Synthesis and antimicrobial activity of α‐aminoboronic‐containing peptidomimetics

A library of 175 dipeptidomimetics and tripeptidomimetics containing an α‐amino boronic acid or boronate has been synthesized, and the activity toward Mycobacterium tuberculosis, Candida albicans, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli and Pseudomonas aeruginosa has been screened. Although there is no clear structure–activity relationship, several compounds exhibit promising activity against different pathogens. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Interference of Seasonal Variation on the Antimicrobial and Cytotoxic Activities of the Essential Oils from the Leaves of Iryanthera polyneura in the Amazon Rain Forest

The essential oils (EOs) obtained from the leaves of Iryanthera polyneura Ducke trees was chemically Assessed and tested for the ability of inhibiting the growth of Candida albicans, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus mutans and S. sanguinis. The oil was also tested against breast (MCF‐7) and prostate (PC‐3) cancer cell lines. Minimum bactericidal concentrations (MBCs) and 50 % inhibition concentrations (IC₅₀) values were obtained. EOs were active against Gram‐positive bacteria. Spathulenol, α‐cadinol and τ‐muurolol were major components of EOs. The oils showed a higher cytotoxicity against PC‐3 than MCF‐7 cells, although the oils were active against both cell types. Oils obtained from leaves collected in the dry season were more active against E. faecalis, S. aureus and PC‐3, while the oils obtained from leaves collected in the rainy season were more active against S. mutans, S. sanguinis and MCF‐7. The antibacterial and cytotoxic activities of the essential oils from the leaves of I. polyneura are related to the seasonal climate variation and are influenced by compounds that are minor components of the oils.     

Evaluation of the efficacy of chemical disinfectants for disinfection of heat‐polymerised acrylic resin

doi: 10.1111/j.1741‐2358.2010.00400.x
Evaluation of the efficacy of chemical disinfectants for disinfection of heat‐polymerised acrylic resin Objective: This study evaluated the efficacy of disinfectants on the internal aspect of heat‐polymerised acrylic resin contaminated with microbial strains. Background: Dentures absorb oral fluids and become contaminated by different microorganisms. Methods: Two hundred and fifty rectangular specimens were made of heat‐polymerised acrylic resin, and then divided into five groups corresponding to the microbial strains (Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, S. mutans and Enterococcus faecalis). After contamination, the specimens were immersed in 1 and 2% sodium hypochlorite and 2% glutaraldehyde for periods of 5, 10 and 15 min. The specimens were placed into tubes containing different broths and incubated at 35°C and then visually analysed. Turbidity in the medium indicated microbial growth. The Fisher’s exact test was used in the analysis of the results. Results: The strain E. faecalis was the most resistant to the disinfectant solutions, and among them, glutaraldehyde was more effective than 2 and 1% hypochlorite for disinfection for 5 min; in the 10‐min period there were no differences between the disinfectants. In 15 min of immersion, 1% hypochlorite and glutaraldehyde were more effective than 2% hypochlorite. Conclusions: Disinfection for 10 min with 1% hypochlorite and glutaraldehyde is effective in disinfecting the internal aspect of heat‐polymerised acrylic resin.     

Colonisation of soft lining materials by micro‐organisms

doi:10.1111/j.1741‐2358.2009.00300.x
Colonisation of soft lining materials by micro‐organisms Objective: This study evaluated the in vitro adherence of pathogenic micro‐organisms, Candida albicans, Staphylococcus aureus and Pseudomonas aeruginosa, to soft lining materials and their inhibitory effect on these micro‐organisms. Materials and Methods: To measure adherence, specimens of Molloplast B and Ufi Gel P were inoculated [10⁷ colony‐forming units per millimetre (cfu/ml)] with TSB media containing the micro‐organisms. To determine the number of micro‐organisms in the 10^?2–10^?5 dilutions, 25 μl of the suspension were transferred to plates of selective media. Colony counts of each specimen were quantified (cfu/ml). The surface roughness was measured with a perfilometer to assess the relationship between the adherence of micro‐organisms and surface roughness of each material. For the inhibition test, specimens of materials were placed in agar plates inoculated individually with the micro‐organisms. After 48 h, the inhibition zones around the specimens were measured. Results: None of the materials exhibited inhibition zones. The number of cfu/ml of S. aureus and P. aeruginosa were significantly greater than C. albicans for both materials. The Ufi Gel P exhibited greater adherence of C. albicans than Molloplast B. No correlation was observed between the adherence of micro‐organisms and surface roughness. Conclusion: The surface roughness of the materials is not the only factor governing micro‐organism adherence.     

Structure of the Escherichia coli ArnA N‐formyltransferase domain in complex with N5‐formyltetrahydrofolate and UDP‐Ara4N

ArnA from Escherichia coli is a key enzyme involved in the formation of 4‐amino‐4‐deoxy‐l ‐arabinose. The addition of this sugar to the lipid A moiety of the lipopolysaccharide of pathogenic Gram‐negative bacteria allows these organisms to evade the cationic antimicrobial peptides of the host immune system. Indeed, it is thought that such modifications may be responsible for the repeated infections of cystic fibrosis patients with Pseudomonas aeruginosa. ArnA is a bifunctional enzyme with the N‐ and C‐terminal domains catalyzing formylation and oxidative decarboxylation reactions, respectively. The catalytically competent cofactor for the formylation reaction is N¹⁰‐formyltetrahydrofolate. Here we describe the structure of the isolated N‐terminal domain of ArnA in complex with its UDP‐sugar substrate and N⁵‐formyltetrahydrofolate. The model presented herein may prove valuable in the development of new antimicrobial therapeutics.     

Time‐kill studies with a ceftazidime‐treated mixed culture consisting of Pseudomonas aeruginosa,Burkholderia cepacia and Staphylococcus aureus

So far, time‐kill analyses were mostly done with isolates of bacteria. Here, we used a mixed culture consisting of Pseudomonas aeruginosa, Burkholderia cepacia, and Staphylococcus aureus to investigate the impact of ceftazidime treatment. Following an earlier study with the same strains, the influence of different ceftazidime concentrations as well as repeated ceftazidime treatment was tested. In order to assess the influence of substrate competition, which might be relevant to interpret mixed‐culture time‐kill studies, the major metabolites of the chemically defined cultivation medium were measured additionally. Time‐kill experiments were conducted in shake flasks with the chemically defined and modified medium M199. The cell concentration in the mixed culture was analyzed on the single‐species level using a quantitative terminal restriction fragment (qT‐RFLP) method. The amount of gluconate produced in mixed culture positively correlated with increased ceftazidime concentrations (5, 15, 30, 60 mg/L). Burkholderia cepacia developed resistance after repeated ceftazidime addition and reached the highest cell concentration of the three cultivated strains. Pseudomonas aeruginosa showed a pronounced regrowth phase after removal of ceftazidime, while growth of S. aureus was not influenced by medium exchange. In conclusion, growth of B. cepacia was dominant in the ceftazidime‐treated mixed culture over the observed time range, due to low susceptibility against ceftazidime as well as advantages in substrate usage.     

Activation of nucleotide‐binding domain‐like receptor containing protein 3 inflammasome in dendritic cells and macrophages by Streptococcus sanguinis

Streptococcus sanguinis is frequently isolated from the blood of patients with infective endocarditis and contributes to the pathology of this disease through induction of interleukin (IL)‐1β responsible for the development of the disease. However, the mechanism of IL‐1β induction remains unknown. In this study, S. sanguinis activated a murine dendritic cell (DC) to induce IL‐1β and this activity was attenuated by silencing the mRNAs of nucleotide‐binding domain‐like receptor containing protein 3 (NLRP3) and caspase‐1. S. sanguinis induced IL‐1β production in murine bone marrow‐derived macrophage, but this activity was significantly reduced in bone marrow‐derived macrophages from NLRP3‐, apoptosis‐associated speck‐like protein containing a caspase‐recruitment domain‐, and caspase‐1‐deficient mice. DC phagocytosed S. sanguinis cells, followed by the release of adenosine triphosphate (ATP). The ATP‐degradating enzyme attenuated the release of ATP and IL‐1β. The inhibitors for ATP receptor reduced IL‐1β release in DC. These results strongly suggest that S. sanguinis has the activity to induce IL‐1β through the NLRP3 inflammasome in macrophage and DC and interaction of purinergic receptors with ATP released is involved in expression of the activity.     

Ribosomal binding and antibacterial activity of ethylene glycol‐bridged apidaecin Api137 and oncocin Onc112 conjugates

Recent surveillance data on antimicrobial resistance predict the beginning of the post‐antibiotic era with pan‐resistant bacteria even overcoming polymyxin as the last available treatment option. Thus, new substances using novel modes of antimicrobial action are urgently needed to reduce this health threat. Antimicrobial peptides are part of the innate immune system of most vertebrates and invertebrates and accepted as valid substances for antibiotic drug development efforts. Especially, short proline‐rich antimicrobial peptides (PrAMP) of insect origin have been optimized for activity against Gram‐negative strains. They inhibit protein expression in bacteria by blocking the 70S ribosome exit tunnel (oncocin‐type) or the assembly of the 50S subunit (apidaecin‐type binding). Thus, apidaecin analog Api137 and oncocin analog Onc112 supposedly bind to different nearby or possibly partially overlapping binding sites. Here, we synthesized Api137/Onc112‐conjugates bridged by ethylene glycol spacers of different length to probe synergistic activities and binding modes. Indeed, the antimicrobial activities against Escherichia coli and Pseudomonas aeruginosa improved for some constructs, although the conjugates did not bind better to the 70S ribosome of E. coli than Api137 and Onc112 using 5(6)‐carboxyfluorescein‐labelled Api137 and Onc112 in a competitive fluorescence polarization assay. In conclusion, Api137/Onc112‐conjugates showed increased antimicrobial activities against P. aeruginosa and PrAMP‐susceptible and ‐resistant E. coli most likely because of improved membrane interactions, whereas the interaction to the 70S ribosome was most likely not improved relying still on the independent apidaecin‐ and oncocin‐type binding modes. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Glutaminyl‐tRNA Synthetase from Pseudomonas aeruginosa: Characterization,structure, and development as a screening platform

Pseudomonas aeruginosa has a high potential for developing resistance to multiple antibiotics. The gene (glnS) encoding glutaminyl‐tRNA synthetase (GlnRS) from P. aeruginosa was cloned and the resulting protein characterized. GlnRS was kinetically evaluated and the K_M and k_cat^obs, governing interactions with tRNA, were 1.0 μM and 0.15 s^?1, respectively. The crystal structure of the α₂ form of P. aeruginosa GlnRS was solved to 1.9 Å resolution. The amino acid sequence and structure of P. aeruginosa GlnRS were analyzed and compared to that of GlnRS from Escherichia coli. Amino acids that interact with ATP, glutamine, and tRNA are well conserved and structure overlays indicate that both GlnRS proteins conform to a similar three‐dimensional structure. GlnRS was developed into a screening platform using scintillation proximity assay technology and used to screen ~2,000 chemical compounds. Three inhibitory compounds were identified and analyzed for enzymatic inhibition as well as minimum inhibitory concentrations against clinically relevant bacterial strains. Two of the compounds, BM02E04 and BM04H03, were selected for further studies. These compounds displayed broad‐spectrum antibacterial activity and exhibited moderate inhibitory activity against mutant efflux deficient strains of P. aeruginosa and E. coli. Growth of wild‐type strains was unaffected, indicating that efflux was likely responsible for the lack of sensitivity. The global mode of action was determined using time‐kill kinetics. BM04H03 did not inhibit the growth of human cell cultures at any concentration and BM02E04 only inhibit cultures at the highest concentration tested (400 μg/ml). In conclusion, GlnRS from P. aeruginosa is shown to have a structure similar to that of E. coli GlnRS and two natural product compounds were identified as inhibitors of P. aeruginosa GlnRS with the potential for utility as lead candidates in antibacterial drug development in a time of increased antibiotic resistance.     

Anti‐Adhesion Activity of Tannins Isolated from the Mangrove Laguncularia racemosa

In the search of new compounds with biofilm‐inhibiting properties, mangroves with their richness of secondary metabolites can be a valuable resource. Crude methanolic leaf extracts from the mangrove Laguncularia racemosa enriched in phenolic substances cause a reduction in initial cell adhesion of Candida glabrata and Candida albicans, but not on Escherichia coli. LC/MS‐guided fractionation of the phenolic compounds resulted in 19 fractions, of which ten were analyzed for their bioactivity against cell adhesion. Effects on cell adhesion and planktonic growth of Escherichia coli, Candida glabrata and Candida albicans were measured in 96‐well microtiter plates in the presence of 0.2 mg ml^?1 of the isolated fractions. Two fractions caused a reduction of cell adhesion of Candida albicans. These fractions containing bioactive compounds were analyzed by LC/MS and NMR spectroscopy. Casuarinin and digalloyl‐hexahydroxydiphenoyl‐glucose were identified in the active fractions, in addition to three signals of ellagitannins. These results indicate a specific mode of action of hydrolysable tannins against cell adhesion of Candida albicans, which needs to be further analyzed.     

Engineering of Phosphoserine Aminotransferase Increases the Conversion of l‐Homoserine to 4‐Hydroxy‐2‐ketobutyrate in a Glycerol‐Independent Pathway of 1,3‐Propanediol Production from Glucose

Phosphoserine aminotransferase (SerC) from Escherichia coli (E. coli) MG1655 is engineered to catalyze the deamination of homoserine to 4‐hydroxy‐2‐ketobutyrate, a key reaction in producing 1,3‐propanediol (1,3‐PDO) from glucose in a novel glycerol‐independent metabolic pathway. To this end, a computation‐based rational approach is used to change the substrate specificity of SerC from l ‐phosphoserine to l ‐homoserine. In this approach, molecular dynamics simulations and virtual screening are combined to predict mutation sites. The enzyme activity of the best mutant, SerC_R42W/R77W, is successfully improved by 4.2‐fold in comparison to the wild type when l ‐homoserine is used as the substrate, while its activity toward the natural substrate l ‐phosphoserine is completely deactivated. To validate the effects of the mutant on 1,3‐PDO production, the “homoserine to 1,3‐PDO” pathway is constructed in E. coli by coexpression of SerC_R42W/R77W with pyruvate decarboxylase and alcohol dehydrogenase. The resulting mutant strain achieves the production of 3.03 g L^?1 1,3‐PDO in fed‐batch fermentation, which is 13‐fold higher than the wild‐type strain and represents an important step forward to realize the promise of the glycerol‐independent synthetic pathway for 1,3‐PDO production from glucose.     

Chemiluminescence of non‐differentiated THP‐1 promonocytes: developing an assay for screening anti‐inflammatory milk proteins and peptides

Human leukemic THP‐1 promonocytes are widely used as a model for peripheral blood monocytes. However, superoxide production during respiratory burst (RB) of non‐differentiated THP‐1 (nd‐THP‐1) cells is very low. Here we present a rapid and low‐cost method for measuring the chemiluminescence (CL) of opsonized zymosan (OZ) induced RB which allows detection of Escherichia coli lipopolysaccharide (LPS) induced priming of nd‐THP‐1 cells on the basis of CL reaction kinetics. Maximum CL intensity obtained was 2.20 ± 0.25 and 1.30 ± 0.11 relative light units, while CL peak time was achieved at 18.1 ± 2.6 and 28.7 ± 1.3 min in primed and non‐primed cells, respectively. The priming of nd‐THP‐1 cells with LPS evoked typical TNF‐α and IL‐6 production. We tested the effects of bovine lactoferrin and protein fractions from Lactobacillus helveticus BGRA43 fermented milk for potential anti‐inflammatory effects on LPS primed nd‐THP‐1 cells. Four fractions were found to inhibit the OZ‐induced CL in a dose‐dependent manner (IC₅₀ 3–30 µg/mL), while lactoferrin inhibited CL to a lesser extent (IC₅₀ 270 µg/mL). These results suggest that measuring CL response of nd‐THP‐1 cells can serve as a method for screening anti‐inflammatory compounds which could be used in reducing the risk of phagocyte‐mediated inflammatory diseases. Copyright © 2010 John Wiley & Sons, Ltd.     

Comparative study of SoxR activation by redox‐active compounds

SoxR from Escherichia coli and related enterobacteria is activated by a broad range of redox‐active compounds through oxidation or nitrosylation of its [2Fe–2S] cluster. Activated SoxR then induces SoxS, which subsequently activates more than 100 genes in response. In contrast, non‐enteric SoxRs directly activate their target genes in response to redox‐active compounds that include endogenously produced metabolites. We compared the responsiveness of SoxRs from Streptomyces coelicolor (ScSoxR), Pseudomonas aeruginosa (PaSoxR) and E. coli (EcSoxR), all expressed in S. coelicolor, towards natural or synthetic redox‐active compounds. EcSoxR responded to all compounds examined, whereas ScSoxR was insensitive to oxidants such as paraquat (Eh ?440 mV) and menadione sodium bisulphite (Eh ?45 mV) and to NO generators. PaSoxR was insensitive only to some NO generators. Whole‐cell EPR analysis of SoxRs expressed in E. coli revealed that the [2Fe–2S]¹⁺ of ScSoxR was not oxidizable by paraquat, differing from EcSoxR and PaSoxR. The mid‐point redox potential of purified ScSoxR was determined to be ?185 ± 10 mV, higher by ～ 100 mV than those of EcSoxR and PaSoxR, supporting its limited response to paraquat. The overall sensitivity profile indicates that both redox potential and kinetic reactivity determine the differential responses of SoxRs towards various oxidants.     

Synthesis of Some Novel 6‐Benzyl(or Substituted Benzyl)‐2‐β‐d‐Glucopyranosyl‐1,2,4‐Triazolo[4,3‐b][1,2,4]Triazines as Potential Antimicrobial Chemotherapeutics

Glucosidation of the new 8‐amino‐6‐benzyl(or substituted benzyl)‐2,8‐dihydro‐1,2,4‐triazolo[4,3‐b][1,2,4]triazin‐7(3H)‐ones (3a–d) with 2,3,4,6‐tetra‐O‐acetyl‐α‐d‐glucopyranosyl bromide 4 gave the corresponding N‐glucosides 5a–d. Chemical transformations leading to new functionalities have also been achieved to give compounds 7–12. Antimicrobial activity of compounds 5a–c against Aspergillus fumigatus, Penicillium italicum, Syncephalastrum racemosum, Candida albicans, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli is described.     

Characterization and structure determination of prolyl‐tRNA synthetase from Pseudomonas aeruginosa and development as a screening platform

Pseudomonas aeruginosa is an opportunistic multi‐drug resistant pathogen implicated as a causative agent in nosocomial and community acquired bacterial infections. The gene encoding prolyl‐tRNA synthetase (ProRS) from P. aeruginosa was overexpressed in Escherichia coli and the resulting protein was characterized. ProRS was kinetically evaluated and the K_M values for interactions with ATP, proline, and tRNA were 154, 122, and 5.5 μM, respectively. The turn‐over numbers, k_cat^obs, for interactions with these substrates were calculated to be 5.5, 6.3, and 0.2 s^?1, respectively. The crystal structure of the α₂ form of P. aeruginosa ProRS was solved to 2.60 Å resolution. The amino acid sequence and X‐ray crystal structure of P. aeruginosa ProRS was analyzed and compared with homologs in which the crystal structures have been solved. The amino acids that interact with ATP and proline are well conserved in the active site region and overlay of the crystal structure with ProRS homologs conforms to a similar overall three‐dimensional structure. ProRS was developed into a screening platform using scintillation proximity assay (SPA) technology and used to screen 890 chemical compounds, resulting in the identification of two inhibitory compounds, BT06A02 and BT07H05. This work confirms the utility of a screening system based on the functionality of ProRS from P. aeruginosa.     

New Pyran‐2‐ones from Alkalophilic Actinomycete,Nocardiopsis alkaliphila sp. Nov. YIM‐80379

Two new pyran‐2‐ones, nocardiopyrones A and B ( 1 and 2 , resp.), along with four known compounds, pyridinols 3 – 5 , and 1‐acetyl‐β‐carboline ( 6 ) were isolated from the alkalophilic actinomycete Nocardiopsis alkaliphila sp. nov. YIM‐80379. Their structures were established on the basis of spectroscopic analysis, CD spectra, and the quantum‐chemical ECD calculation. Pyridinols 3 – 5 were isolated from a natural source for the first time. Compounds 1 and 2 showed weak antibacterial activities against Pseudomonas aeruginosa, Enterobacter aerogenes, and Escherichia coli with MIC values of 20–48 μM . Compound 2 showed weak antimicrobial activities against Candida albicans and Staphylococcus aureus with MIC values of 24 and 48 μM , respectively.     

In vitro antiseptic properties of an ammonium compound combined with denture base acrylic resin

Background: Denture base acrylic resin is easily colonised by oral endogenous bacteria and Candida spp., and eventually by extra‐oral species such as Staphylococcus spp., Pseudomonadaceae or members of Enterobacteriaceae. This microbial reservoir can be responsive for denture related stomatitis and aspiration pneumonia, a life‐threatening infection especially in geriatric patients. However, the oral and denture hygiene of dependant elderly individuals is extremely poor. Objective: This in vitro study aimed to determine the per cent of a quaternary ammonium compound heat‐polymerised in acrylic resin necessary to obtain denture base displaying antiseptic properties. Design: Acrylic resin discs containing 2–50% ammonium polymer (Poly 202063A; 0% control) were soaked in artificial saliva for 4 weeks. Resin discs were incubated for 24 hours with Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa [37°C, brain–heart infusion (BHI) broth and phosphate‐buffered saline (PBS) buffer] and Candida albicans (30°C, Schaedler broth), in 15 ml (168 discs) and 600 μl (168 discs) of inoculum. Microbial growth was verified at t 0 hours and t 24 hours. Data were recorded as the mean of three colony forming unit (CFU) numerations. The borderline of antimicrobial effect was determined at 0.1% viable cells. Results: In 600 μl of PBS inoculum, resin specimens had a bactericidal effect (E. coli and S. aureus: 2%; P. aeruginosa: 10%) and a fungicidal effect (C. albicans: 50%). Long‐term stability and toxicity in vivo studies are now required. Conclusion: A 2% quaternary ammonium compound polymerised with a denture acrylic resin displayed antiseptic properties after a 4‐week soaking period in artificial saliva. Such antiseptic denture base could help geriatric patients to improve their oral health.     

Enhanced diastereoselective synthesis of t‐Butyl 6‐cyano‐(3R,5R)‐dihydroxyhexanoate by using aldo‐keto reductase and glucose dehydrogenase co‐producing engineered Escherichia coli

t‐Butyl 6‐cyano‐(3R,5R)‐dihydroxyhexanoate ((3R,5R)‐ 2 ) is a key chiral diol precursor of atorvastatin calcium (Lipitor®). We have constructed a Kluyveromyces lactis aldo‐keto reductase mutant KlAKR‐Y295W/W296L (KlAKRm) by rational design in previous research, which displayed high activity and excellent diastereoselectivity (de_p > 99.5%) toward t‐butyl 6‐cyano‐(5R)‐hydroxy‐3‐oxohexanoate ((5R)‐ 1 ). To realize in situ cofactor regeneration, a robust KlAKRm and Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) co‐producer E. coli BL 21(DE3) pETDuet‐esgdh (MCS1)/pET‐28b (+)‐klakrm was constructed in this work. Under the optimized conditions, AKR and GDH activities of E. coli BL 21(DE3) pETDuet‐esgdh (MCS1)/pET‐28b (+)‐klakrm peaked at 249.9 U/g DCW (dry cellular weight) and 29100 U/g DCW, respectively. It completely converted (5R)‐ 1 at substrate loading size of up to 60.0 g/L (5R)‐ 1 in the absence of exogenous NADH, which was one‐fifth higher than that of the separately prepared KlAKRm and EsGDH under the same conditions. In this manner, a biocatalytic process for (3R,5R)‐ 2 with productivity of 243.2 kg/m³ d was developed. Compared with the combination of separate expressed KlAKRm with EsGDH, co‐expression of KlAKRm and EsGDH has the advantages of alleviating cell cultivation burden and elevating substrate load. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1235–1242, 2017     

Unique coenzyme binding mode of hyperthermophilic archaeal sn‐glycerol‐1‐phosphate dehydrogenase from Pyrobaculum calidifontis

A gene encoding an sn‐glycerol‐1‐phosphate dehydrogenase (G1PDH) was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. In contrast to conventional G1PDHs, the expressed enzyme showed strong preference for NADH: the reaction rate (V_max) with NADPH was only 2.4% of that with NADH. The crystal structure of the enzyme was determined at a resolution of 2.45 Å. The asymmetric unit consisted of one homohexamer. Refinement of the structure and HPLC analysis showed the presence of the bound cofactor NADPH in subunits D, E, and F, even though it was not added in the crystallization procedure. The phosphate group at C2’ of the adenine ribose of NADPH is tightly held through the five biased hydrogen bonds with Ser40 and Thr42. In comparison with the known G1PDH structure, the NADPH molecule was observed to be pushed away from the normal coenzyme binding site. Interestingly, the S40A/T42A double mutant enzyme acquired much higher reactivity than the wild‐type enzyme with NADPH, which suggests that the biased interactions around the C2’‐phosphate group make NADPH binding insufficient for catalysis. Our results provide a unique structural basis for coenzyme preference in NAD(P)‐dependent dehydrogenases. Proteins 2016; 84:1786–1796. © 2016 Wiley Periodicals, Inc.