Conserved water mediated recognition and the dynamics of active site Cys 331 and Tyr 411 in hydrated structure of human IMPDH‐II

Inosine monophosphate dehydrogenase (IMPDH) of human is involved in GMP biosynthesis pathway, increased level of IMPDH‐II (an isoform of enzyme) activity have found in leukemic and sarcoma cells. Modeling and extensive molecular dynamics simulation (15 ns) studies of IMPDH‐II (1B3O PDB structure) have indicated the intricate involvement of four conserved water molecules (W 1, W 2, W 3, and W 4) in the conformational transition or the mobilities of “flap” (residues 400–450) and “loop” (residues 325–342) regions in enzyme. The stabilization of active site residues Asn 303, Gly 324, Ser 329, Cys 331, Asp 364, and Tyr 411 through variable H‐bonding coordination from the conserved water molecular center seems interesting in the uninhibited hydrated form of human IMPDH‐II structures. This conformational transition or the flexibility of mobile regions, water molecular recognition to active site residues Cys 331 and Tyr 411, and the presence of a hydrophilic cavity ～540 ų (enclaved by the loop and flap region) near the C‐terminal surface of this enzyme may explore a rational hope toward the water mimic inhibitor or anticancer agent design for human. Copyright © 2010 John Wiley & Sons, Ltd.     

Kinetic and structural analysis of mutant Escherichia coli dihydroorotases: a flexible loop stabilizes the transition state

Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamyl-l-aspartate (CA-asp) to l-dihydroorotate (DHO) in the de novo biosynthesis of pyrimidine nucleotides. Two different conformations of the surface loop (residues 105-115) were found in the dimeric Escherichia coli DHOase crystallized in the presence of DHO (PDB code 1XGE). The loop asymmetry reflected that of the active site contents of the two subunits: the product, DHO, was bound in the active site of one subunit and the substrate, CA-asp, in the active site of the other. In the substrate- (CA-asp-) bound subunit, the surface loop reaches in toward the active site and makes hydrogen bonds with the bound CA-asp via two threonine residues (Thr109 and Thr110), whereas the loop forms part of the surface of the protein in the product- (DHO-) bound subunit. To investigate the relationship between the structural states of this loop and the catalytic mechanism of the enzyme, a series of mutant DHOases including deletion of the flexible loop were generated and characterized kinetically and structurally. Disruption of the hydrogen bonds between the surface loop and the substrate results in significant loss of catalytic activity. Furthermore, structures of these mutants with low catalytic activity have no interpretable electron density for parts of the flexible loop. The structure of the mutant (Delta107-116), in which the flexible loop is deleted, shows only small differences in positions of other substrate binding residues and in the binuclear zinc center compared with the native structure, yet the enzyme has negligible activity. The kinetic and structural analyses suggest that Thr109 and Thr110 in the flexible loop provide productive binding of substrate and stabilize the transition-state intermediate, thereby increasing catalytic activity.     

Solution structure of a low-molecular-weight protein tyrosine phosphatase from Bacillus subtilis

The low-molecular-weight (LMW) protein tyrosine phosphatases (PTPs) exist ubiquitously in prokaryotes and eukaryotes and play important roles in cellular processes. We report here the solution structure of YwlE, an LMW PTP identified from the gram-positive bacteria Bacillus subtilis. YwlE consists of a twisted central four-stranded parallel beta-sheet with seven alpha-helices packing on both sides. Similar to LMW PTPs from other organisms, the conformation of the YwlE active site is favorable for phosphotyrosine binding, indicating that it may share a common catalytic mechanism in the hydrolysis of phosphate on tyrosine residue in proteins. Though the overall structure resembles that of the eukaryotic LMW PTPs, significant differences were observed around the active site. Residue Asp115 is likely interacting with residue Arg13 through electrostatic interaction or hydrogen bond interaction to stabilize the conformation of the active cavity, which may be a unique character of bacterial LMW PTPs. Residues in the loop region from Phe40 to Thr48 forming a wall of the active cavity are more flexible than those in other regions. Ala41 and Gly45 are located near the active cavity and form a noncharged surface around it. These unique properties demonstrate that this loop may be involved in interaction with specific substrates. In addition, the results from spin relaxation experiments elucidate further insights into the mobility of the active site. The solution structure in combination with the backbone dynamics provides insights into the mechanism of substrate specificity of bacterial LMW PTPs.     

Analysis of the substrate specificity loop of the HAD superfamily cap domain

The haloacid dehalogenase (HAD) superfamily includes a variety of enzymes that catalyze the cleavage of substrate C-Cl, P-C, and P-OP bonds via nucleophilic substitution pathways. All members possess the alpha/beta core domain, and many also possess a small cap domain. The active site of the core domain is formed by four loops (corresponding to sequence motifs 1-4), which position substrate and cofactor-binding residues as well as the catalytic groups that mediate the "core" chemistry. The cap domain is responsible for the diversification of chemistry within the family. A tight beta-turn in the helix-loop-helix motif of the cap domain contains a stringently conserved Gly (within sequence motif 5), flanked by residues whose side chains contribute to the catalytic site formed at the domain-domain interface. To define the role of the conserved Gly in the structure and function of the cap domain loop of the HAD superfamily members phosphonoacetaldehyde hydrolase and beta-phosphoglucomutase, the Gly was mutated to Pro, Val, or Ala. The catalytic activity was severely reduced in each mutant. To examine the impact of Gly substitution on loop 5 conformation, the X-ray crystal structure of the Gly50Pro phosphonoacetaldehyde hydrolase mutant was determined. The altered backbone conformation at position 50 had a dramatic effect on the spatial disposition of the side chains of neighboring residues. Lys53, the Schiff Base forming lysine, had rotated out of the catalytic site and the side chain of Leu52 had moved to fill its place. On the basis of these studies, it was concluded that the flexibility afforded by the conserved Gly is critical to the function of loop 5 and that it is a marker by which the cap domain substrate specificity loop can be identified within the amino acid sequence of HAD family members.     

The mobility of an HIV-1 integrase active site loop is correlated with catalytic activity.

Replication of HIV-1 requires the covalent integration of the viral cDNA into the host chromosomal DNA directed by the virus-encoded integrase protein. Here we explore the importance of a protein surface loop near the integrase active site using protein engineering and X-ray crystallography. We have redetermined the structure of the integrase catalytic domain (residues 50-212) using an independent phase set at 1.7 A resolution. The structure extends helix alpha4 on its N-terminal side (residues 149-154), thus defining the position of the three conserved active site residues. Evident in this and in previous structures is a conformationally flexible loop composed of residues 141-148. To probe the role of flexibility in this loop, we replaced Gly 140 and Gly 149, residues that appear to act as conformational hinges, with Ala residues. X-ray structures of the catalytic domain mutants G149A and G140A/G149A show further rigidity of alpha4 and the adjoining loop. Activity assays in vitro revealed that these mutants are impaired in catalysis. The DNA binding affinity, however, is minimally affected by these mutants as assayed by UV cross-linking. We propose that the conformational flexibility of this active site loop is important for a postbinding catalytic step.     

Mutational analysis of active site residues in the Staphylococcus aureus transpeptidase SrtA

In Staphylococcus aureus, virulence and colonization-associated surface proteins are covalently anchored to the cell wall by the transpeptidase Sortase A (SrtA). In order to better understand the contribution of specific active site residues to substrate recognition and catalysis, we performed mutational analysis of several key residues in the SrtA active site. Analysis of protein stability, kinetic parameters, solvent isotope effects, and pH-rate profiles for key SrtA variants are consistent with a reverse protonated Cys184-His120 catalytic dyad, and implicate a role for Arg197 in formation of an oxyanion hole to stabilize the transition state. In contrast, mutation of Asp185 and Asp186 produced negligible effects on catalysis, and no evidence was found to support the existence of a functional catalytic triad. Mutation of Thr180, Leu181, and Ile182 to alanine produced modest decreases in SrtA activity and led to substrate inhibition. Thermodynamic stability measurements by SUPREX (stability of unpurified proteins from rates of H/D exchange) revealed decreases in conformational stability that correlate with the observed substrate inhibition for each variant, signifying a potential role for the conserved 180TLITC184 motif in defining the active-site architecture of SrtA. In contrast, mutation of Thr183 to alanine led to a significant 1200-fold decrease in kcat, which appears to be unrelated to conformational stability. Potential explanations for these results are discussed, and a revised model for SrtA catalysis is presented.     

Crystal structure determination of cholesterol oxidase from Streptomyces and structural characterization of key active site mutants

Cholesterol oxidase is a monomeric flavoenzyme which catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. The enzyme interacts with lipid bilayers in order to bind its steroid substrate. The X-ray structure of the enzyme from Brevibacterium sterolicum revealed two loops, comprising residues 78-87 and residues 433-436, which act as a lid over the active site and facilitate binding of the substrate [Vrielink et al. (1991) J. Mol. Biol. 219, 533-554; Li et al. (1993) Biochemistry 32, 11507-11515]. It was postulated that these loops must open, forming a hydrophobic channel between the membrane and the active site of the protein and thus sequestering the cholesterol substrate from the aqueous environment. Here we describe the three-dimensional structure of the homologous enzyme from Streptomyces refined to 1.5 A resolution. Structural comparisons to the enzyme from B. sterolicum reveal significant conformational differences in these loop regions; in particular, a region of the loop comprising residues 78-87 adopts a small amphipathic helical turn with hydrophobic residues directed toward the active site cavity and hydrophilic residues directed toward the external surface of the molecule. It seems reasonable that this increased rigidity reduces the entropy loss that occurs upon binding substrate. Consequently, the Streptomyces enzyme is a more efficient catalyst. In addition, we have determined the structures of three active site mutants which have significantly reduced activity for either the oxidation (His447Asn and His447Gln) or the isomerization (Glu361Gln). Our structural and kinetic data indicate that His447 and Glu361 act as general base catalysts in association with conserved water H2O541 and Asn485. The His447, Glu361, H2O541, and Asn485 hydrogen bond network is conserved among other oxidoreductases. This catalytic tetrad appears to be a structural motif that occurs in flavoenzymes that catalyze the oxidation of unactivated alcohols.     

Crystal structure of the stromelysin catalytic domain at 2.0 A resolution: inhibitor-induced conformational changes.

Matrix metalloproteinases are believed to play an important role in pathological conditions such as osteoarthritis, rheumatoid arthritis and tumor invasion. Stromelysin is a zinc-dependent proteinase and a member of the matrix metalloproteinase family. We have solved the crystal structure of an active uninhibited form of truncated stromelysin and a complex with a hydroxamate-based inhibitor. The catalytic domain of the enzyme of residues 83-255 is an active fragment. Two crystallographically independent molecules, A and B, associate as a dimer in the crystals. There are three alpha-helices and one twisted, five-strand beta-sheet in each molecule, as well as one catalytic Zn, one structural Zn and three structural Ca ions. The active site of stromelysin is located in a large, hydrophobic cleft. In particular, the S1' specificity site is a deep and highly hydrophobic cavity. The structure of a hydroxamate-phosphinamide-type inhibitor-bound stromelysin complex, formed by diffusion soaking, has been solved as part of our structure-based design strategy. The most important feature we observed is an inhibitor-induced conformational change in the S1' cavity which is triggered by Tyr223. In the uninhibited enzyme structure, Tyr223 completely covers the S1' cavity, while in the complex, the P1' group of the inhibitor displaces the Tyr223 in order to fit into the S1' cavity. Furthermore, the displacement of Tyr223 induces a major conformational change of the entire loop from residue 222 to residue 231. This finding provides direct evidence that Tyr223 plays the role of gatekeeper of the S1' cavity. Another important intermolecular interaction occurs at the active sit of molecule A, in which the C-terminal tail (residues 251-255) from molecule B inserts. The C-terminal tail interacts extensively with the active site of molecule A, and the last residue (Thr255) coordinated to the catalytic zinc as the fourth ligand, much like a product inhibitor would. The inhibitor-induced conformational change and the intermolecular C-terminal-zinc coordination are significant in understanding the structure-activity relationships of the enzyme.     

Structural basis for the catalytic mechanism of aspartate ammonia lyase

Aspartate ammonia lyases (or aspartases) catalyze the reversible deamination of L-aspartate into fumarate and ammonia. The lack of crystal structures of complexes with substrate, product, or substrate analogues so far precluded determination of their precise mechanism of catalysis. Here, we report crystal structures of AspB, the aspartase from Bacillus sp. YM55-1, in an unliganded state and in complex with L-aspartate at 2.4 and 2.6 ? resolution, respectively. AspB forces the bound substrate to adopt a high-energy, enediolate-like conformation that is stabilized, in part, by an extensive network of hydrogen bonds between residues Thr101, Ser140, Thr141, and Ser319 and the substrate's β-carboxylate group. Furthermore, substrate binding induces a large conformational change in the SS loop (residues G(317)SSIMPGKVN(326)) from an open conformation to one that closes over the active site. In the closed conformation, the strictly conserved SS loop residue Ser318 is at a suitable position to act as a catalytic base, abstracting the Cβ proton of the substrate in the first step of the reaction mechanism. The catalytic importance of Ser318 was confirmed by site-directed mutagenesis. Site-directed mutagenesis of SS loop residues, combined with structural and kinetic analysis of a stable proteolytic AspB fragment, further suggests an important role for the small C-terminal domain of AspB in controlling the conformation of the SS loop and, hence, in regulating catalytic activity. Our results provide evidence supporting the notion that members of the aspartase/fumarase superfamily use a common catalytic mechanism involving general base-catalyzed formation of a stabilized enediolate intermediate.     

Structural constraints on protein self-processing in L-aspartate-alpha-decarboxylase

Aspartate decarboxylase, which is translated as a pro-protein, undergoes intramolecular self-cleavage at Gly24-Ser25. We have determined the crystal structures of an unprocessed native precursor, in addition to Ala24 insertion, Ala26 insertion and Gly24-->Ser, His11-->Ala, Ser25-->Ala, Ser25-->Cys and Ser25-->Thr mutants. Comparative analyses of the cleavage site reveal specific conformational constraints that govern self-processing and demonstrate that considerable rearrangement must occur. We suggest that Thr57 Ogamma and a water molecule form an 'oxyanion hole' that likely stabilizes the proposed oxyoxazolidine intermediate. Thr57 and this water molecule are probable catalytic residues able to support acid-base catalysis. The conformational freedom in the loop preceding the cleavage site appears to play a determining role in the reaction. The molecular mechanism of self-processing, presented here, emphasizes the importance of stabilization of the oxyoxazolidine intermediate. Comparison of the structural features shows significant similarity to those in other self-processing systems, and suggests that models of the cleavage site of such enzymes based on Ser-->Ala or Ser-->Thr mutants alone may lead to erroneous interpretations of the mechanism.     

Homology modelling and site-directed mutagenesis studies of the epoxide hydrolase from Phanerochaete chrysosporium

Three-dimensional structural model of epoxide hydrolase (PchEHA) from Phanerochaete chrysosporium was constructed based on X-ray structure of Agrobacterium radiobacter AD1 sEH using SWISS-MODEL server. Conserved residues constituting the active site cavity were identified, of which the functional roles of 14 residues were determined by site-directed mutagenesis. In catalytic triad, Asp105 and His308 play a leading role in alkylation and hydrolysis steps, respectively. Distance between Asp105 and epoxide ring of substrate may determine the regiospecificity in the substrate docking model. Asp277 located at the entrance of substrate tunnel is concerned with catalysis but not essential. D307E had the highest activity and lower enantioselectivity among 14 mutants, suggesting Asp307 may be involved in choice of substrate configuration. Y159F and Y241F almost exhibited no activity, indicating that they are essential to bind substrate and facilitate opening of epoxide ring. Besides, His35-Gly36-Asn37-Pro38, Trp106 and Trp309 surrounding Asp105, may coordinate the integration of active site cavity and influence substrate binding. Especially, W106I reversed the enantioselectivity, perhaps due to more deteriorative impact on the preferred (R)-styrene oxide. Gly65 and Gly67 occurring at β-turns and Gly36 are vital in holding protein conformation. Conclusively, single conserved residue around the active sites has an important impact on catalytic properties.     

Crystal structures of a poxviral glutaredoxin in the oxidized and reduced states show redox-correlated structural changes

Glutaredoxins act as reducing agents for the large subunit of ribonucleotide reductase (R1) in many prokaryotes and eukaryotes, including humans. The same relationship has been proposed for the glutaredoxin and R1 proteins expressed by all orthopoxviruses, including vaccinia, variola, and ectromelia virus. Interestingly, the orthopoxviral proteins share 45% and 78% sequence identity with human glutaredoxin-1 (Grx-1) and R1, respectively. To study structure-function relationships of the vertebrate Grx-1 family, and reveal potential viral adaptations, we have determined crystal structures of the ectromelia virus glutaredoxin, EVM053, in the oxidized and reduced states. The structures show a large redox-induced conformational rearrangement of Tyr21 and Thr22 near the active site. We predict that the movement of Tyr21 is a viral-specific adaptation that increases the redox potential by stabilizing the reduced state. The conformational switch of Thr22 appears to be shared by vertebrate Grx-1 and may affect the strictly conserved Lys20. A crystal packing-induced structural change in residues 68-70 affects the GSH-binding loop, and our structures reveal a potential interaction network that connects the GSH-binding loop and the active site. EVM053 also exhibits a novel cis-proline (Pro53) in a loop that has been shown to contribute to R1-binding in Escherichia coli Grx-1. The cis-peptide bond of Pro53 may be required to promote electrostatic interactions between Lys52 and the C-terminal carboxylate of R1. Finally, dimethylarsenite was covalently attached to Cys23 in one reduced EVM053 structure and our preliminary data show that EVM053 has dimethylarsenate reductase activity.     

Charged residues on a flap-loop structure of Lactococcus lactis prolidase play critical roles in allosteric behavior and substrate inhibition

Allosteric behavior and substrate inhibition are unique characteristics of Lactococcus lactis prolidase. We hypothesized that charged residues (Asp36, His38, Glu39, and Arg40), present on one loop essential for catalysis, interact with residues in or near the active site to impart these unique characteristics. Asp36 has a predominant role in the allosteric behavior, as demonstrated through the non-allosteric behavior of the D36S mutant enzyme. In contrast, a double mutant (D36E/R293K) maintained the allostery, indicating that this aspartic acid residue interacts with Arg293, previously shown to be critical in the allostery. Substitution of His38 drastically reduced the substrate inhibition, and substrate specificity of the mutant at Asp36 or His38 showed the influence of these residues to the substrate specificity. These findings confirm the importance of the loop in the enzymatic reaction mechanism and suggest the existence of conformational changes of the loop structure between open and closed states. A variety of mutations at Glu39 and Arg40 showed that these residues influence roles of the loop in the enzyme reaction. On the basis of these results and combined with observations of molecular models of this prolidase, we concluded that Asp36 and His38 interact with the residues in the active site to generate an allosteric subsite and a pseudo-S(1)' site, which are responsible for the allosteric behavior and substrate inhibition.     

The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin

Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (M_{r ca.} 29,000), and the S-peptide (M_r 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH₂-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH₂-terminal 60-amino acid peptide.     

Recognition site for the side chain of 2-ketoacid substrate in d-lactate dehydrogenase

Replacement of Tyr52 with Val or Ala in Lactobacillus pentosus d-lactate dehydrogenase induced high activity and preference for large aliphatic 2-ketoacids and phenylpyruvate. On the other hand, replacements with Arg, Thr or Asp severely reduced the enzyme activity, and the Tyr52Arg enzyme, the only one that exhibited significant enzyme activity, showed a similar substrate preference to the Tyr52Val and Tyr52Ala enzymes. Replacement of Phe299 with Gly or Ser greatly reduced the enzyme activity with less marked change in the substrate preference. Except for the Phe299Ser enzyme, these mutant enzymes with low catalytic activity consistently stimulated NADH oxidation in the absence of 2-ketoacid substrates. However, the double mutant enzymes, Tyr52Arg/Phe299Gly and Tyr52Thr/Phe299Ser, did not exhibit synergically decreased enzyme activity or the substrate-independent NADH oxidation, but rather increased activities toward certain 2-ketoacid substrates. These results indicate that the coordinative combination of amino acid residues at two positions is pivotal in both the functional recognition of the 2-ketoacid side chain and the protection of the bound NADH molecule from the solvent. Multiplicity in such combinations appears to provide d-LDH-related 2-hydroxyacid dehydrogenases with a great variety of catalytic and physiological functions.     

Starter substrate specificities of wild-type and mutant polyketide synthases from Rutaceae

Chalcone synthases (CHSs) and acridone synthases (ACSs) belong to the superfamily of type III polyketide synthases (PKSs) and condense the starter substrate 4-coumaroyl-CoA or N-methylanthraniloyl-CoA with three malonyl-CoAs to produce flavonoids and acridone alkaloids, respectively. ACSs which have been cloned exclusively from Ruta graveolens share about 75-85% polypeptide sequence homology with CHSs from other plant families, while 90% similarity was observed with CHSs from Rutaceae, i.e., R. graveolens, Citrus sinensis and Dictamnus albus. CHSs cloned from many plants do not accept N-methylanthraniloyl-CoA as a starter substrate, whereas ACSs were shown to possess some side activity with 4-coumaroyl-CoA. The transformation of an ACS to a functional CHS with 10% residual ACS activity was accomplished previously by substitution of three amino acids through the corresponding residues from Ruta-CHS1 (Ser132Thr, Ala133Ser and Val265Phe). Therefore, the reverse triple mutation of Ruta-CHS1 (mutant R2) was generated, which affected only insignificantly the CHS activity and did not confer ACS activity. However, competitive inhibition of CHS activity by N-methylanthraniloyl-CoA was observed for the mutant in contrast to wild-type CHSs. Homology modeling of ACS2 with docking of 1,3-dihydroxy-N-methylacridone suggested that the starter substrates for CHS or ACS reaction are placed in different topographies in the active site pocket. Additional site specific substitutions (Asp205Pro/Thr206Asp/His207Ala or Arg60Thr and Val100Ala/Gly218Ala, respectively) diminished the CHS activity to 75-50% of the wild-type CHS1 without promoting ACS activity. The results suggest that conformational changes in the periphery beyond the active site cavity volumes determine the product formation by ACSs vs. CHSs in R. graveolens. It is likely that ACS has evolved from CHS, but the sole enlargement of the active site pocket as in CHS1 mutant R2 is insufficient to explain this process.     

Molecular modeling of the dimeric structure of human lipoprotein lipase and functional studies of the carboxyl-terminal domain.

Lipoprotein lipase (LPL) plays a key role in lipid metabolism. Molecular modeling of dimeric LPL was carried out using insight ii based upon the crystal structures of human, porcine, and horse pancreatic lipase. The dimeric model reveals a saddle-shaped structure and the key heparin-binding residues in the amino-terminal domain located on the top of this saddle. The models of two dimeric conformations - a closed, inactive form and an open, active form - differ with respect to how surface-loop positions affect substrate access to the catalytic site. In the closed form, the surface loop covers the catalytic site, which becomes inaccessible to solvent. Large conformational changes in the open form, especially in the loop and carboxyl-terminal domain, allow substrate access to the active site. To dissect the structure-function relationships of the LPL carboxyl-terminal domain, several residues predicted by the model structure to be essential for the functions of heparin binding and substrate recognition were mutagenized. Arg405 plays an important role in heparin binding in the active dimer. Lys413/Lys414 or Lys414 regulates heparin affinity in both monomeric and dimeric forms. To evaluate the prediction that LPL forms a homodimer in a 'head-to-tail' orientation, two inactive LPL mutants - a catalytic site mutant (S132T) and a substrate-recognition mutant (W390A/W393A/W394A) - were cotransfected into COS7 cells. Lipase activity could be recovered only when heterodimerization occurred in a head-to-tail orientation. After cotransfection, 50% of the wild-type lipase activity was recovered, indicating that lipase activity is determined by the interaction between the catalytic site on one subunit and the substrate-recognition site on the other.     

Topological role of cytochrome P450 2D6 active site residues

Recent reports have identified Phe120, Asp301, Thr309, and Glu216 as important residues in cytochrome P450 2D6 (CYP2D6) substrate binding and catalysis. Complementary homology models have located these amino acids within the binding pocket of CYP2D6 and in the present study we have used aryldiazenes to test these models and gain further insight in the role these amino acids have in maintaining the integrity of the active site cavity. When Phe120 was replaced to alanine, there was a significant increase in probe migration to pyrrole nitrogens C and D, in agreement with homology models which have located the phenyl side-chain of Phe120 above these two pyrrole rings. No changes in topology were observed with the D301Q mutant, supporting claims that in this mutant the electrostatic interactions with the B/C-loop are largely maintained and the loop retains its native orientation. The T309V mutation resulted in significant topological alteration suggesting that, in addition to its potential role in dioxygen activation, Thr309 plays an important structural role within the active site crevice. Replacement of Ile106 with Glu, engineered to cause electrostatic repulsion with Glu216, had a profound topological effect in the higher region within the active site cavity and impaired the catalytic activity towards CYP2D6 probe substrates.     

Cosubstrate-induced dynamics of D-3-hydroxybutyrate dehydrogenase from Pseudomonas putida

D-3-Hydroxybutyrate dehydrogenase from Pseudomonas putida belongs to the family of short-chain dehydrogenases/reductases. We have determined X-ray structures of the D-3-hydroxybutyrate dehydrogenase from Pseudomonas putida, which was recombinantly expressed in Escherichia coli, in three different crystal forms to resolutions between 1.9 and 2.1 A. The so-called substrate-binding loop (residues 187-210) was partially disordered in several subunits, in both the presence and absence of NAD(+). However, in two subunits, this loop was completely defined in an open conformation in the apoenzyme and in a closed conformation in the complex structure with NAD(+). Structural comparisons indicated that the loop moves as a rigid body by about 46 degrees . However, the two small alpha-helices (alphaFG1 and alphaFG2) of the loop also re-orientated slightly during the conformational change. Probably, the interactions of Val185, Thr187 and Leu189 with the cosubstrate induced the conformational change. A model of the binding mode of the substrate D-3-hydroxybutyrate indicated that the loop in the closed conformation, as a result of NAD(+) binding, is positioned competent for catalysis. Gln193 is the only residue of the substrate-binding loop that interacts directly with the substrate. A translation, libration and screw (TLS) analysis of the rigid body movement of the loop in the crystal showed significant librational displacements, describing the coordinated movement of the substrate-binding loop in the crystal. NAD(+) binding increased the flexibility of the substrate-binding loop and shifted the equilibrium between the open and closed forms towards the closed form. The finding that all NAD(+) -bound subunits are present in the closed form and all NAD(+) -free subunits in the open form indicates that the loop closure is induced by cosubstrate binding alone. This mechanism may contribute to the sequential binding of cosubstrate followed by substrate.