Reaction mechanism of the farnesyl pyrophosphate C-methyltransferase towards the biosynthesis of pre-sodorifen pyrophosphate by Serratia plymuthica 4Rx13

AbstractClassical terpenoid biosynthesis involves the cyclization of the linear prenyl pyrophosphate precursors geranyl-, farnesyl-, or geranylgeranyl pyrophosphate (GPP, FPP, GGPP) and their isomers, to produce a huge number of natural compounds. Recently, it was shown for the first time that the biosynthesis of the unique homo-sesquiterpene sodorifen by Serratia plymuthica 4Rx13 involves a methylated and cyclized intermediate as the substrate of the sodorifen synthase. To further support the proposed biosynthetic pathway, we now identified the cyclic prenyl pyrophosphate intermediate pre-sodorifen pyrophosphate (PSPP). Its absolute configuration (6R,7S,9S) was determined by comparison of calculated and experimental CD-spectra of its hydrolysis product and matches with those predicted by semi-empirical quantum calculations of the reaction mechanism. In silico modeling of the reaction mechanism of the FPP C-methyltransferase (FPPMT) revealed a SN2 mechanism for the methyl transfer followed by a cyclization cascade. The cyclization of FPP to PSPP is guided by a catalytic dyad of H191 and Y39 and involves an unprecedented cyclopropyl intermediate. W46, W306, F56, and L239 form the hydrophobic binding pocket and E42 and H45 complex a magnesium cation that interacts with the diphosphate moiety of FPP. Six additional amino acids turned out to be essential for product formation and the importance of these amino acids was subsequently confirmed by site-directed mutagenesis. Our results reveal the reaction mechanism involved in methyltransferase-catalyzed cyclization and demonstrate that this coupling of C-methylation and cyclization of FPP by the FPPMT represents an alternative route of terpene biosynthesis that could increase the terpenoid diversity and structural space.

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Site-directed mutagenesis at aspartate and glutamate residues of xylanase from Bacillus pumilus

Biochemical Journal ◽

10.1042/bj2880117 ◽

1992 ◽

Vol 288 (1) ◽

pp. 117-121 ◽

Cited By ~ 48

Author(s):

E P Ko ◽

H Akatsuka ◽

H Moriyama ◽

A Shinmyo ◽

Y Hata ◽

...

Keyword(s):

Amino Acids ◽

Reaction Mechanism ◽

Amino Acid ◽

Bacillus Pumilus ◽

Specific Activity ◽

Sequence Similarity ◽

Site Directed Mutagenesis ◽

Dimensional Structure ◽

Amino Acid Sequence Similarity ◽

Directed Mutagenesis

To elucidate the reaction mechanism of xylanase, the identification of amino acids essential for its catalysis is of importance. Studies have indicated the possibility that the reaction mechanism of xylanase is similar to that of hen's egg lysozyme, which involves acidic amino acid residues. On the basis of this assumption, together with the three-dimensional structure of Bacillus pumilus xylanase and its amino acid sequence similarity to other xylanases of different origins, three acidic amino acids, namely Asp-21, Glu-93 and Glu-182, were selected for site-directed mutagenesis. The Asp residue was altered to either Ser or Glu, and the Glu residues to Ser or Asp. The purified mutant xylanases D21E, D21S, E93D, E93S, E182D and E182S showed single protein bands of about 26 kDa on SDS/PAGE. C.d. spectra of these mutant enzymes show no effect on the secondary structure of xylanase, except that of D21E, which shows a little variation. Furthermore, mutations of Glu-93 and Glu-182 resulted in a drastic decrease in the specific activity of xylanase as compared with mutation of Asp-21. On the basis of these results we propose that Glu-93 and Glu-182 are the best candidates for the essential catalytic residues of xylanase.

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Probing the Molecular Determinant for the Catalytic Efficiency of l-Arabinose Isomerase from Bacillus licheniformis

Applied and Environmental Microbiology ◽

10.1128/aem.02254-09 ◽

2010 ◽

Vol 76 (5) ◽

pp. 1653-1660 ◽

Cited By ~ 23

Author(s):

Ponnandy Prabhu ◽

Marimuthu Jeya ◽

Jung-Kul Lee

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Bacillus Licheniformis ◽

Catalytic Efficiency ◽

Homology Model ◽

Binding Pocket ◽

Site Directed Mutagenesis ◽

Dynamics Simulation ◽

High Turnover ◽

Arabinose Isomerase

ABSTRACT Bacillus licheniformis l-arabinose isomerase (l-AI) is distinguished from other l-AIs by its high degree of substrate specificity for l-arabinose and its high turnover rate. A systematic strategy that included a sequence alignment-based first screening of residues and a homology model-based second screening, followed by site-directed mutagenesis to alter individual screened residues, was used to study the molecular determinants for the catalytic efficiency of B. licheniformis l-AI. One conserved amino acid, Y333, in the substrate binding pocket of the wild-type B. licheniformis l-AI was identified as an important residue affecting the catalytic efficiency of B. licheniformis l-AI. Further insights into the function of residue Y333 were obtained by replacing it with other aromatic, nonpolar hydrophobic amino acids or polar amino acids. Replacing Y333 with the aromatic amino acid Phe did not alter catalytic efficiency toward l-arabinose. In contrast, the activities of mutants containing a hydrophobic amino acid (Ala, Val, or Leu) at position 333 decreased as the size of the hydrophobic side chain of the amino acid decreased. However, mutants containing hydrophilic and charged amino acids, such as Asp, Glu, and Lys, showed almost no activity with l-arabinose. These data and a molecular dynamics simulation suggest that Y333 is involved in the catalytic efficiency of B. licheniformis l-AI.

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Molecular sampling of the allosteric binding pocket of the TSH receptor provides discriminative pharmacophores for antagonist and agonists

Biochemical Society Transactions ◽

10.1042/bst20120319 ◽

2013 ◽

Vol 41 (1) ◽

pp. 213-217 ◽

Cited By ~ 16

Author(s):

Inna Hoyer ◽

Ann-Karin Haas ◽

Annika Kreuchwig ◽

Ralf Schülein ◽

Gerd Krause

Keyword(s):

Amino Acids ◽

Structural Model ◽

Transmembrane Domain ◽

Binding Pocket ◽

Trigger Points ◽

Antagonistic Effect ◽

Site Directed Mutagenesis ◽

Detailed Knowledge ◽

Contact Points ◽

Allosteric Binding Pocket

The TSHR (thyrotropin receptor) is activated endogenously by the large hormone thyrotropin and activated pathologically by auto-antibodies. Both activate and bind at the extracellular domain. Recently, SMLs (small-molecule ligands) have been identified, which bind in an allosteric binding pocket within the transmembrane domain. Modelling driven site-directed mutagenesis of amino acids lining this pocket led to the delineation of activation and inactivation sensitive residues. Modified residues showing CAMs (constitutively activating mutations) indicate signalling-sensitive positions and mark potential trigger points for agonists. Silencing mutations lead to an impairment of basal activity and mark contact points for antagonists. Mapping these residues on to a structural model of TSHR indicates locations where an SML may switch the receptor to an inactive or active conformation. In the present article, we report the effects of SMLs on these signalling-sensitive amino acids at the TSHR. Surprisingly, the antagonistic effect of SML compound 52 was reversed to an agonistic effect, when tested at the CAM Y667A. Switching agonism to antagonism and the reverse by changing either SMLs or residues covering the binding pocket provides detailed knowledge about discriminative pharmacophores. It prepares the basis for rational optimization of new high-affinity antagonists to interfere with the pathogenic activation of the TSHR.

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Molecular Determinants of the Cofactor Specificity of Ribitol Dehydrogenase, a Short-Chain Dehydrogenase/Reductase

Applied and Environmental Microbiology ◽

10.1128/aem.07751-11 ◽

2012 ◽

Vol 78 (9) ◽

pp. 3079-3086 ◽

Cited By ~ 17

Author(s):

Hee-Jung Moon ◽

Manish Kumar Tiwari ◽

Ranjitha Singh ◽

Yun Chan Kang ◽

Jung-Kul Lee

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Binding Pocket ◽

Site Directed Mutagenesis ◽

Titration Calorimetry ◽

Content Type ◽

Cofactor Specificity ◽

Charged Amino Acids ◽

Molecular Determinants ◽

Ribitol Dehydrogenase

ABSTRACTRibitol dehydrogenase fromZymomonas mobilis(ZmRDH) catalyzes the conversion of ribitol tod-ribulose and concomitantly reduces NAD(P)+to NAD(P)H. A systematic approach involving an initial sequence alignment-based residue screening, followed by a homology model-based screening and site-directed mutagenesis of the screened residues, was used to study the molecular determinants of the cofactor specificity of ZmRDH. A homologous conserved amino acid, Ser156, in the substrate-binding pocket of the wild-type ZmRDH was identified as an important residue affecting the cofactor specificity of ZmRDH. Further insights into the function of the Ser156 residue were obtained by substituting it with other hydrophobic nonpolar or polar amino acids. Substituting Ser156 with the negatively charged amino acids (Asp and Glu) altered the cofactor specificity of ZmRDH toward NAD+(S156D, [kcat/Km,NAD]/[kcat/Km,NADP] = 10.9, whereKm,NADis theKmfor NAD+andKm,NADPis theKmfor NADP+). In contrast, the mutants containing positively charged amino acids (His, Lys, or Arg) at position 156 showed a higher efficiency with NADP+as the cofactor (S156H, [kcat/Km,NAD]/[kcat/Km,NADP] = 0.11). These data, in addition to those of molecular dynamics and isothermal titration calorimetry studies, suggest that the cofactor specificity of ZmRDH can be modulated by manipulating the amino acid residue at position 156.

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Mutagenesis of the l-Amino Acid Ligase RizA Increased the Production of Bioactive Dipeptides

Catalysts ◽

10.3390/catal11111385 ◽

2021 ◽

Vol 11 (11) ◽

pp. 1385

Author(s):

Sven Bordewick ◽

Ralf G. Berger ◽

Franziska Ersoy

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Aspartic Acid ◽

Product Yield ◽

Binding Pocket ◽

Site Directed Mutagenesis ◽

Product Concentration ◽

Substrate Binding Pocket ◽

E Coli ◽

Set Up

The l-amino acid ligase RizA from B. subtilis selectively synthesizes dipeptides containing an N-terminal arginine. Many arginyl dipeptides have salt-taste enhancing properties while Arg-Phe has been found to have an antihypertensive effect. A total of 21 RizA variants were created by site-directed mutagenesis of eight amino acids in the substrate binding pocket. The variants were recombinantly produced in E. coli and purified by affinity chromatography. Biocatalytic reactions were set up with arginine and four amino acids differing in size and polarity (aspartic acid, serine, alanine, and phenylalanine) and were analyzed by RP-HPLC with fluorescence detection. Variant T81F significantly improved the yield in comparison to wild type RizA for aspartic acid (7 to 17%), serine (33 to 47%) and alanine (12 to 17%). S84F increased product yield similarly for aspartic acid (7 to 17%) and serine (33 to 42%). D376E increased the yield with alanine (12 to 19%) and phenylalanine (11 to 26%). The largest change was observed for S156A, which showed a yield for Arg-Phe of 40% corresponding to a 270% increase in product concentration. This study expands the knowledge about positions governing the substrate specificity of RizA and may help to inform future protein engineering endeavors.

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Substrate Specificity of Naphthalene Dioxygenase: Effect of Specific Amino Acids at the Active Site of the Enzyme

Journal of Bacteriology ◽

10.1128/jb.182.6.1641-1649.2000 ◽

2000 ◽

Vol 182 (6) ◽

pp. 1641-1649 ◽

Cited By ~ 132

Author(s):

Rebecca E. Parales ◽

Kyoung Lee ◽

Sol M. Resnick ◽

Haiyan Jiang ◽

Daniel J. Lessner ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Active Site ◽

Product Formation ◽

Site Directed Mutagenesis ◽

Dimensional Structure ◽

Amino Acid Substitutions ◽

Active Site Residues ◽

Naphthalene Dioxygenase ◽

Wide Range

ABSTRACT The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene byPseudomonas sp. strain NCIB 9816-4. The three-dimensional structure of NDO revealed that several of the amino acids at the active site of the oxygenase are hydrophobic, which is consistent with the enzyme's preference for aromatic hydrocarbon substrates. Although NDO catalyzes cis-dihydroxylation of a wide range of substrates, it is highly regio- and enantioselective. Site-directed mutagenesis was used to determine the contributions of several active-site residues to these aspects of catalysis. Amino acid substitutions at Asn-201, Phe-202, Val-260, Trp-316, Thr-351, Trp-358, and Met-366 had little or no effect on product formation with naphthalene or biphenyl as substrates and had slight but significant effects on product formation from phenanthrene. Amino acid substitutions at Phe-352 resulted in the formation ofcis-naphthalene dihydrodiol with altered stereochemistry [92 to 96% (+)-1R,2S], compared to the enantiomerically pure [>99% (+)-1R,2S] product formed by the wild-type enzyme. Substitutions at position 352 changed the site of oxidation of biphenyl and phenanthrene. Substitution of alanine for Asp-362, a ligand to the active-site iron, resulted in a completely inactive enzyme.

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Characterization of Recombinant Human Coagulation Factor XFriuli

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650267 ◽

1996 ◽

Vol 75 (02) ◽

pp. 313-317 ◽

Cited By ~ 8

Author(s):

D J Kim ◽

A Girolami ◽

H L James

Keyword(s):

Catalytic Domain ◽

Coagulation Factor ◽

Molecular Size ◽

Binding Pocket ◽

Site Directed Mutagenesis ◽

Bleeding Tendency ◽

Factor X ◽

Amino Terminal ◽

Normal Plasma ◽

Plasma Factor

SummaryNaturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational y-carboxy-glutamic acid (Gla) and (β-hydroxy aspartic acid (β-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel (3-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.

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