Homology modeling of the structure of tobacco acetohydroxy acid synthase and examination of the active site by site-directed mutagenesis

A reliable model of tobacco acetohydroxy acid synthase (AHAS) was obtained by homology modeling based on a yeast AHAS X-ray structure using the Swiss-Model server. Conserved residues at the dimer interface were identified, of which the functional roles of four residues, namely H142, E143, M489, and M542, were determined by site-directed mutagenesis. Eight mutants were successfully generated and purified, five of which (H142T, M489V, M542C, M542I, and M542V) were found to be inactive under various assay conditions. The H142K mutant was moderately altered in all kinetic parameters to a similar extent. In addition, the mutant was more thermo-labile than wild type enzyme. The E143A mutant increased the Km value more than 20-fold while other parameters were not significantly changed. All mutations carried out on residue M542 inactivated the enzyme. Though showing a single band on SDS-PAGE, the M542C mutant lost its native tertiary structure and was aggregated. Except M542C, each of the other mutants showed a secondary structure similar to that of wild type enzyme. Although all the inactive mutants were able to bind FAD, the mutants M489V and M542C showed a very low affinity for FAD. None of the active mutants constructed was strongly resistant to three tested herbicides. Taken together, the results suggest that the residues of H142, E143, M489, and M542 are essential for catalytic activity. Furthermore, it seems that H142 residue is involved in stabilizing the dimer interaction, while E143 residue may be involved in binding with substrate pyruvate. The data from the site-directed mutagenesis imply that the constructed homology model of tobacco AHAS is realistic.     

Structural rationalization for the lack of stereospecificity in coenzyme B12-dependent diol dehydratase

Adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca is apparently not stereospecific and catalyzes the conversion of both (R)- and (S)-1,2-propanediol to propionaldehyde. To explain this unusual property of the enzyme, we analyzed the crystal structures of diol dehydratase in complexes with cyanocobalamin and (R)- or (S)-1,2-propanediol. (R)- and (S)-isomers are bound in a symmetrical manner, although the hydrogen-bonding interactions between the substrate and the active-site residues are the same. From the position of the adenosyl radical in the modeled "distal" conformation, it is reasonable for the radical to abstract the pro-R and pro-S hydrogens from (R)- and (S)-isomers, respectively. The hydroxyl groups in the substrate radicals would migrates from C(2) to C(1) by a suprafacial shift, resulting in the stereochemical inversion at C(1). This causes 60 degrees clockwise and 70 degrees counterclockwise rotations of the C(1)-C(2) bond of the (R)- and (S)-isomers, respectively, if viewed from K+. A modeling study of 1,1-gem-diol intermediates indicated that new radical center C(2) becomes close to the methyl group of 5'-deoxyadenosine. Thus, the hydrogen back-abstraction (recombination) from 5'-deoxyadenosine by the product radical is structurally feasible. It was also predictable that the substitution of the migrating hydroxyl group by a hydrogen atom from 5'-deoxyadenosine takes place with the inversion of the configuration at C(2) of the substrate. Stereospecific dehydration of the 1,1-gem-diol intermediates can also be rationalized by assuming that Asp-alpha335 and Glu-alpha170 function as base catalysts in the dehydration of the (R)- and (S)-isomers, respectively. The structure-based mechanism and stereochemical courses of the reaction are proposed.     

Identification of catalytic residues of pepstatin-insensitive carboxyl proteinases from prokaryotes by site-directed mutagenesis.

Pepstatin-insensitive carboxyl proteinases from Pseudomonas sp. (PCP) and Xanthomonas sp. (XCP) have no conserved catalytic residue sequences, -Asp*-Thr-Gly- (Asp is the catalytic residue) for aspartic proteinases. To identify the catalytic residues of PCP and XCP, we selected presumed catalytic residues based on their high sequence similarity, assuming that such significant sites as catalytic residues will be generally conserved. Several Ala mutants of Asp or Glu residues were constructed and analyzed. The D170A, E222A, and D328A mutants for PCP and XD79A, XD169A, and XD348A mutants for XCP were not converted to mature protein after activation, and no catalytic activity could be detected in these mutants. The specificity constants toward chromogenic substrate of the other PCP and XCP mutants, except for the D84A mutant of PCP, were similar to that of wild-type PCP or XCP. Coupled with the result of chemical modification (Ito, M., Narutaki, S., Uchida, K., and Oda, K. (1999) J. Biochem. (Tokyo) 125, 210-216), a pair of Asp residues (170 and 328) for PCP and a pair of Asp residues (169 and 348) for XCP were elucidated to be their catalytic residues, respectively. The Glu(222) residue in PCP or Asp(79) residue in XCP was excluded from the candidates as catalytic residues, since the corresponding mutant retained its original activity.     

Histidine-alpha143 assists 1,2-hydroxyl group migration and protects radical intermediates in coenzyme B12-dependent diol dehydratase

Diol dehydratase of Klebsiella oxytoca contains an essential histidine residue. Its X-ray structure revealed that the migrating hydroxyl group on C2 of substrate is hydrogen-bonded to Hisalpha143. Mutant enzymes in which Hisalpha143 was mutated to another amino acid residue were expressed in Escherichia coli, purified, and examined for enzymatic activity. The Halpha143Q mutant was 34% as active as the wild-type enzyme. Halpha143A and Halpha143L showed only a trace of activity. Kinetic analyses indicated that the hydrogen bonding interaction between the hydroxyl group on C2 of substrate and the side chain of residue alpha143 is important not only for catalysis but also for protecting radical intermediates. Halpha143E and Halpha143K that did not exist as (alphabetagamma) 2 complexes were inactive. The deuterium kinetic isotope effect on the overall reaction suggested that a hydrogen abstraction step is fully rate-determining for the wild type and Halpha143Q and partially rate-determining for Halpha143A. The preference for substrate enantiomers was reversed by the Halpha143Q mutation in both substrate binding and catalysis. Upon the inactivation of the Halpha143A holoenzyme by 1,2-propanediol, cob(II)alamin without an organic radical coupling partner accumulated, 5'-deoxyadenosine was quantitatively formed from the coenzyme adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus concluded to be a mechanism-based inactivation. The holoenzyme of Halpha143Q underwent irreversible inactivation by O 2 in the absence of substrate at a much lower rate than the wild type.     

Mutational Analysis of Highly Conserved Residues in the Phage PhiC31 Integrase Reveals Key Amino Acids Necessary for the DNA Recombination

Background

Amino acid sequence alignment of phage phiC31 integrase with the serine recombinases family revealed highly conserved regions outside the catalytic domain. Until now, no system mutational or biochemical studies have been carried out to assess the roles of these conserved residues in the recombinaton of phiC31 integrase.

Methodology/Principal Findings

To determine the functional roles of these conserved residues, a series of conserved residues were targeted by site-directed mutagenesis. Out of the 17 mutants, 11 mutants showed impaired or no recombination ability, as analyzed by recombination assay both in vivo and in vitro. Results of DNA binding activity assays showed that mutants (R18A, I141A, L143A,E153A, I432A and V571A) exhibited a great decrease in DNA binding affinity, and mutants (G182A/F183A, C374A, C376A/G377A, Y393A and V566A) had completely lost their ability to bind to the specific target DNA attB as compared with wild-type protein. Further analysis of mutants (R18A, I141A, L143A and E153A) synapse and cleavage showed that these mutants were blocked in recombination at the stage of strand cleavage.

Conclusions/Significance

This data reveals that some of the highly conserved residues both in the N-terminus and C-terminus region of phiC31 integrase, play vital roles in the substrate binding and cleavage. The cysteine-rich motif and the C-tail val-rich region of phiC31 integrase may represent the major DNA binding domains of phiC31 integrase.     

Further characterization of the putative human isopeptidase T catalytic site

The human isopeptidase T (isoT) is a zinc-binding deubiquitinating enzyme involved in the disassembly of free K48-linked polyubiquitin chains into ubiquitin monomers. The catalytic site of this enzyme is thought to be composed of Cys335, Asp435, His786 and His795. These four residues were site-directed mutagenized. None of the mutants were able to cleave a peptide-linked ubiquitin dimer. Similarly, C335S, D435N and H795N mutants had virtually no activity against a K48-linked isopeptide ubiquitin dimer, which is an isoT-specific substrate that mimics the K48-linked polyubiquitin chains. On the other hand, the H786N mutant retained a partial activity toward the K48-linked substrate, suggesting that the His786 residue might not be part of the catalytic site. None of the mutations significantly affected the capacity of isoT to bind ubiquitin and zinc. Thus, the catalytic site of UBPs could resemble that of other cysteine proteases, which contain one Cys, one Asp and one His.     

The fourth transmembrane domain of the Helicobacter pylori Na+/H+ antiporter NhaA faces a water-filled channel required for ion transport

Cysteine-scanning mutagenesis was performed from Ser-130 to Leu-160 in the fourth transmembrane domain (TM4) of the Na+/H+ antiporter NhaA from Helicobacter pylori to determine the topology of each residue and to identify functionally important residues. All of the mutants were based on cysteine-less NhaA (Cys-less NhaA), which functions very similarly to the wild-type protein, and were expressed at a level similar to Cys-less NhaA. Discontinuity of [14C]N-ethylmaleimide (NEM)-reactive residues suggested that TM4 comprises residues Gly-135 to Val-156. Even within TM4, NEM reactivity was high for I136C, D141C to A143C, L146C, M150C, and G153C to R155C. These residues are thought to be located on one side of the -helical structure of TM4 and to face a putative water-filled channel. Pretreatment of intact cells with membrane-impermeable maleimide did not inhibit [14C]NEM binding to the NEM-reactive residues within TM4, suggesting that the putative channel opens toward the cytoplasm. NEM reactivity of the A143C mutant was significantly inhibited by Li+. The T140C and D141C mutants showed lower affinity for Na+ and Li+ as transport substrates, but their maximal antiporter velocities (Vmax) were relatively unaffected. Whereas the I142C and F144C mutants completely lost their Li+/H+ antiporter activity, I142C had a lower Vmax for the Na+/H+ antiporter. F144C exhibited a markedly lower Vmax and a partially reduced affinity for Na+. These results suggest that Thr-140, Asp-141, and Phe-144 are located in the end portion of a putative water-filled channel and may provide the binding site for Na+, Li+, and/or H+. Furthermore, residues Ile-142 to Phe-144 may be important for the conformational change that accompanies ion transport in NhaA.     

Structural and functional roles of highly conserved serines in human lipoprotein lipase. Evidence that serine 132 is essential for enzyme catalysis

The structure of human lipoprotein lipase was recently deduced from its cDNA sequence. It contains 8 serine residues (residues 45, 132, 143, 172, 193, 244, 251, and 363) that are absolutely conserved in both lipoprotein lipase and hepatic lipase across all species studied. The high homology between lipoprotein lipase, hepatic lipase, and pancreatic lipase suggests that the catalytic functions of these enzymes share a common mechanism and that one of the 8 conserved serines in human lipoprotein lipase must play a catalytic role as does serine 152 in the case of pancreatic lipase (Winkler, F. K., D'Arcy, A., and Hunziker, W. Nature 343, 771-774). We expressed wild-type and site-specific mutants of human lipoprotein lipase in COS cells in vitro. We produced two to four substitution mutants involving each of the 8 serines and assayed a total of 22 mutants for both enzyme activity and the amount of immunoreactive enzyme mass produced. Immunoreactive lipase was detected in all cases. With the exception of Ser132, for each of the 8 serine mutants we studied, at least one of several mutants at each position showed detectable enzyme activity. All three substitution mutants at Ser132, Ser----Thr, Ser----Ala, and Ser----Asp, were totally inactive. Ser132 occurs in the consensus sequence Gly-Xaa-Ser-Xaa-Gly present in all serine proteinases and in human pancreatic lipase. The x-ray crystallography structure of human pancreatic lipase suggests that the analogous serine residue in human pancreatic lipase, Ser152, is the nucleophilic residue essential for catalysis. Our biochemical data strongly support the conclusion that Ser132 in human lipoprotein lipase is the crucial residue required for enzyme catalysis. The observed specific activities of the variants involving the other seven highly conserved serines in human lipoprotein lipase are consistent with the interpretation that this enzyme has a three-dimensional structure very similar to that of human pancreatic lipase.     

The crystal structure of l-sorbose reductase from Gluconobacter frateurii complexed with NADPH and l-sorbose

l-Sorbose reductase from Gluconobacter frateurii (SR) is an NADPH-dependent oxidoreductase. SR preferentially catalyzes the reversible reaction between d-sorbitol and l-sorbose with high substrate specificity. To elucidate the structural basis of the catalytic mechanism and the substrate specificity of SR, we have determined the structures of apo-SR, SR in complex with NADPH, and the inactive mutant (His116Leu) of SR in complex with NADPH and l-sorbose at 2.83 Å, 1.90 Å, and 1.80 Å resolutions, respectively. Our results show that SR belongs to the short-chain dehydrogenase/reductase (SDR) family and forms a tetrameric structure. Although His116 is not conserved among SDR family enzymes, the structures of SR have revealed that His116 is important for the stabilization of the proton relay system and for active-site conformation as a fourth catalytic residue. In the ternary complex structure, l-sorbose is recognized by 11 hydrogen bonds. Site-directed mutagenesis of residues around the l-sorbose-binding site has shown that the loss of almost full enzymatic activity was caused by not only the substitution of putative catalytic residues but also the substitution of the residue used for the recognition of the C4 hydroxyl groups of l-sorbose (Glu154) and of the residues used for the construction of the substrate-binding pocket (Cys146 and Gly188). The recognition of the C4 hydroxyl group of l-sorbose would be indispensable for the substrate specificity of SR, which recognizes only l-sorbose and d-sorbitol but not other sugars. Our results indicated that these residues were crucial for the substrate recognition and specificity of SR.     

Reassessing the type I dehydroquinate dehydratase catalytic triad: Kinetic and structural studies of Glu86 mutants

Dehydroquinate dehydratase (DHQD) catalyzes the third reaction in the biosynthetic shikimate pathway. Type I DHQDs are members of the greater aldolase superfamily, a group of enzymes that contain an active site lysine that forms a Schiff base intermediate. Three residues (Glu86, His143, and Lys170 in the Salmonella enterica DHQD) have previously been proposed to form a triad vital for catalysis. While the roles of Lys170 and His143 are well defined—Lys170 forms the Schiff base with the substrate and His143 shuttles protons in multiple steps in the reaction—the role of Glu86 remains poorly characterized. To probe Glu86′s role, Glu86 mutants were generated and subjected to biochemical and structural study. The studies presented here demonstrate that mutant enzymes retain catalytic proficiency, calling into question the previously attributed role of Glu86 in catalysis and suggesting that His143 and Lys170 function as a catalytic dyad. Structures of the Glu86Ala (E86A) mutant in complex with covalently bound reaction intermediate reveal a conformational change of the His143 side chain. This indicates a predominant steric role for Glu86, to maintain the His143 side chain in position consistent with catalysis. The structures also explain why the E86A mutant is optimally active at more acidic conditions than the wild‐type enzyme. In addition, a complex with the reaction product reveals a novel, likely nonproductive, binding mode that suggests a mechanism of competitive product inhibition and a potential strategy for the design of therapeutics.     

Conformational dynamics of threonine 195 and the S1 subsite in functional trypsin variants

Replacing the catalytic serine in trypsin with threonine (S195T variant) leads to a nearly complete loss of catalytic activity, which can be partially restored by eliminating the C42-C58 disulfide bond. The 0.69 μs of combined explicit solvent molecular dynamics (MD) simulations revealed continuous rearrangement of T195 with different conformational preferences between five trypsin variants tested. Among three conformational families observed for the T195 residue, one showed the T195 hydroxyl in a conformation analogous to that of the serine residue in wild-type trypsin, positioning the hydroxyl oxygen atom for attack on the carbonyl carbon of the peptide substrate. MD simulations demonstrated that this conformation was more populated for the C42A/C58V/S195T and C42A/C58A/S195T triple variants than for the catalytically inactive S195T variant and correlated with restored enzymatic activities for triple variants. In addition, observation of the increased motion of the S214-G219 segment in the S195T substituted variants suggested an existence of open and closed conformations for the substrate binding pocket. The closed conformation precludes access to the S1 binding site and could further reduce enzymatic activities for triple variants. Double variants with intact serine residues (C42A/C58A/S195 and C42A/C58V/S195) also showed interchange between closed and open conformations for the S214-G219 segment, but to a lesser extent than the triple variants. The increased conformational flexibility of the S1 subsite, which was not observed for the wild-type, correlated with reduced enzymatic activities and suggested a possible mode of substrate regulation for the trypsin variants tested.     

The role of factor XIa (FXIa) catalytic domain exosite residues in substrate catalysis and inhibition by the Kunitz protease inhibitor domain of protease nexin 2

To select residues in coagulation factor XIa (FXIa) potentially important for substrate and inhibitor interactions, we examined the crystal structure of the complex between the catalytic domain of FXIa and the Kunitz protease inhibitor (KPI) domain of a physiologically relevant FXIa inhibitor, protease nexin 2 (PN2). Six FXIa catalytic domain residues (Glu(98), Tyr(143), Ile(151), Arg(3704), Lys(192), and Tyr(5901)) were subjected to mutational analysis to investigate the molecular interactions between FXIa and the small synthetic substrate (S-2366), the macromolecular substrate (factor IX (FIX)) and inhibitor PN2KPI. Analysis of all six Ala mutants demonstrated normal K(m) values for S-2366 hydrolysis, indicating normal substrate binding compared with plasma FXIa; however, all except E98A and K192A had impaired values of k(cat) for S-2366 hydrolysis. All six Ala mutants displayed deficient k(cat) values for FIX hydrolysis, and all were inhibited by PN2KPI with normal values of K(i) except for K192A, and Y5901A, which displayed increased values of K(i). The integrity of the S1 binding site residue, Asp(189), utilizing p-aminobenzamidine, was intact for all FXIa mutants. Thus, whereas all six residues are essential for catalysis of the macromolecular substrate (FIX), only four (Tyr(143), Ile(151), Arg(3704), and Tyr(5901)) are important for S-2366 hydrolysis; Glu(98) and Lys(192) are essential for FIX but not S-2366 hydrolysis; and Lys(192) and Tyr(5901) are required for both inhibitor and macromolecular substrate interactions.     

Mutagenesis of conserved residues within the active site of Escherichia coli alkaline phosphatase yields enzymes with increased kcat.

The likelihood for improvement in the catalytic properties of Escherichia coli alkaline phosphatase was examined using site-directed mutagenesis. Mutants were constructed by introducing sequence changes into nine preselected amino acid sites within 10 A of the catalytic residue serine 102. When highly conserved residues in the family of alkaline phosphatases were mutated, many of the resulting enzymes not only maintained activity, but also exhibited greatly improved kcat. Of approximately 170 mutant enzymes screened, 5% (eight mutants) exhibited significant increases in specific activity. In particular, a substitution by serine of a totally invariant Asp101 resulted in a 35-fold increase of specific activity over wild-type at pH 10.0. Up to 6-fold increases of the kcat/Km ratio were observed.     

Insight into the binding of the wild type and mutated alginate lyase (AlyVI) with its substrate: a computational and experimental study

The homology model of the wild type alginate lyase (AlyVI) marine bacterium Vibrio sp. protein, was built using the crystal structure of the Family 7 alginate lyase from Sphingomonas sp. A1. To rationalize the observed structure-affinity relationships of aliginate lyase alyVI with its (GGG) substrate, molecular docking, MD imulations and binding free energy calculations followed by site-directed mutagenesis and alyVI activity assays were carried out. Per-residue decomposition of the (GGG) binding energy revealed that the most important contributions were from polar and charged residues, such as Asn138, Arg143, Asn217, and Lys308, while van der Waals interactions were responsible for binding with the catalytic His200 and Tyr312 residues. The mutants H200A, K308A, Y312A, Y312F, and W165A were found to be inactive or almost inactive. However, the catalytic efficiency (k(cat)/K(m)) of the double mutant L224V/D226G increased by two-fold compared to the wild type enzyme. This first structural model with its substrate binding mode and the agreement with experimental results provide a suitable base for the future rational design of new mutated alyVI structures with improved catalytic activity.     

Site-directed mutagenesis of glutamate 317 of bovine alpha-1,3Galactosyltransferase and its effect on enzyme activity: implications for reaction mechanism

Bovine alpha1,3galactosyltransferase (alpha1,3GalT) transfers galactose from UDP-alpha-galactose to terminal beta-linked galactosyl residues with retention of configuration of the incoming galactose residue. The epitope synthesized has been shown to be critical for xenotransplantation. According to a proposed double-displacement reaction mechanism, glutamate-317 (E317) is thought to be the catalytic nucleophile. The proposed catalytic role of E317 involves an initial nucleophilic attack with inversion of configuration and formation of a covalent sugar-enzyme intermediate between E317 and galactose from the donor substrate, followed by a second nucleophilic attack performed by the acceptor substrate with a second inversion of configuration. To determine whether E317 of alpha1,3GalT is critical for enzyme activity, site-directed mutagenesis was used to substitute alanine, aspartic acid, cysteine and histidine for E317. If the proposed reaction mechanism for the alpha1,3GalT enzyme is correct, E317D and E317H would produce active enzymes since they can act as nucleophiles. The non-conservative mutation E317A and conservative mutation E317C are predicted to produce inactive or very low activity enzymes since the E317A mutant cannot engage in a nucleophilic attack, and the E317C mutant would trap the galactose residue. The results obtained demonstrate that E317D and E317H mutants retained activity, albeit significantly less than the wild-type enzyme. Additionally, both E317A and E317C mutant also retained enzyme activity, suggesting that E317 is not the catalytic nucleophile proposed in the double-displacement mechanism. Therefore, either a different amino acid may act as the catalytic nucleophile or the reaction must proceed by a different mechanism.     

The contribution of adjacent subunits to the active sites of D-3-phosphoglycerate dehydrogenase

D-3-Phosphoglycerate dehydrogenase (PGDH) from Escherichia coli is allosterically inhibited by L-serine, the end product of its metabolic pathway. Previous results have shown that inhibition by serine has a large effect on Vmax and only a small or negligible effect on Km. PGDH is thus classified as a V-type allosteric enzyme. In this study, the active site of PGDH has been studied by site-directed mutagenesis to assess the role of certain residues in substrate binding and catalysis. These consist of a group of cationic residues (Arg-240, Arg-60, Arg-62, Lys-39, and Lys-141') that potentially form an electrostatic environment for the binding of the negatively charged substrate, as well as the only tryptophan residue found in PGDH and which fits into a hydrophobic pocket immediately adjacent to the active site histidine residue. Interestingly, Trp-139' and Lys-141' are part of the polypeptide chain of the subunit that is adjacent to the active site. The results of mutating these residues show that Arg-240, Arg-60, Arg-62, and Lys-141' play distinct roles in the binding of the substrate to the active site. Mutants of Trp-139' show that this residue may play a role in stabilizing the catalytic center of the enzyme. Furthermore, these mutants appear to have a significant effect on the cooperativity of serine inhibition and suggest a possible role for Trp-139' in the cooperative interactions between subunits.     

Atomic-resolution crystal structure of the proteolytic domain of Archaeoglobus fulgidus lon reveals the conformational variability in the active sites of lon proteases

The atomic-resolution crystal structure of the proteolytic domain (P-domain, residues 415-621) of Archaeoglobus fulgidus B-type Lon protease (wtAfLonB) and the structures of several mutants have revealed significant differences in the conformation of the active-site residues when compared to other known Lon P-domains, despite the conservation of the overall fold. The catalytic Ser509 is facing the solvent and is distant from Lys552, the other member of the catalytic dyad. Instead, the adjacent Asp508 forms an ion pair with the catalytic lysine residue. Glu506, an analog of the putative third catalytic residue from a related Methanococcus jannaschii LonB, also faces the solvent and does not interact with the catalytic dyad. We have established that full-length wtAfLonB is proteolytically active in an ATP-dependent manner. The loss of enzymatic activity of the S509A mutant confirms the functional significance of this residue, while retention of considerable level of activity by the D508A and E506A mutants rules out their critical involvement in catalysis. In contrast to the full-length enzymes, all individually purified P-domains (wild-type and mutants) were inactive, and the mutations had no influence on the active-site structure. These findings raise the possibility that, although isolated proteolytic domains of both AfLonB and E.coli LonA are able to assemble into expected functional hexamers, the presence of the other domains, as well as substrate binding, may be needed to stabilize the productive conformation of their active sites. Thus, the observed conformational variability may reflect the differences in the stability of active-site structures for the proteolytic counterparts of single-chain Lon versus independently folded proteolytic subunits of two-chain AAA+ proteases.     

Mutation of a unique aspartate residue abolishes the catalytic activity but not substrate binding of the mouse N-methylpurine-DNA glycosylase (MPG)

N-Methylpurine-DNA glycosylase (MPG) initiates base excision repair in DNA by removing a variety of alkylated purine adducts. Although Asp was identified as the active site residue in various DNA glycosylases based on the crystal structure, Glu-125 in human MPG (Glu-145 in mouse MPG) was recently proposed to be the catalytic residue. Mutational analysis for all Asp residues in a truncated, fully active MPG protein showed that only Asp-152 (Asp-132 in the human protein), which is located near the active site, is essential for catalytic activity. However, the substrate binding was not affected in the inactive Glu-152, Asn-152, and Ala-152 mutants. Furthermore, mutation of Asp-152 did not significantly affect the intrinsic tryptophan fluorescence of the enzyme and the far UV CD spectra, although a small change in the near UV CD spectra of the mutants suggests localized conformational change in the aromatic residues. We propose that in addition to Glu-145 in mouse MPG, which functions as the activator of a water molecule for nucleophilic attack, Asp-152 plays an essential role either by donating a proton to the substrate base and, thus, facilitating its release or by stabilizing the steric configuration of the active site pocket.     

Mechanistic details of the actinobacterial lyase-catalyzed degradation reaction of 2-hydroxyisobutyryl-CoA

Actinobacterial 2-hydroxyacyl-CoA lyase reversibly catalyzes the thiamine diphosphate-dependent cleavage of 2-hydroxyisobutyryl-CoA to formyl-CoA and acetone. This enzyme has great potential for use in synthetic one-carbon assimilation pathways for sustainable production of chemicals, but lacks details of substrate binding and reaction mechanism for biochemical reengineering. We determined crystal structures of the tetrameric enzyme in the closed conformation with bound substrate, covalent postcleavage intermediate, and products, shedding light on active site architecture and substrate interactions. Together with molecular dynamics simulations of the covalent precleavage complex, the complete catalytic cycle is structurally portrayed, revealing a proton transfer from the substrate acyl Cβ hydroxyl to residue E493 that returns it subsequently to the postcleavage Cα-carbanion intermediate. Kinetic parameters obtained for mutants E493A, E493Q, and E493K confirm the catalytic role of E493 in the WT enzyme. However, the 10- and 50-fold reduction in lyase activity in the E493A and E493Q mutants, respectively, compared with WT suggests that water molecules may contribute to proton transfer. The putative catalytic glutamate is located on a short α-helix close to the active site. This structural feature appears to be conserved in related lyases, such as human 2-hydroxyacyl-CoA lyase 2. Interestingly, a unique feature of the actinobacterial 2-hydroxyacyl-CoA lyase is a large C-terminal lid domain that, together with active site residues L127 and I492, restricts substrate size to ≤C5 2-hydroxyacyl residues. These details about the catalytic mechanism and determinants of substrate specificity pave the ground for designing tailored catalysts for acyloin condensations for one-carbon and short-chain substrates in biotechnological applications.     

Dissecting substrate specificity of two rice BADH isoforms: Enzyme kinetics, docking and molecular dynamics simulation studies

Fragrance rice (Oryza sativa) contains two isoforms of BADH, named OsBADH1 and OsBADH2. OsBADH1 is implicated in acetaldehyde oxidation in rice plant peroxisomes, while the non-functional OsBADH2 is believed to be involved in the accumulation of 2-acetyl-1-pyrroline, the major compound of aroma in fragrance rice. In the present study, site-directed mutagenesis, molecular docking and molecular dynamics simulation studies were used to investigate the substrate specificity towards Bet-ald and GAB-ald. Consistent with our previous study, kinetics data indicated that the enzymes catalyze the oxidation of GAB-ald more efficiently than Bet-ald and the OsBADH1 W172F and OsBADH2 W170F mutants displayed a higher catalytic efficiency towards GAB-ald. Molecular docking analysis and molecular dynamics simulations for the first time provided models for aldehyde substrate-bound complexes of OsBADHs. The amino acid residues, E262, L263, C296 and W461 of OsBADH1 and E260, L261, C294 and W459 of OsBADH2 located within 5 Å of the OsBADH active site mainly interacted with GAB-ald forming strong hydrogen bonds in both OsBADH isoforms. Residues W163, N164, Q294, C296 and F397 of OsBADH1–Bet-ald and Y163, M167, W170, E260, S295 and C453 of OsBADH2–Bet-ald formed the main interaction sites while E260 showed an interaction energy of −14.21 kcal/mol. Unconserved A290 in OsBADH1 and W288 in OsBADH2 appeared to be important for substrate recognition similar to that observed in PsAMADHs. Overall, the results here help to explain how two homologous rice BADHs recognize the aldehyde substrate differently, a key property to their biological role.