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

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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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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Structure-Activity Relationship of Synthetic Variants of the Milk-Derived Antimicrobial Peptide αs2-Casein f(183–207)

Applied and Environmental Microbiology ◽

10.1128/aem.01394-13 ◽

2013 ◽

Vol 79 (17) ◽

pp. 5179-5185 ◽

Cited By ~ 18

Author(s):

Avelino Alvarez-Ordóñez ◽

Máire Begley ◽

Tanya Clifford ◽

Thérèse Deasy ◽

Kiera Considine ◽

...

Keyword(s):

Amino Acids ◽

Biological Activity ◽

Amino Acid ◽

Antimicrobial Peptide ◽

Peptide Sequence ◽

Content Type ◽

Charged Amino Acids ◽

Food Borne ◽

Relationship Of ◽

Positively Charged

ABSTRACTTemplate-based studies on antimicrobial peptide (AMP) derivatives obtained through manipulation of the amino acid sequence are helpful to identify properties or residues that are important for biological activity. The present study sheds light on the importance of specific amino acids of the milk-derived αs2-casein f(183–207) peptide to its antibacterial activity against the food-borne pathogensListeria monocytogenesandCronobacter sakazakii. Trimming of the peptide revealed that residues at the C-terminal end of the peptide are important for activity. Removal of the last 5 amino acids at the C-terminal end and replacement of the Arg at position 23 of the peptide sequence by an Ala residue significantly decreased activity. These findings suggest that Arg23 is very important for optimal activity of the peptide. Substitution of the also positively charged Lys residues at positions 15 and 17 of the αs2-casein f(183–207) peptide also caused a significant reduction of the effectiveness againstC. sakazakii, which points toward the importance of the positive charge of the peptide for its biological activity. Indeed, simultaneous replacement of various positively charged amino acids was linked to a loss of bactericidal activity. On the other hand, replacement of Pro residues at positions 14 and 20 resulted in a significantly increased antibacterial potency, and hydrophobic end tagging of αs2-casein f(193–203) and αs2-casein f(197–207) peptides with multiple Trp or Phe residues significantly increased their potency againstL. monocytogenes. Finally, the effect of pH (4.5 to 7.4), temperature (4°C to 37°C), and addition of sodium and calcium salts (1% to 3%) on the activity of the 15-amino-acid αs2-casein f(193–207) peptide was also determined, and its biological activity was shown to be completely abolished in high-saline environments.

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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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A l-Lysine Transporter of High Stereoselectivity of the Amino Acid-Polyamine-Organocation (APC) Superfamily

Journal of Biological Chemistry ◽

10.1074/jbc.m113.510743 ◽

2013 ◽

Vol 289 (3) ◽

pp. 1377-1387 ◽

Cited By ~ 8

Author(s):

Jagdeep Kaur ◽

Elena Olkhova ◽

Viveka Nand Malviya ◽

Ernst Grell ◽

Hartmut Michel

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Ligand Binding ◽

Isothermal Titration Calorimetry ◽

Functional Characterization ◽

Homology Model ◽

Docking Studies ◽

Site Directed Mutagenesis ◽

Titration Calorimetry ◽

Amino Acid Residues

Membrane proteins of the amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play an important role in the regulation of cellular processes. We report the heterologous production of the LysP-related transporter STM2200 from Salmonella typhimurium in Escherichia coli, its purification, and functional characterization. STM2200 is assumed to be a proton-dependent APC transporter of l-lysine. The functional interaction between basic amino acids and STM2200 was investigated by thermoanalytical methods, i.e. differential scanning and isothermal titration calorimetry. Binding of l-lysine to STM2200 in its solubilized monomer form is entropy-driven. It is characterized by a dissociation constant of 40 μm at pH 5.9 and is highly selective; no evidence was found for the binding of l-arginine, l-ornithine, l-2,4-diaminobutyric acid, and l-alanine. d-Lysine is bound 45 times more weakly than its l-chiral form. We thus postulate that STM2200 functions as a specific transport protein. Based on the crystal structure of ApcT (Shaffer, P. L., Goehring, A., Shankaranarayanan, A., and Gouaux, E. (2009) Science 325, 1010–1014), a proton-dependent amino acid transporter of the APC superfamily, a homology model of STM2200 was created. Docking studies allowed identification of possible ligand binding sites. The resulting predictions indicated that Glu-222 and Arg-395 of STM2200 are markedly involved in ligand binding, whereas Lys-163 is suggested to be of structural and functional relevance. Selected variants of STM2200 where these three amino acid residues were substituted using single site-directed mutagenesis showed no evidence for l-lysine binding by isothermal titration calorimetry, which confirmed the predictions. Molecular aspects of the observed ligand specificity are discussed.

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Binding of Aldolase and Glyceraldehyde-3-Phosphate Dehydrogenase to the Cytoplasmic Tails of Plasmodium falciparum Merozoite Duffy Binding-Like and Reticulocyte Homology Ligands

mBio ◽

10.1128/mbio.00292-12 ◽

2012 ◽

Vol 3 (5) ◽

Cited By ~ 15

Author(s):

Ipsita Pal-Bhowmick ◽

John Andersen ◽

Prakash Srinivasan ◽

David L. Narum ◽

Jürgen Bosch ◽

...

Keyword(s):

Amino Acids ◽

Plasmodium Falciparum ◽

Amino Acid ◽

Aromatic Amino Acid ◽

Cytoplasmic Tail ◽

Content Type ◽

Aromatic Residues ◽

Charged Amino Acids ◽

Cytoplasmic Tails ◽

Erythrocyte Binding

ABSTRACTInvasion of erythrocytes byPlasmodium falciparumrequires a connection between the cytoplasmic tail of the parasite’s ligands for its erythrocyte receptors and the actin-myosin motor of the parasite. For the thromobospondin-related anonymous protein (TRAP) ligand onPlasmodiumsporozoites, aldolase forms this connection and requires tryptophan and negatively charged amino acids in the ligand’s cytoplasmic tail. Because of the importance of the Duffy binding-like (DBL) and the reticulocyte homology (RH) ligand families in erythrocyte binding and merozoite invasion, we characterized the ability of their cytoplasmic tails to bind aldolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), both of which bind actin. We tested the binding of the cytoplasmic peptides of the two ligand families to aldolase and GAPDH. Only the cytoplasmic peptides of some RH ligands showed strong binding to aldolase, and the binding depended on the presence of an aromatic amino acid (phenylalanine or tyrosine), rather than tryptophan, in the context of negatively charged amino acids. The binding was confirmed by surface plasmon resonance analysis and was found to represent affinity similar to that seen with TRAP. An X-ray crystal structure of aldolase at 2.5 Å in the presence of RH2b peptide suggested that the binding site location was near the TRAP-binding site. GAPDH bound to some of the cytoplasmic tails of certain RH and DBL ligands in an aromatic amino acid-dependent manner. Thus, the connection betweenPlasmodiummerozoite ligands and erythrocyte receptors and the actin motor can be achieved through the activity of either aldolase or GAPDH by mechanisms that do not require tryptophan but, rather, other aromatic amino acids.IMPORTANCEThe invasion of thePlasmodiummerozoite into erythrocytes is a critical element in malaria pathogenesis. It is important to understand the molecular details of this process, as this machinery can be a target for both vaccine and drug development. InPlasmodiumsporozoites andToxoplasmatachyzoites, invasion involves a glycolytic enzyme aldolase, linking the cytoplasmic tail domains of the parasite ligands to the actin-myosin motor that drives invasion. This binding requires a tryptophan that cannot be replaced by other aromatic residues. Here we show that aldolase binds the cytoplasmic tails of someP. falciparummerozoite erythrocyte-binding ligands but that the binding involves aromatic residues other than tryptophan. The biological relevance of aldolase binding to cytoplasmic tails of parasite ligands in invasion is demonstrated by our observation that RH2b but not RH2a binds to aldolase and, as previously shown, that RH2b but not RH2a is required forP. falciparuminvasion of erythrocytes.

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Structure-Function Relationships in Hydrophobins: Probing the Role of Charged Side Chains

Applied and Environmental Microbiology ◽

10.1128/aem.01493-13 ◽

2013 ◽

Vol 79 (18) ◽

pp. 5533-5538 ◽

Cited By ~ 16

Author(s):

Michael Lienemann ◽

Julie-Anne Gandier ◽

Jussi J. Joensuu ◽

Atsushi Iwanaga ◽

Yoshiyuki Takatsuji ◽

...

Keyword(s):

Amino Acids ◽

Self Assembly ◽

Fluorescent Protein ◽

Site Directed Mutagenesis ◽

Phase System ◽

Surface Binding ◽

Two Phase ◽

Content Type ◽

Charged Amino Acids

ABSTRACTHydrophobins are small fungal proteins that are amphiphilic and have a strong tendency to assemble at interfaces. By taking advantage of this property, hydrophobins have been used for a number of applications: as affinity tags in protein purification, for protein immobilization, such as in foam stabilizers, and as dispersion agents for insoluble drug molecules. Here, we used site-directed mutagenesis to gain an understanding of the molecular basis of their properties. We especially focused on the role of charged amino acids in the structure of hydrophobins. For this purpose, fusion proteins consisting ofTrichoderma reeseihydrophobin I (HFBI) and the green fluorescent protein (GFP) that contained various combinations of substitutions of charged amino acids (D30, K32, D40, D43, R45, K50) in the HFBI structure were produced. The effects of the introduced mutations on binding, oligomerization, and partitioning were characterized in an aqueous two-phase system. It was found that some substitutions caused better surface binding and reduced oligomerization, while some showed the opposite effects. However, all mutations decreased partitioning in surfactant systems, indicating that the different functions are not directly correlated and that partitioning is dependent on finely tuned properties of hydrophobins. This work shows that not all functions in self-assembly are connected in a predictable way and that a simple surfactant model for hydrophobin function is insufficient.

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Tailoring a Global Iron Regulon to a Uropathogen

mBio ◽

10.1128/mbio.00351-20 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 1

Author(s):

Rajdeep Banerjee ◽

Erin Weisenhorn ◽

Kevin J. Schwartz ◽

Kevin S. Myers ◽

Jeremy D. Glasner ◽

...

Keyword(s):

Amino Acids ◽

Urinary Tract ◽

Amino Acid ◽

Regulatory Network ◽

Iron Homeostasis ◽

Iron Limitation ◽

Content Type ◽

E Coli ◽

General Stress ◽

K 12

ABSTRACT Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O2-dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O2 to produce aerobactin. IMPORTANCE Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes.

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The Carboxy-Terminal Region ofFlavobacterium johnsoniaeSprB Facilitates Its Secretion by the Type IX Secretion System and Propulsion by the Gliding Motility Machinery

Journal of Bacteriology ◽

10.1128/jb.00218-19 ◽

2019 ◽

Vol 201 (19) ◽

Cited By ~ 5

Author(s):

Surashree S. Kulkarni ◽

Joseph J. Johnston ◽

Yongtao Zhu ◽

Zachary T. Hying ◽

Mark J. McBride

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Cell Surface ◽

Type A ◽

Secretion System ◽

Gliding Motility ◽

Foreign Protein ◽

Type B ◽

Content Type ◽

Type Ix Secretion System

ABSTRACTFlavobacterium johnsoniaeSprB moves rapidly along the cell surface, resulting in gliding motility. SprB secretion requires the type IX secretion system (T9SS). Proteins secreted by the T9SS typically have conserved C-terminal domains (CTDs) belonging to the type A CTD or type B CTD family. Attachment of 70- to 100-amino-acid type A CTDs to a foreign protein allows its secretion. Type B CTDs are common but have received little attention. Secretion of the foreign protein superfolder green fluorescent protein (sfGFP) fused to regions spanning the SprB type B CTD (sfGFP-CTDSprB) was analyzed. CTDs of 218 amino acids or longer resulted in secretion of sfGFP, whereas a 149-amino-acid region did not. Some sfGFP was secreted in soluble form, whereas the rest was attached on the cell surface. Surface-attached sfGFP was rapidly propelled along the cell, suggesting productive interaction with the motility machinery. This did not result in rapid cell movement, which apparently requires additional regions of SprB. Secretion of sfGFP-CTDSprBrequired coexpression withsprF, which lies downstream ofsprB. SprF is similar in sequence toPorphyromonas gingivalisPorP. MostF. johnsoniaegenes encoding proteins with type B CTDs lie immediately upstream ofporP/sprF-like genes. sfGFP was fused to the type B CTD from one such protein (Fjoh_3952). This resulted in secretion of sfGFP only when it was coexpressed with its cognate PorP/SprF-like protein. These results highlight the need for extended regions of type B CTDs and for coexpression with the appropriate PorP/SprF-like protein for efficient secretion and cell surface localization of cargo proteins.IMPORTANCETheF. johnsoniaegliding motility adhesin SprB is delivered to the cell surface by the type IX secretion system (T9SS) and is rapidly propelled along the cell by the motility machinery. How this 6,497-amino-acid protein interacts with the secretion and motility machines is not known. Fusion of the C-terminal 218 amino acids of SprB to a foreign cargo protein resulted in its secretion, attachment to the cell surface, and rapid movement by the motility machinery. Efficient secretion of SprB required coexpression with the outer membrane protein SprF. Secreted proteins that have sequence similarity to SprB in their C-terminal regions are common in the phylumBacteroidetesand may have roles in adhesion, motility, and virulence.

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Negatively-charged amino acids at the peptide-binding pocket of HLA-DPB1 alleles are associated with susceptibility to anti-topoisomerase I-positive systemic sclerosis

Human Immunology ◽

10.1016/j.humimm.2016.05.012 ◽

2016 ◽

Vol 77 (7) ◽

pp. 550-554 ◽

Cited By ~ 2

Author(s):

Jeong Seok Lee ◽

Jin Kyun Park ◽

Heung Jae Kim ◽

Hyung Ki Lee ◽

Yeong Wook Song ◽

...

Keyword(s):

Amino Acids ◽

Systemic Sclerosis ◽

Topoisomerase I ◽

Peptide Binding ◽

Binding Pocket ◽

Charged Amino Acids

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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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