effect. S4 and the S4 mutants had no effect on β1-integrin expression, but increased the cell membrane localization of β1-integrin and PKCα. Furthermore, cell apoptosis and vinculin containing focal adhesions were not affected by S4 and its mutants. The results suggest that S4 and its side chains play important roles in regulating FAK activity, and PKCα and β1-integrin cell membrane localization, but not cell apoptosis and vinculin-containing focal adhesion formation.     

Protein-protein interactions between cytochrome b and the Fe-S protein subunits during QH2 oxidation and large-scale domain movement in the bc1 complex

The ubihydroquinone:cytochrome (cyt) c oxidoreductase, or bc(1) complex, and its homologue the b(6)f complex are key components of respiratory and photosynthetic electron transport chains as they contribute to the generation of an electrochemical gradient used by the ATP synthase to produce ATP. The bc(1) complex has two catalytic domains, ubihydroquinone oxidation (Q(o)) and ubiquinone reduction (Q(i)) sites, that are located on each side of the membrane. The key to the energetic efficiency of this enzyme relies upon the occurrence of a unique electron bifurcation reaction at its Q(o) site. Recently, several lines of evidence have converged to establish that in the bc(1) complex the extrinsic domain of the Fe-S subunit that contains a [2Fe2S] metal cluster moves during catalysis to shuttle electrons between the Q(o) site and c(1) heme. While this step is required for electron bifurcation, available data also suggest that the movement might be controlled to ensure maximal energetic efficiency [Darrouzet et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 4567-4572]. To gain insight into the plausible control mechanism, we used a biochemical genetic approach to define the different regions of the bc(1) complex that might interact with each other. Previously, we found that a mutation located at position L286 of the ef loop of Rhodobacter capsulatus cyt b could alleviate movement impairment resulting from a mutation in the hinge region, linking the [2Fe2S] cluster domain to the membrane anchor of the Fe-S subunit. Here we report that various substitutions at position 288 on the opposite side of the ef loop also impair Q(o) site catalysis. In particular, we note that while most of the substitutions affect only QH(2) oxidation, yet others like T288S also hinder the rate of the movement of the Fe-S subunit. Thus, position 288 of cyt b appears to be important for both the QH(2) oxidation and the movement of the Fe-S subunit. Moreover, we found that, upon substitution of T288 by other amino acids, additional compensatory mutations located at the [2Fe2S] cluster or the hinge domains of the Fe-S subunit, or on the cd loop of cyt b, arise readily to alleviate these defects. These studies indicate that intimate protein-protein interactions occur between cyt b and the Fe-S subunits to sustain fast movement and efficient QH(2) oxidation and highlight the critical dual role the ef loop of cyt b to fine-tune the docking and movement of the Fe-S subunit during Q(o) site catalysis.     

Three-dimensional structure of a barley beta-D-glucan exohydrolase, a family 3 glycosyl hydrolase.

BACKGROUND: Cell walls of the starchy endosperm and young vegetative tissues of barley (Hordeum vulgare) contain high levels of (1-->3,1-->4)-beta-D-glucans. The (1-->3,1-->4)-beta-D-glucans are hydrolysed during wall degradation in germinated grain and during wall loosening in elongating coleoptiles. These key processes of plant development are mediated by several polysaccharide endohydrolases and exohydrolases. RESULTS:. The three-dimensional structure of barley beta-D-glucan exohydrolase isoenzyme ExoI has been determined by X-ray crystallography. This is the first reported structure of a family 3 glycosyl hydrolase. The enzyme is a two-domain, globular protein of 605 amino acid residues and is N-glycosylated at three sites. The first 357 residues constitute an (alpha/beta)8 TIM-barrel domain. The second domain consists of residues 374-559 arranged in a six-stranded beta sandwich, which contains a beta sheet of five parallel beta strands and one antiparallel beta strand, with three alpha helices on either side of the sheet. A glucose moiety is observed in a pocket at the interface of the two domains, where Asp285 and Glu491 are believed to be involved in catalysis. CONCLUSIONS: The pocket at the interface of the two domains is probably the active site of the enzyme. Because amino acid residues that line this active-site pocket arise from both domains, activity could be regulated through the spatial disposition of the domains. Furthermore, there are sites on the second domain that may bind carbohydrate, as suggested by previously published kinetic data indicating that, in addition to the catalytic site, the enzyme has a second binding site specific for (1-->3, 1-->4)-beta-D-glucans.     

Zinc-finger hydrolase: Computational selection of a linker and a sequence towards metal activation with a synthetic αββ protein

The zinc-finger protein is targeted for computational redesign as a hydrolase enzyme. Successful in having zinc activated for hydrolase function, the study validates the stepwise approach to having the protein tuned in main-chain structure stereochemically and over side chains chemically. A leucine homopolypeptide, harboring histidines to tri coordinate zinc and d-amino-acid-nucleated α-helix and β-hairpin building blocks of an αββ protein, is taken up for modeling, first with cyana, in a mixed-chirality linker between the building blocks, and then with IDeAS, in a sequence over side chains. The designed mixed-chirality polypeptide structure is proven to order as an intended αββ fold and capture zinc to activate its role as a hydrolase catalyst. The design approach to have protein folds defined stereochemically and receptor and catalysis functions defined chemically is presented, and illustrates L- and D-α-amino-acid structures as the alphabet integrating chemical- and stereochemical-structure variables as its letters.     

The crystal structure of spermidine/spermine N1-acetyltransferase in complex with spermine provides insights into substrate binding and catalysis

The enzyme spermidine/spermine N (1)-acetyltransferase (SSAT) catalyzes the transfer of acetyl groups from acetylcoenzyme A to spermidine and spermine, as part of a polyamine degradation pathway. This work describes the crystal structure of SSAT in complex with coenzyme A, with and without bound spermine. The complex with spermine provides a direct view of substrate binding by an SSAT and demonstrates structural plasticity near the active site of the enzyme. Associated water molecules bridge several of the intermolecular contacts between spermine and the enzyme and form a "proton wire" between the side chain of Glu92 and the N1 amine of spermine. A single water molecule can also be seen forming hydrogen bonds with the side chains of Glu92, Asp93, and the N4 amine of spermine. Site-directed mutation of Glu92 to glutamine had a detrimental effect on both substrate binding and catalysis and shifted the optimal pH for enzyme activity further into alkaline solution conditions, while mutation of Asp93 to asparagine affected both substrate binding and catalysis without changing the pH dependence of the enzyme. Considered together, the structural and kinetic data suggest that Glu92 functions as a catalytic base to drive an otherwise unfavorable deprotonation step at physiological pH.