Mortensen UH

References (17)

Title : Acurin A, a novel hybrid compound, biosynthesized by individually translated PKS- and NRPS-encoding genes in Aspergillus aculeatus - Wolff_2020_Fungal.Genet.Biol_139_103378
Author(s) : Wolff PB , Nielsen ML , Slot JC , Andersen LN , Petersen LM , Isbrandt T , Holm DK , Mortensen UH , Nodvig CS , Larsen TO , Hoof JB
Ref : Fungal Genet Biol , 139 :103378 , 2020
Abstract : This work presents the identification and proposed biosynthetic pathway for a compound of mixed polyketide-nonribosomal peptide origin that we named acurin A. The compound was isolated from an extract of the filamentous fungus Aspergillus aculeatus, and its core structure resemble that of the mycotoxin fusarin C produced by several Fusarium species. Based on bioinformatics in combination with RT-qPCR experiments and gene-deletion analysis, we identified a biosynthetic gene cluster (BGC) in A. aculeatus responsible for the biosynthesis of acurin A. Moreover, we were able to show that a polyketide synthase (PKS) and a nonribosomal peptide synthetase (NRPS) enzyme separately encoded by this BGC are responsible for the synthesis of the PK-NRP compound, acurin A, core structure. In comparison, the production of fusarin C is reported to be facilitated by a linked PKS-NRPS hybrid enzyme. Phylogenetic analyses suggest the PKS and NRPS in A. aculeatus resulted from a recent fission of an ancestral hybrid enzyme followed by gene duplication. In addition to the PKS- and NRPS-encoding genes of acurin A, we show that six other genes are influencing the biosynthesis including a regulatory transcription factor. Altogether, we have demonstrated the involvement of eight genes in the biosynthesis of acurin A, including an in-cluster transcription factor. This study highlights the biosynthetic capacity of A. aculeatus and serves as an example of how the CRISPR/Cas9 system can be exploited for the construction of fungal strains that can be readily engineered.
ESTHER : Wolff_2020_Fungal.Genet.Biol_139_103378
PubMedSearch : Wolff_2020_Fungal.Genet.Biol_139_103378
PubMedID: 32234543
Gene_locus related to this paper: aspa1-acrc

Title : Novofumigatonin biosynthesis involves a non-heme iron-dependent endoperoxide isomerase for orthoester formation - Matsuda_2018_Nat.Commun_9_2587
Author(s) : Matsuda Y , Bai T , Phippen CBW , Nodvig CS , Kjaerbolling I , Vesth TC , Andersen MR , Mortensen UH , Gotfredsen CH , Abe I , Larsen TO
Ref : Nat Commun , 9 :2587 , 2018
Abstract : Novofumigatonin (1), isolated from the fungus Aspergillus novofumigatus, is a heavily oxygenated meroterpenoid containing a unique orthoester moiety. Despite the wide distribution of orthoesters in nature and their biological importance, little is known about the biogenesis of orthoesters. Here we show the elucidation of the biosynthetic pathway of 1 and the identification of key enzymes for the orthoester formation by a series of CRISPR-Cas9-based gene-deletion experiments and in vivo and in vitro reconstitutions of the biosynthesis. The novofumigatonin pathway involves endoperoxy compounds as key precursors for the orthoester synthesis, in which the Fe(II)/alpha-ketoglutarate-dependent enzyme NvfI performs the endoperoxidation. NvfE, the enzyme catalyzing the orthoester synthesis, is an Fe(II)-dependent, but cosubstrate-free, endoperoxide isomerase, despite the fact that NvfE shares sequence homology with the known Fe(II)/alpha-ketoglutarate-dependent dioxygenases. NvfE thus belongs to a class of enzymes that gained an isomerase activity by losing the alpha-ketoglutarate-binding ability.
ESTHER : Matsuda_2018_Nat.Commun_9_2587
PubMedSearch : Matsuda_2018_Nat.Commun_9_2587
PubMedID: 29968715
Gene_locus related to this paper: aspn1-nvfd

Title : Linking secondary metabolites to gene clusters through genome sequencing of six diverse Aspergillus species - Kjaerbolling_2018_Proc.Natl.Acad.Sci.U.S.A_115_E753
Author(s) : Kjaerbolling I , Vesth TC , Frisvad JC , Nybo JL , Theobald S , Kuo A , Bowyer P , Matsuda Y , Mondo S , Lyhne EK , Kogle ME , Clum A , Lipzen A , Salamov A , Ngan CY , Daum C , Chiniquy J , Barry K , LaButti K , Haridas S , Simmons BA , Magnuson JK , Mortensen UH , Larsen TO , Grigoriev IV , Baker SE , Andersen MR
Ref : Proc Natl Acad Sci U S A , 115 :E753 , 2018
Abstract : The fungal genus of Aspergillus is highly interesting, containing everything from industrial cell factories, model organisms, and human pathogens. In particular, this group has a prolific production of bioactive secondary metabolites (SMs). In this work, four diverse Aspergillus species (A. campestris, A. novofumigatus, A. ochraceoroseus, and A. steynii) have been whole-genome PacBio sequenced to provide genetic references in three Aspergillus sections. A. taichungensis and A. candidus also were sequenced for SM elucidation. Thirteen Aspergillus genomes were analyzed with comparative genomics to determine phylogeny and genetic diversity, showing that each presented genome contains 15-27% genes not found in other sequenced Aspergilli. In particular, A. novofumigatus was compared with the pathogenic species A. fumigatus This suggests that A. novofumigatus can produce most of the same allergens, virulence, and pathogenicity factors as A. fumigatus, suggesting that A. novofumigatus could be as pathogenic as A. fumigatus Furthermore, SMs were linked to gene clusters based on biological and chemical knowledge and analysis, genome sequences, and predictive algorithms. We thus identify putative SM clusters for aflatoxin, chlorflavonin, and ochrindol in A. ochraceoroseus, A. campestris, and A. steynii, respectively, and novofumigatonin, ent-cycloechinulin, and epi-aszonalenins in A. novofumigatus Our study delivers six fungal genomes, showing the large diversity found in the Aspergillus genus; highlights the potential for discovery of beneficial or harmful SMs; and supports reports of A. novofumigatus pathogenicity. It also shows how biological, biochemical, and genomic information can be combined to identify genes involved in the biosynthesis of specific SMs.
ESTHER : Kjaerbolling_2018_Proc.Natl.Acad.Sci.U.S.A_115_E753
PubMedSearch : Kjaerbolling_2018_Proc.Natl.Acad.Sci.U.S.A_115_E753
PubMedID: 29317534
Gene_locus related to this paper: 9euro-a0a0f8xhh7 , 9euro-a0a2t5ll04 , aspn1-nvfd

Title : Investigation of a 6-MSA Synthase Gene Cluster in Aspergillus aculeatus Reveals 6-MSA-derived Aculinic Acid, Aculins A-B and Epi-Aculin A - Petersen_2015_Chembiochem_16_2200
Author(s) : Petersen LM , Holm DK , Gotfredsen CH , Mortensen UH , Larsen TO
Ref : Chembiochem , 16 :2200 , 2015
Abstract : Aspergillus aculeatus, a filamentous fungus belonging to the Aspergillus clade Nigri, is an industrial workhorse in enzyme production. Recently we reported a number of secondary metabolites from this fungus; however, its genetic potential for the production of secondary metabolites is vast. In this study we identified a 6-methylsalicylic acid (6-MSA) synthase from A. aculeatus, and verified its functionality by episomal expression in A. aculeatus and heterologous expression in A. nidulans. Feeding studies with fully (13) C-labeled 6-MSA revealed that 6-MSA is incorporated into aculinic acid, which further incorporates into three compounds that we name aculins A and B, and epi-aculin A, described here for the first time. Based on NMR data and bioinformatic studies we propose the structures of the compounds as well as a biosynthetic pathway leading to formation of aculins from 6-MSA.
ESTHER : Petersen_2015_Chembiochem_16_2200
PubMedSearch : Petersen_2015_Chembiochem_16_2200
PubMedID: 26374386
Gene_locus related to this paper: aspa1-acui

Title : Reconstruction of the biosynthetic pathway for the core fungal polyketide scaffold rubrofusarin in Saccharomyces cerevisiae - Rugbjerg_2013_Microb.Cell.Fact_12_31
Author(s) : Rugbjerg P , Naesby M , Mortensen UH , Frandsen RJ
Ref : Microb Cell Fact , 12 :31 , 2013
Abstract : BACKGROUND: Fungal polyketides include commercially important pharmaceuticals and food additives, e.g. the cholesterol-lowering statins and the red and orange monascus pigments. Presently, production relies on isolation of the compounds from the natural producers, and systems for heterologous production in easily fermentable and genetically engineerable organisms, such as Saccharomyces cerevisiae and Escherichia coli are desirable. Rubrofusarin is an orange polyketide pigment that is a common intermediate in many different fungal biosynthetic pathways. RESULTS: In this study, we established a biosynthetic pathway for rubrofusarin in S. cerevisiae. First, the Fusarium graminearum gene encoding polyketide synthase 12 (PKS12) was heterologously co-expressed with the Aspergillus fumigatus gene encoding phosphopantetheinyl transferase (npgA) resulting in production of YWA1. This aromatic heptaketide intermediate was converted into nor-rubrofusarin upon expression of the dehydratase gene aurZ from the aurofusarin gene cluster of F. graminearum. Final conversion into rubrofusarin was achieved by expression of the O-methyltransferase encoding gene aurJ, also obtained from the aurofusarin gene cluster, resulting in a titer of 1.1 mg/L. Reduced levels of rubrofusarin were detected when expressing PKS12, npgA, and aurJ alone, presumably due to spontaneous conversion of YWA1 to nor-rubrofusarin. However, the co-expression of aurZ resulted in an approx. six-fold increase in rubrofusarin production. CONCLUSIONS: The reconstructed pathway for rubrofusarin in S. cerevisiae allows the production of a core scaffold molecule with a branch-point role in several fungal polyketide pathways, thus paving the way for production of further natural pigments and bioactive molecules. Furthermore, the reconstruction verifies the suggested pathway, and as such, it is the first example of utilizing a synthetic biological "bottom up" approach for the validation of a complex fungal polyketide pathway.
ESTHER : Rugbjerg_2013_Microb.Cell.Fact_12_31
PubMedSearch : Rugbjerg_2013_Microb.Cell.Fact_12_31
PubMedID: 23557488
Gene_locus related to this paper: gibze-q66sy0

Title : Involvement of a natural fusion of a cytochrome P450 and a hydrolase in mycophenolic acid biosynthesis - Hansen_2012_Appl.Environ.Microbiol_78_4908
Author(s) : Hansen BG , Mnich E , Nielsen KF , Nielsen JB , Nielsen MT , Mortensen UH , Larsen TO , Patil KR
Ref : Applied Environmental Microbiology , 78 :4908 , 2012
Abstract : Mycophenolic acid (MPA) is a fungal secondary metabolite and the active component in several immunosuppressive pharmaceuticals. The gene cluster coding for the MPA biosynthetic pathway has recently been discovered in Penicillium brevicompactum, demonstrating that the first step is catalyzed by MpaC, a polyketide synthase producing 5-methylorsellinic acid (5-MOA). However, the biochemical role of the enzymes encoded by the remaining genes in the MPA gene cluster is still unknown. Based on bioinformatic analysis of the MPA gene cluster, we hypothesized that the step following 5-MOA production in the pathway is carried out by a natural fusion enzyme MpaDE, consisting of a cytochrome P450 (MpaD) in the N-terminal region and a hydrolase (MpaE) in the C-terminal region. We verified that the fusion gene is indeed expressed in P. brevicompactum by obtaining full-length sequence of the mpaDE cDNA prepared from the extracted RNA. Heterologous coexpression of mpaC and the fusion gene mpaDE in the MPA-nonproducer Aspergillus nidulans resulted in the production of 5,7-dihydroxy-4-methylphthalide (DHMP), the second intermediate in MPA biosynthesis. Analysis of the strain coexpressing mpaC and the mpaD part of mpaDE shows that the P450 catalyzes hydroxylation of 5-MOA to 4,6-dihydroxy-2-(hydroxymethyl)-3-methylbenzoic acid (DHMB). DHMB is then converted to DHMP, and our results suggest that the hydrolase domain aids this second step by acting as a lactone synthase that catalyzes the ring closure. Overall, the chimeric enzyme MpaDE provides insight into the genetic organization of the MPA biosynthesis pathway.
ESTHER : Hansen_2012_Appl.Environ.Microbiol_78_4908
PubMedSearch : Hansen_2012_Appl.Environ.Microbiol_78_4908
PubMedID: 22544261
Gene_locus related to this paper: penbr-mpac

Title : Molecular basis for mycophenolic acid biosynthesis in Penicillium brevicompactum - Regueira_2011_Appl.Environ.Microbiol_77_3035
Author(s) : Regueira TB , Kildegaard KR , Hansen BG , Mortensen UH , Hertweck C , Nielsen J
Ref : Applied Environmental Microbiology , 77 :3035 , 2011
Abstract : Mycophenolic acid (MPA) is the active ingredient in the increasingly important immunosuppressive pharmaceuticals CellCept (Roche) and Myfortic (Novartis). Despite the long history of MPA, the molecular basis for its biosynthesis has remained enigmatic. Here we report the discovery of a polyketide synthase (PKS), MpaC, which we successfully characterized and identified as responsible for MPA production in Penicillium brevicompactum. mpaC resides in what most likely is a 25-kb gene cluster in the genome of Penicillium brevicompactum. The gene cluster was successfully localized by targeting putative resistance genes, in this case an additional copy of the gene encoding IMP dehydrogenase (IMPDH). We report the cloning, sequencing, and the functional characterization of the MPA biosynthesis gene cluster by deletion of the polyketide synthase gene mpaC of P. brevicompactum and bioinformatic analyses. As expected, the gene deletion completely abolished MPA production as well as production of several other metabolites derived from the MPA biosynthesis pathway of P. brevicompactum. Our work sets the stage for engineering the production of MPA and analogues through metabolic engineering.
ESTHER : Regueira_2011_Appl.Environ.Microbiol_77_3035
PubMedSearch : Regueira_2011_Appl.Environ.Microbiol_77_3035
PubMedID: 21398490
Gene_locus related to this paper: penbr-mpac

Title : Versatile enzyme expression and characterization system for Aspergillus nidulans, with the Penicillium brevicompactum polyketide synthase gene from the mycophenolic acid gene cluster as a test case - Hansen_2011_Appl.Environ.Microbiol_77_3044
Author(s) : Hansen BG , Salomonsen B , Nielsen MT , Nielsen JB , Hansen NB , Nielsen KF , Regueira TB , Nielsen J , Patil KR , Mortensen UH
Ref : Applied Environmental Microbiology , 77 :3044 , 2011
Abstract : Assigning functions to newly discovered genes constitutes one of the major challenges en route to fully exploiting the data becoming available from the genome sequencing initiatives. Heterologous expression in an appropriate host is central in functional genomics studies. In this context, filamentous fungi offer many advantages over bacterial and yeast systems. To facilitate the use of filamentous fungi in functional genomics, we present a versatile cloning system that allows a gene of interest to be expressed from a defined genomic location of Aspergillus nidulans. By a single USER cloning step, genes are easily inserted into a combined targeting-expression cassette ready for rapid integration and analysis. The system comprises a vector set that allows genes to be expressed either from the constitutive PgpdA promoter or from the inducible PalcA promoter. Moreover, by using the vector set, protein variants can easily be made and expressed from the same locus, which is mandatory for proper comparative analyses. Lastly, all individual elements of the vectors can easily be substituted for other similar elements, ensuring the flexibility of the system. We have demonstrated the potential of the system by transferring the 7,745-bp large mpaC gene from Penicillium brevicompactum to A. nidulans. In parallel, we produced defined mutant derivatives of mpaC, and the combined analysis of A. nidulans strains expressing mpaC or mutated mpaC genes unequivocally demonstrated that mpaC indeed encodes a polyketide synthase that produces the first intermediate in the production of the medically important immunosuppressant mycophenolic acid.
ESTHER : Hansen_2011_Appl.Environ.Microbiol_77_3044
PubMedSearch : Hansen_2011_Appl.Environ.Microbiol_77_3044
PubMedID: 21398493
Gene_locus related to this paper: penbr-mpac

Title : Reversed-flow affinity elution applied to the purification of carboxypeptidase Y - Mortensen_1998_Anal.Biochem_258_236
Author(s) : Mortensen UH , Stennicke HR , Breddam K
Ref : Analytical Biochemistry , 258 :236 , 1998
Abstract : In the present study we describe a novel method for obtaining highly pure carboxypeptidase Y, or derivatives thereof, in a single-step purification procedure. The method is based on affinity chromatography and the results demonstrate that an efficient method is obtained only when the affinity gel is fully saturated with enzyme. Thus, pilot experiments are required to determine the binding capacity of the resin with respect to a given enzyme. To avoid this additional experimental effort, we have developed a method utilizing reversed-flow affinity elution. The method has been successfully employed to purify hundreds of carboxypeptidase Y mutant enzymes.
ESTHER : Mortensen_1998_Anal.Biochem_258_236
PubMedSearch : Mortensen_1998_Anal.Biochem_258_236
PubMedID: 9570835
Gene_locus related to this paper: yeast-cbpy1

Title : Studies on the hydrolytic properties of (serine) carboxypeptidase Y - Stennicke_1996_Biochemistry_35_7131
Author(s) : Stennicke HR , Mortensen UH , Breddam K
Ref : Biochemistry , 35 :7131 , 1996
Abstract : The activity of serine carboxypeptidases is dependent on a catalytic triad, an oxyanion hole, and a binding site equivalent to those found in the serine endopeptidases. The action of carboxypeptidase Y on substrates containing amino acids, alcohols, and amines as leaving groups is described. It is demonstrated that the features common to serine endopeptidases and carboxypeptidases are sufficient for hydrolysis of ester bonds. However, rapid hydrolysis of amide bonds is dependent on interactions between the C-terminal carboxylate group of the substrate and the C-terminal recognition site of the enzyme. Furthermore, on the basis of the pH dependencies of wild-type and mutant enzyme, combined with the ability of the enzyme to utilize binding energy to promote catalysis, alternative models for the high activity of carboxypeptidase Y at low pH are discussed. They describe how the catalytically essential histidine is maintained in its active deprotonated state through perturbation of its pKa value in the enzyme-substrate complex.
ESTHER : Stennicke_1996_Biochemistry_35_7131
PubMedSearch : Stennicke_1996_Biochemistry_35_7131
PubMedID: 8679540

Title : Site-Directed Mutagenesis on (Serine) Carboxypeptidase Y from Yeast. The Significance of Thr60 and Met398 in Hydrolysis and Aminolysis Reactions - Sorensen_1995_J.Am.Chem.Soc_117_5944
Author(s) : Sorensen SB , Raaschou-Nielsen M , Mortensen UH , Remington SJ , Breddam K
Ref : Journal of the American Chemical Society , 117 :5944 , 1995
Abstract : In (serine)carboxypeptidase Y, the flexible side chain of Met 398 forms one side of the Si' binding pocket and the beta -and gamma-carbon atoms of Thr60 form the opposite side. Met398 has been substituted with the residues Gly,Ala,Val,lie,Leu,Phe,and Tyr while Thr60 has been substituted with the residues Ala,Val,Leu,Met,Phe,and Tyr by site-directed mutagenesis,and the resulting enzymes have been characterized with respect to their Pi' substrate preferences using thes ubstrate series FA-Phe-Xaa-OH (Xaa=Gly,Ala,Val,orLeu) and FA-Ala-Yaa-OH (Yaa=Leu,Gin,Glu,Lys,or Arg). The results show that Met398 is much more important for transition state stabilization than Thr60 although itappears that the selected non bulky amino acid residue(Thr) at position 60 is important for high Kcat values. The results further suggest that bulky amino acid side chains at position 398 are able to adjust the size of the Si' pocket such that favorable interactions with the substrate can be obtained with even small Pi' side chains,e.g., Gly. Accordingly,the hydrolysis of substrates with bulky/hydrophobic Pi' side chains is less dependent on the nature of the amino acid residue at position 398 than that of a substrate with a non bulky Pi' sid echain.The three-dimensional structure oft hemutant enzymeE65A+E145A has been determined, and it provides support for the high mobility of the Met398 side chain. In transpeptidation reactions the substitutions at position 398 also influence the interactions between the binding pocket and the amino acid leaving group as well as the added nucleophile competing with water in the deacylation reaction.Much higher aminolysis was obtained with some of the mutant enzymes, presumably due to a changed accessibility of water to the acyl-enzyme intermediate while the nucleophile/leaving group isbound at the Si' binding site.
ESTHER : Sorensen_1995_J.Am.Chem.Soc_117_5944
PubMedSearch : Sorensen_1995_J.Am.Chem.Soc_117_5944
PubMedID:
Gene_locus related to this paper: yeast-cbpy1

Title : Effects of introduced aspartic and glutamic acid residues on the P'1 substrate specificity, pH dependence and stability of carboxypeptidase Y - Stennicke_1994_Protein.Eng_7_911
Author(s) : Stennicke HR , Mortensen UH , Christensen U , Remington SJ , Breddam K
Ref : Protein Engineering , 7 :911 , 1994
Abstract : Carboxypeptidase Y is a serine carboxypeptidase isolated from Saccharomyces cerevisiae with a preference for C-terminal hydrophobic amino acid residues. In order to alter the inherent substrate specificity of CPD-Y into one for basic amino acid residues in P'1, we have introduced Asp and/or Glu residues at a number of selected positions within the S'1 binding site. The effects of these substitutions on the substrate specificity, pH dependence and protein stability have been evaluated. The results presented here demonstrate that it is possible to obtain significant changes in the substrate preference by introducing charged amino acids into the framework provided by an enzyme with a quite different specificity. The introduced acidic amino acid residues provide a marked pH dependence of the (kcat/Km)FA-A-R-OH/(kcat/Km)FA-A-L-OH ratio. The change in stability upon introduction of Asp/Glu residues can be correlated to the difference in the mean buried surface area between the substituted and the substituting amino acid. Thus, the effects of acidic amino acid residues on the protein stability depend upon whether the introduced amino acid protrudes from the solvent accessible surface as defined by the surrounding residues in the wild type enzyme or is submerged below.
ESTHER : Stennicke_1994_Protein.Eng_7_911
PubMedSearch : Stennicke_1994_Protein.Eng_7_911
PubMedID: 7971953
Gene_locus related to this paper: yeast-cbpy1

Title : Recognition of C-terminal amide groups by (serine) carboxypeptidase Y investigated by site-directed mutagenesis - Mortensen_1994_J.Biol.Chem_269_15528
Author(s) : Mortensen UH , Raaschou-Nielsen M , Breddam K
Ref : Journal of Biological Chemistry , 269 :15528 , 1994
Abstract : Serine carboxypeptidases have the ability to hydrolyze peptides as well as peptide amides. Previously, it has been demonstrated that Asn51 and Glu145 (in the protonated form) each donate a hydrogen bond to the alpha-carboxylate of peptide substrate. It is here demonstrated by characterization of carboxypeptidase Y derivatives, mutationally altered at positions 51 and 145, that the same groups are involved in the interaction with the C-terminal carboxyamide group of peptide amides. Asn51 donates a hydrogen bond to the C = O group of the substrate, and Glu145 (in the charged form) accepts one from the NH2 group of the substrate. Thus, the ionic state of Glu145 is different when peptides are hydrolyzed as compared with when peptide amides are hydrolyzed. This explains why Km for the hydrolysis of peptides increases with pH, whereas it remains constant for peptide amides. As a consequence, kcat/Km for the hydrolysis of peptide amides is higher than for the hydrolysis of peptides at pH > 8. At physiological pH, peptides and peptide amides are hydrolyzed with rates of the same order of magnitude; this is in accordance with reports describing that serine carboxypeptidases are involved in the degradation of biologically active peptide amides.
ESTHER : Mortensen_1994_J.Biol.Chem_269_15528
PubMedSearch : Mortensen_1994_J.Biol.Chem_269_15528
PubMedID: 8195197
Gene_locus related to this paper: yeast-cbpy1

Title : Improvement of the applicability of carboxypeptidase Y in peptide synthesis by protein engineering - Raaschou-Nielsen_1994_Pept.Res_7_132
Author(s) : Raaschou-Nielsen M , Mortensen UH , Olesen K , Breddam K
Ref : Pept Res , 7 :132 , 1994
Abstract : Asn51 and Glu145 of (serine) carboxypeptidase Y function as binding sites for the C-terminal carboxylate group of peptide substrates, and Glu65 is involved in orienting these two amino acid residues. A series of mutants of carboxypeptidase Y where these three amino acid residues have been replaced were investigated for their applicability in transacylation reactions with amino acid esters as acceptors. With H-Val-OMethyl as the nucleophile, the fraction of aminolysis is significantly higher than with the corresponding amino acid, suggesting a beneficial effect of blocking the alpha-carboxylate group. Increasing the size of the alcohol moiety, i.e., -OEthyl, -OPropyl or OButyl, has an adverse effect on the binding of the nucleophile and on the maximum yield of aminolysis. Replacement of Asn51 and Glu145 with Ala or Gly has a pronounced beneficial effect both on binding and the maximum fraction of aminolysis. However, the results do not establish a specific type of interaction between the enzyme and these valine esters. It is probable that the rotational freedom around the ester bond allows multiple binding modes, depending on both the leaving group and type of structural change within the binding site. From a synthetic point of view, some of the mutant enzymes are much better than the wildtype enzyme when amino acid esters are used as nucleophiles.
ESTHER : Raaschou-Nielsen_1994_Pept.Res_7_132
PubMedSearch : Raaschou-Nielsen_1994_Pept.Res_7_132
PubMedID: 8081068
Gene_locus related to this paper: yeast-cbpy1

Title : Site-directed mutagenesis on (serine) carboxypeptidase Y. A hydrogen bond network stabilizes the transition state by interaction with the C-terminal carboxylate group of the substrate - Mortensen_1994_Biochemistry_33_508
Author(s) : Mortensen UH , Remington SJ , Breddam K
Ref : Biochemistry , 33 :508 , 1994
Abstract : The three-dimensional structure of (serine) carboxypeptidase Y suggests that the side chains of Trp49, Asn51, Glu65, and Glu145 could be involved in the recognition of the C-terminal carboxylate group of peptide substrates. The mutations Trp49-->Phe; Asn51-->Ala, Asp, Glu, Gln, Ser, or Thr; Glu65-->Ala; and Glu145-->Ala, Asp, Asn, Gln, or Ser have been performed. Enzymes with Ala at these positions were also produced as double and triple mutations. These mutations have only little effect on the esterase activity of the enzyme, consistent with the absence of a hydrogen bond acceptor in the P1' position of such substrates. On the other hand, removal of the hydrogen-bonding capacity by incorporation of Ala at any of these four positions results in reduced peptidase activity, in particular when Asn51 and Glu145 are replaced. The results are consistent with Trp49 and Glu65 orienting Asn51 and Glu145 by hydrogen bonds, such that these can function as hydrogen bond donors (Glu145 only in its protonated carboxylic acid form) with the C-terminal alpha-carboxylate group of the peptide substrate as acceptor. However, it appears that strong interactions are formed only in the transition state since the combined removal of Asn51 and Glu145 reduces kcat about 100-fold and leaves KM practically unchanged. The results obtained with enzymes in which Asn51 or Glu145 has been replaced with other residues possessing the capacity to donate a hydrogen bond demonstrate that there is no flexibility with respect to the nature of the hydrogen bond donor at position 145, whereas enzymes with Gln, Ser, or Thr at position 51 exhibit much higher activity than N51A, although none of them reaches the wild-type level. With carboxypeptidase Y as well as other serine carboxypeptidases the binding of peptide substrates in the ground state (KM) is adversely affected by an increase in pH. It is shown that deprotonation of a single ionizable group with a pKa of 4.3 on the enzyme is responsible for this pH effect. The results show that the group involved is either Glu65 or Glu145, the latter being the more probable. The effect of this ionization on KM is explained by charge repulsion between the carboxylate group of the substrate and that of Glu145, hence preventing substrate from binding.
ESTHER : Mortensen_1994_Biochemistry_33_508
PubMedSearch : Mortensen_1994_Biochemistry_33_508
PubMedID: 7904479
Gene_locus related to this paper: yeast-cbpy1

Title : The activity of carboxypeptidase Y toward substrates with basic P1 amino acid residues is drastically increased by mutational replacement of leucine 178 - Olesen_1994_Biochemistry_33_11121
Author(s) : Olesen K , Mortensen UH , Aasmul-Olsen S , Kielland-Brandt MC , Remington SJ , Breddam K
Ref : Biochemistry , 33 :11121 , 1994
Abstract : A random mutagenesis study on carboxypeptidase Y has previously suggested that Leu178 is situated in the S1 binding pocket, and this has later been confirmed by the three-dimensional structure. We here report the mutational replacement of Leu178 with Trp, Phe, Ala, Ser, Cys, Asn, Asp, or Lys and the kinetic characterization of each mutant, using substrates systematically varied at the P1 position. The general effect of these substitutions is a reduced kcat/Km for substrates with uncharged amino acid residues in the P1 position, little effect on those with acidic residues, and an increased kcat/Km for those with basic amino acid residues. There is a clear correlation between the reduction in kcat/Km for substrates with uncharged P1 side chains and the nature of the residue at position 178. A small reduction is observed when Leu178 is replaced by another hydrophobic amino acid residue, a larger reduction when it is replaced by a polar residue, and a very large reduction when it is replaced by a charged residue. When Leu178 is replaced by Asp, kcat/Km is reduced by a factor of 2200 for a substrate with Val in the P1 position. The kcat/Km values for the hydrolysis of substrates with charged P1 side chains are increased when Leu178 is replaced by an amino acid residue with the opposite charge, and they are decreased when it is replaced by a residue with the same charge. Surprisingly, all mutants (except L178K) exhibit increased activity with substrates with basic P1 side chains.(ABSTRACT TRUNCATED AT 250 WORDS)
ESTHER : Olesen_1994_Biochemistry_33_11121
PubMedSearch : Olesen_1994_Biochemistry_33_11121
PubMedID: 7727363

Title : A conserved glutamic acid bridge in serine carboxypeptidases, belonging to the alpha\/beta hydrolase fold, acts as a pH-dependent protein-stabilizing element - Mortensen_1994_Protein.Sci_3_838
Author(s) : Mortensen UH , Breddam K
Ref : Protein Science , 3 :838 , 1994
Abstract : Serine endopeptidases of the chymotrypsin family contain a salt bridge situated centrally within the active site, the acidic component of the salt bridge being adjacent to the catalytically essential serine. Serine carboxypeptidases also contain an acidic residue in this position but it interacts through a short hydrogen bond, probably of low-barrier type, with another acidic residue, hence forming a "glutamic acid bridge." In this study, the residues constituting this structural element in carboxypeptidase Y have been replaced by site-specific mutagenesis. It is demonstrated that the glutamic acid bridge contributes significantly to the stability of the enzyme below pH 6.5 and has an adverse effect at pH 9.5. Carboxypeptidase WII from wheat contains 2 such bridges, and it is more stable than carboxypeptidase Y at acidic pH.
ESTHER : Mortensen_1994_Protein.Sci_3_838
PubMedSearch : Mortensen_1994_Protein.Sci_3_838
PubMedID: 7914789
Gene_locus related to this paper: yeast-cbpy1