The PHY Domain Dimer Interface of Bacteriophytochromes Mediates Cross-talk between Photosensory Modules and Output Domains.

Protein dynamics play a major role for the catalytic function of enzymes, the interaction of protein complexes or signal integration in regulatory proteins. In the context of multi-domain proteins involved in light-regulation of enzymatic effectors, the central role of conformational dynamics is well established. Light activation of sensory modules is followed by long-range signal transduction to different effectors; rather than domino-style structural rearrangements, a complex interplay of functional elements is required to maintain functionality. One family of such sensor-effector systems are red-light-regulated phytochromes that control diguanylate cyclases involved in cyclic-dimeric-GMP formation. Based on structural and functional studies of one prototypic family member, the central role of the coiled-coil sensor-effector linker was established. Interestingly, subfamilies with different linker lengths feature strongly varying biochemical characteristics. The dynamic interplay of the domains involved, however, is presently not understood. Here we show that the PHY domain dimer interface plays an essential role in signal integration, and that a functional coupling with the coiled-coil linker element is crucial. Chimaeras of two biochemically different family members highlight the phytochrome-spanning helical spine as an essential structural element involved in light-dependent upregulation of enzymatic turnover. However, isolated structural elements can frequently not be assigned to individual characteristics, which further emphasises the importance of global conformational dynamics. Our results provide insights into the intricate processes at play during light signal integration and transduction in these photosensory systems and thus provide additional guidelines for a more directed design of novel sensor-effector combinations with potential applications as optogenetic tools.

Protective effects of culture extracts (CB08035-SCA and CB08035-SYP) from Marinobacter hydrocarbonoclasticus (strain CB08035) against oxidant-induced stress in human colon carcinoma Caco-2 cells.

The present study investigated the effect of culture extracts (CB08035-SCA and CB08035-SYP) from Marinobacter hydrocarbonoclasticus (strain CB08035) on cell viability and the potential protective effects attributed to molecular mechanisms underlying antioxidant response to survive oxidative stress injuries. Caco-2 cells were submitted to oxidative stress by treatment with tert-butyl hydroperoxide (t-BOOH). Both extracts prevented cell damage and enhanced activity of antioxidant defenses (NQO1 and GST activities and GSH levels) reduced by treatment with t-BOOH. Increased ROS and caspase 3/7 activity induced by t-BOOH were dose-dependently prevented when cells were treated with the extracts. CB08035-SCA caused up-regulation of Nrf2, AKT1 and Bcl-2 gene expressions. Moreover, CB08035-SCA and CB08035-SYP treatments reduced significantly Bax, BNIP3, APAF1, ERK1, JNK1, MAPK1, NFκB1, TNFα, IL-6, IL-1ß and HO-1 gene expressions of apoptosis, proinflammation and oxidative stress induced by t-BOOH. CB08035-SCA and CB08035-SYP CPE extracts confer a significant protection against oxidative insults to cells. Our results show that culture extracts CB08035-SCA and CB08035-SYP from M. hydrocarbonoclasticus (strain CB08035) appeared to have antioxidant potential, based on their ability to protect antioxidant enzymes and mRNA gene expressions linked to apoptosis/oxidative pathways. These results suggest that culture extracts CB08035-SCA and CB08035-SYP can be a potential ingredient in the pharmaceutical and cosmeceutical industries.

Proton-coupled electron transfer mechanisms of the copper centres of nitrous oxide reductase from Marinobacter hydrocarbonoclasticus - An electrochemical study.

Reduction of N2O to N2 is catalysed by nitrous oxide reductase in the last step of the denitrification pathway. This multicopper enzyme has an electron transferring centre, CuA, and a tetranuclear copper-sulfide catalytic centre, "CuZ", which exists as CuZ*(4Cu1S) or CuZ(4Cu2S). The redox behaviour of these metal centres in Marinobacter hydrocarbonoclasticus nitrous oxide reductase was investigated by potentiometry and for the first time by direct electrochemistry. The reduction potential of CuA and CuZ(4Cu2S) was estimated by potentiometry to be +275 ± 5 mV and +65 ± 5 mV vs SHE, respectively, at pH 7.6. A proton-coupled electron transfer mechanism governs CuZ(4Cu2S) reduction potential, due to the protonation/deprotonation of Lys397 with a pKox of 6.0 ± 0.1 and a pKred of 9.2 ± 0.1. The reduction potential of CuA, in enzyme samples with CuZ*(4Cu1S), is controlled by protonation of the coordinating histidine residues in a two-proton coupled electron transfer process. In the cyclic voltammograms, two redox pairs were identified corresponding to CuA and CuZ(4Cu2S), with no additional signals being detected that could be attributed to CuZ*(4Cu1S). However, an enhanced cathodic signal for the activated enzyme was observed under turnover conditions, which is explained by the binding of nitrous oxide to CuZ0(4Cu1S), an intermediate species in the catalytic cycle.

An induced corrosion inhibition of X80 steel by using marine bacterium Marinobacter salsuginis.

The corrosion behaviour of X80 pipeline steel was studied in a simulated marine environment inoculated with marine bacterium Marinobacter salsuginis. The electrochemical results showed that the increase in linear polarization resistance, charge transfer resistance, and the decrease in corrosion current density of the X80 pipeline steel immersed in the biotic medium indicated its high corrosion resistance compared to those in the abiotic medium. Surface morphological techniques including scanning electron microscopy, confocal laser scanning microscopy and live/dead cells staining were employed to observe the biofilm morphology and bacterial viability after different immersion times. X-ray photoelectron spectroscopy was used to analyse the oxides film formed on the steel surface. The obtained results indicated that the corrosion inhibition efficiency was obviously higher in the biotic medium compared to that in the abiotic medium. The high corrosion resistance of X80 steel in biotic medium was attributed to the formation of biofilm and the development of extracellular polymeric substances (EPS) layer on its surface.

Bacterial sugar-binding protein as a one-step affinity purification tag on dextran-containing resins.

Marinobacter hydrocarbonoclasticus is an oil-eating bacterium that possesses a large adhesion protein (MhLap) with the potential to bind extracellular ligands. One of these ligand-binding modules is the ~20-kDa PA14 domain (MhPA14) that has affinity for glucose-based carbohydrates. Previous studies showed this sugar-binding domain is retained on dextran-based size-exclusion resins during chromatography, requiring the introduction of glucose or EDTA to remove the protein from the column. Given the ready availability of such size-exclusion resins in biochemistry laboratories, this study explores the use of MhPA14 as an affinity tag for recombinant protein purification. Two different fusion proteins were tested: 1) Green fluorescent protein (GFP) linked to the N-terminus of the MhPA14 tag; and 2) the ice-binding domain from the Marinomonas primoryensis ice-binding protein (MpIBD) linked to the MhPA14 C-terminus by a TEV cut site. The GFP_MhPA14 fusion visibly bound to Superdex, Sephadex, and Sephacryl resins, but did not bind to Sepharose. Using Superdex resin, dextran-affinity purification proved to be an effective one-step purification strategy for both proteins, superior to even nickel-affinity chromatography. Dextran-affinity chromatography was also the most effective method of separating the MhPA14 tag from MpIBD following TEV proteolysis, as compared to both nickel-affinity and ice-affinity methods. These results indicate that MhPA14 has potential for widespread use in recombinant protein purification.

Marinobacter sp. Stable Hydrous Titanium Oxide-Functionalized Bovine Serum Albumin Nanospheres for Uranium Capture from Spiked Seawater.

A novel nanospherical hydrous titanium oxide adsorbent (hydrous titanium oxide-immobilized bovine serum albumin nanospheres, HTO-BSA-NSs) was prepared by immobilizing HTOs with a manipulated molecular mass and number of active sites for uranium on the surface of BSA-NSs. The adsorption performances of HTO-BSA-NSs were investigated in spiked natural seawater with extra 8 ppm uranium. The results demonstrated that HTO-BSA-NSs are capable of uranium capture from a complex aqueous matrix with a low uranium concentration. Meanwhile, the microbial stability of HTO-BSA-NSs in sterilized natural seawater with Marinobacter sp. was investigated and observed through an optical microscope and TEM, revealing that the wrapped HTOs could protect the BSA-NSs from the decomposition of microorganisms, and the structure and functional groups of HTO-BSA-NSs remain stable compared with the BSA-NSs. In addition, the uranium adsorption mechanism of HTO-BSA-NSs is mainly recognized as dehydrated complexation, which was concluded from characterization analysis, adsorption model fitting, and theoretical calculations based on density functional theory. The remarkable uranium adsorption performance and microbial stability of HTO-BSA-NSs indicated that they have the potential to be a low-cost and environmentally friendly adsorbent for uranium extraction from complex environments such as seawater or uranium-containing industrial wastewater.

Structure and functional analysis of a bacterial adhesin sugar-binding domain.

Bacterial adhesins attach their hosts to surfaces through one or more ligand-binding domains. In RTX adhesins, which are localized to the outer membrane of many Gram-negative bacteria via the type I secretion system, we see several examples of a putative sugar-binding domain. Here we have recombinantly expressed one such ~20-kDa domain from the ~340-kDa adhesin found in Marinobacter hydrocarbonoclasticus, an oil-degrading bacterium. The sugar-binding domain was purified from E. coli with a yield of 100 mg/L of culture. Circular dichroism analysis showed that the protein was rich in beta-structure, was moderately heat resistant, and required Ca2+ for proper folding. A crystal structure was obtained in Ca2+ at 1.2-Å resolution, which showed the presence of three Ca2+ ions, two of which were needed for structural integrity and one for binding sugars. Glucose was soaked into the crystal, where it bound to the sugar's two vicinal hydroxyl groups attached to the first and second (C1 and C2) carbons in the pyranose ring. This attraction to glucose caused the protein to bind certain polysaccharide-based column matrices and was used in a simple competitive binding assay to assess the relative affinity of sugars for the protein's ligand-binding site. Fucose, glucose and N-acetylglucosamine bound most tightly, and N-acetylgalactosamine hardly bound at all. Isothermal titration calorimetry was used to determine specific binding affinities, which lie in the 100-µM range. Glycan arrays were tested to expand the range of ligand sugars assayed, and showed that MhPA14 bound preferentially to branched polymers containing terminal sugars highlighted as strong binders in the competitive binding assay. Some of these binders have vicinal hydroxyl groups attached to the C3 and C4 carbons that are sterically equivalent to those presented by the C1 and C2 carbons of glucose.

Distribution of dissolved iron and bacteria producing the photoactive siderophore, vibrioferrin, in waters off Southern California and Northern Baja.

Phytoplankton blooms can cause acute effects on marine ecosystems due either to their production of endogenous toxins or to their enormous biomass leading to major impacts on local economies and public health. Despite years of effort, the causes of these Harmful Algal Blooms are still not fully understood. Our hypothesis is that bacteria that produce photoactive siderophores may provide a bioavailable source of iron for phytoplankton which could in turn stimulate algal growth and support bloom dynamics. Here we correlate iron concentrations, phytoplankton cell counts, bacterial cell abundance, and copy numbers for a photoactive siderophore vibrioferrin biosynthesis gene in water samples taken from 2017 cruises in the Gulf of California, and the Pacific Ocean off the coast of northern Baja California as well as during a multiyear sampling at Scripps Pier in San Diego, CA. We find that bacteria producing the photoactive siderophore vibrioferrin, make up a surprisingly high percentage of total bacteria in Pacific/Gulf of California coastal waters (up to 9%). Vibroferrin's unique properties and the widespread prevalence of its bacterial producers suggest that it may contribute significantly to generating bioavailability of iron via photoredox reactions.

Direct Evidence for Ferrous Ion Oxidation and Incorporation in the Absence of Oxidants by Dps from Marinobacter hydrocarbonoclasticus.

Dps proteins (DNA-binding protein from starved cells) are hollow-sphere-shaped, dodecameric enzymes found in bacteria and archaeal species. They can oxidize ferrous iron in a controlled manner using hydrogen peroxide or molecular oxygen as co-substrate, and most of them confer physical protection through DNA binding. Oxidized iron is stored, as a mineral core, in a central cavity. Direct evidence is now provided that, furthermore, Dps proteins containing small mineral cores can oxidize and mineralize toxic ferrous ions in anaerobic conditions and in the absence of any additional aqueous oxidant co-substrate. Dps proteins containing cores of 24 irons per dodecamer can oxidize about 5 ferrous irons per dodecamer, with that number approximately doubling for protein particles containing in average 96 irons per protein. This additional activity carries importance as it can be a detoxification mechanism present during anaerobic or oxygen-limited growth conditions.

Adhesion of Marinobacter hydrocarbonoclasticus to Surfactant-Decorated Dodecane Droplets.

We investigate the effect of interfacial properties on the adhesion of bacteria at oil/water interfaces using confocal microscopy. Surfactant-decorated dodecane droplets of diameter 20-60 µm are generated using a coflow microfluidic device, introduced into an aqueous saline suspension of Marinobacter hydrocarbonoclasticus bacteria, and imaged in 3-D over time. Using image analysis algorithms, we determine the number of bacteria adhering at oil/water interfaces over time in the presence of dioctyl sodium succinate (DOSS), a component of the dispersant used in oil-spill recovery. The adsorption of bacteria at the oil/water interface follows Langmuir first-order kinetics for all droplet sizes, with the greatest areal number density of bacteria adhered to the smallest droplets. We vary the surfactant type [DOSS, dicyclohexyl sodium sulfosuccinate, dibutyl sodium sulfosuccinate, cetyltrimethylammonium bromide, and Tween 20] and concentration and examine the effects on long-time adhesion of bacteria. For a fixed droplet size, the areal density of bacteria at the interface decreases with increasing surfactant concentration because of a reduction in oil/water interfacial tension that increases the free energy of adhesion of the bacterium.

Salt Adaptation and Evolutionary Implication of a Nah-related PAHs Dioxygenase cloned from a Halophilic Phenanthrene Degrading Consortium.

Polycyclic aromatic hydrocarbons (PAHs) pollutions often occur in marine and other saline environment, largely due to anthropogenic activities. However, study of the PAHs-degradation genotypes in halophiles is limited, compared with the mesophilic terrestrial PAHs degraders. In this study, a bacterial consortium (CY-1) was enriched from saline soil contaminated with crude oil using phenanthrene as the sole carbon source at 10% salinity. CY-1 was dominated by the moderate halophilic Marinobacter species, and its dominant PAHs ring-hydroxylating dioxygenase (RHD) genotypes shared high identity to the classic nah-related RHDs found in the mesophilic species. Further cloning of a 5.6-kb gene cluster from CY-1 unveiled the existence of a new type of PAHs degradation gene cluster (hpah), which most probably evolves from the nah-related gene clusters. Expression of the RHD in this gene cluster in E. coli lead to the discovery of its prominent salt-tolerant properties compared with two RHDs from mesophiles. As a common structural feature shared by all halophilic and halotolerant enzymes, higher abundance of acidic amino acids was also found on the surface of this RHD than its closest nah-related alleles. These results suggest evolution towards saline adaptation occurred after horizontal transfer of this hpah gene cluster into the halophiles.

Marinobacter lipolyticus from Red Sea for lipase production and modulation of silver nanomaterials for anti-candidal activities.

In this study, the bacterial strain CEES 33 was isolated from the coastal area of the Red Sea, Jeddah, Kingdom of Saudi Arabia. The bacterium isolate was identified and characterized by using biochemical and molecular methods. The isolate CEES 33 has been identified as Gram-negative rod shaped and cream pigmented spherical colonies. It also demonstrated a positive result for nitrate reduction, oxidase, catalase, citrate utilization, lipase and exopolysaccharide production. Strain CEES 33 was characterized at the molecular level by partial 16S rRNA sequencing and it has been identified as Marinobacter lipolyticus (EMBL|LN835275.1). The lipolytic activity of the isolate was also observed 2.105 nkatml-1. Furthermore, the bacterial aqueous extract was used for green synthesis of silver nanoparticles (AgNPs), which was further confirmed by UV-visible spectra (430 nm), XRD and SEM analysis. Moreover, the biological functional group that involved in AgNPs synthesis was confirmed by FTIR spectra. The biological activities of AgNPs were also investigated, which showed a significant growth inhibition of Candida albicans with 16 ± 2 mm zone of inhibition at 10 µg dose/wells. Therefore, bacterium Marinobacter lipolyticus might be used in future for lipase production and nanoparticles fabrication for biomedical application, to control fungal diseases caused by C. albicans.

Unusually high mechanical stability of bacterial adhesin extender domains having calcium clamps.

To gain insight into the relationship between protein structure and mechanical stability, single molecule force spectroscopy experiments on proteins with diverse structure and topology are needed. Here, we measured the mechanical stability of extender domains of two bacterial adhesins MpAFP and MhLap, in an atomic force microscope. We find that both proteins are remarkably stable to pulling forces between their N- and C- terminal ends. At a pulling speed of 1 µm/s, the MpAFP extender domain fails at an unfolding force Fu = 348 ± 37 pN and MhLap at Fu = 306 ± 51 pN in buffer with 10 mM Ca2+. These forces place both extender domains well above the mechanical stability of many other ß-sandwich domains in mechanostable proteins. We propose that the increased stability of MpAFP and MhLap is due to a combination of both hydrogen bonding between parallel terminal strands and intra-molecular coordination of calcium ions.

Five Fatty Aldehyde Dehydrogenase Enzymes from Marinobacter and Acinetobacter spp. and Structural Insights into the Aldehyde Binding Pocket.

Enzymes involved in lipid biosynthesis and metabolism play an important role in energy conversion and storage and in the function of structural components such as cell membranes. The fatty aldehyde dehydrogenase (FAldDH) plays a central function in the metabolism of lipid intermediates, oxidizing fatty aldehydes to the corresponding fatty acid and competing with pathways that would further reduce the fatty aldehydes to fatty alcohols or require the fatty aldehydes to produce alkanes. In this report, the genes for four putative FAldDH enzymes from Marinobacter aquaeolei VT8 and an additional enzyme from Acinetobacter baylyi were heterologously expressed in Escherichia coli and shown to display FAldDH activity. Five enzymes (Maqu_0438, Maqu_3316, Maqu_3410, Maqu_3572, and the enzyme reported under RefSeq accession no. WP_004927398) were found to act on aldehydes ranging from acetaldehyde to hexadecanal and also acted on the unsaturated long-chain palmitoleyl and oleyl aldehydes. A comparison of the specificities of these enzymes with various aldehydes is presented. Crystallization trials yielded diffraction-quality crystals of one particular FAldDH (Maqu_3316) from M. aquaeolei VT8. Crystals were independently treated with both the NAD+ cofactor and the aldehyde substrate decanal, revealing specific details of the likely substrate binding pocket for this class of enzymes. A likely model for how catalysis by the enzyme is accomplished is also provided.IMPORTANCE This study provides a comparison of multiple enzymes with the ability to oxidize fatty aldehydes to fatty acids and provides a likely picture of how the fatty aldehyde and NAD+ are bound to the enzyme to facilitate catalysis. Based on the information obtained from this structural analysis and comparisons of specificities for the five enzymes that were characterized, correlations to the potential roles played by specific residues within the structure may be drawn.

Carbofuran Degradation by Biogenic Manganese Oxides.

This work studied the effect of biogenic manganese oxides (Bio-MnOx) on carbofuran degradation.The results showed that 21.05 % and 90.63 % carbofuran, respectively, were degraded in 4 days by Bio-MnOx with and without NaN3 at initial pH 4.80, whereas carbofuran was hardly degraded by chemical manganese oxides in the same condition. Bio-MnOx promoted carbofuran hydrolysis by changing the pH of the environment and encouraged carbofuran phenol cleavage by its oxidization. Both the oxidation of carbofuran phenol by Bio-MnOx and the reoxidation of the released Mn(II) by Mn(II)-oxidizing microorganisms ensured the continuous reactivity of Bio-MnOx and prevented the secondary pollution of Mn(II). Carbofuran phenol was the major transformation product in the degradation and was further oxidized into small organic molecules as monitored by a GC/MS analyzer. This report offers an efficient, feasible, and no-secondary-pollution approach to controlling carbofuran pollution.

Probing the metabolic water contribution to intracellular water using oxygen isotope ratios of PO4.

Knowledge of the relative contributions of different water sources to intracellular fluids and body water is important for many fields of study, ranging from animal physiology to paleoclimate. The intracellular fluid environment of cells is challenging to study due to the difficulties of accessing and sampling the contents of intact cells. Previous studies of multicelled organisms, mostly mammals, have estimated body water composition-including metabolic water produced as a byproduct of metabolism-based on indirect measurements of fluids averaged over the whole organism (e.g., blood) combined with modeling calculations. In microbial cells and aquatic organisms, metabolic water is not generally considered to be a significant component of intracellular water, due to the assumed unimpeded diffusion of water across cell membranes. Here we show that the (18)O/(16)O ratio of PO4 in intracellular biomolecules (e.g., DNA) directly reflects the O isotopic composition of intracellular water and thus may serve as a probe allowing direct sampling of the intracellular environment. We present two independent lines of evidence showing a significant contribution of metabolic water to the intracellular water of three environmentally diverse strains of bacteria. Our results indicate that ∼30-40% of O in PO4 comprising DNA/biomass in early stationary phase cells is derived from metabolic water, which bolsters previous results and also further suggests a constant metabolic water value for cells grown under similar conditions. These results suggest that previous studies assuming identical isotopic compositions for intracellular/extracellular water may need to be reconsidered.

Flagellin from Marinobacter algicola and Vibrio vulnificus activates the innate immune response of gilthead seabream.

Adjuvants have emerged as the best tools to enhance the efficacy of vaccination. However, the traditional adjuvants used in aquaculture may cause adverse alterations in fish making necessary the development of new adjuvants able to stimulate the immune system and offer strong protection against infectious pathogens with minimal undesirable effects. In this respect, flagellin seems an attractive candidate due to its ability to strongly stimulate the immune response of fish. In the present study, we have evaluated the ability of recombinant flagellin from Marinobacter algicola (MA) and Vibrio vulnificus (Vvul), a non-pathogenic and a pathogenic bacteria, respectively, to stimulate the innate immune system of gilthead seabream (Sparus aurata L.) and compare the effect with that of the classical flagellin from Salmonella enterica serovar Typhimurium (Salmonella Typhimurium, STF). Intraperitoneal injection of MA and Vvul resulted in a strong inflammatory response characterized by increased reactive oxygen species production and the infiltration of acidophilic granulocytes at the injection site. Interestingly, however, only flagellin from MA consistently induced the expression of the gene encoding pro-inflammatory interleukin-1ß. These effects were further confirmed in vitro, where a dose-dependent activation of macrophages and acidophilic granulocytes by MA and Vvul flagellins was observed. In contrast, STF flagellin was found to be less potent in both in vivo and in vitro experiments. Our results suggest the potential use of MA and Vvul flagellins as immunostimulants and adjuvants for fish vaccination.

Fatty alcohols for wax esters in Marinobacter aquaeolei VT8: two optional routes in the wax biosynthesis pathway.

The biosynthesis of wax esters in bacteria is accomplished by a unique pathway that combines a fatty alcohol and a fatty acyl coenzyme A substrate. Previous in vitro enzymatic studies indicated that two different enzymes could be involved in the synthesis of the required fatty alcohol in Marinobacter aquaeolei VT8. In this study, we demonstrate through a series of gene deletions and transcriptional analysis that either enzyme is capable of fulfilling the role of providing the fatty alcohol required for wax ester biosynthesis in vivo, but evolution has clearly selected one of these, a previously characterized fatty aldehyde reductase, as the preferred enzyme to perform this reaction under typical wax ester-accumulating conditions. These results complement previous in vitro studies and provide the first glimpse into the role of each enzyme in vivo in the native organism.

Crystal structure of a catalytically active GG(D/E)EF diguanylate cyclase domain from Marinobacter aquaeolei with bound c-di-GMP product.

Recent studies of signal transduction in bacteria have revealed a unique second messenger, bis-(3'-5')-cyclic dimeric GMP (c-di-GMP), which regulates transitions between motile states and sessile states, such as biofilms. C-di-GMP is synthesized from two GTP molecules by diguanylate cyclases (DGC). The catalytic activity of DGCs depends on a conserved GG(D/E)EF domain, usually part of a larger multi-domain protein organization. The domains other than the GG(D/E)EF domain often control DGC activation. This paper presents the 1.83 Å crystal structure of an isolated catalytically competent GG(D/E)EF domain from the A1U3W3_MARAV protein from Marinobacter aquaeolei. Co-crystallization with GTP resulted in enzymatic synthesis of c-di-GMP. Comparison with previously solved DGC structures shows a similar orientation of c-di-GMP bound to an allosteric regulatory site mediating feedback inhibition of the enzyme. Biosynthesis of c-di-GMP in the crystallization reaction establishes that the enzymatic activity of this DGC domain does not require interaction with regulatory domains.

Identification of amino acids involved in the hydrolytic activity of lipase LipBL from Marinobacter lipolyticus.

The lipolytic enzyme family VIII currently includes only seven members but represents a group of lipolytic enzymes with interesting properties. Recently, we identified a gene encoding the family VIII lipase LipBL from the halophilic bacterium Marinobacter lipolyticus. This enzyme, like most lipolytic enzymes from family VIII, possesses two possible nucleophilic serines located in an S-X-X-K ß-lactamase motif and a G-X-S-X-G lipase motif. The serine in the S-X-X-K motif is a catalytic residue, but the role of serine within the common lipase consensus sequence G-X-S-X-G has not yet been systematically studied. Here, the previously reported time-intensive procedure for purification of recombinant LipBL was replaced by one-step metal-affinity chromatography purification in the presence of ATP. Heterologous co-expression of His(6)-tagged LipBL with the cytoplasmic molecular chaperones GroEL/GroES was necessary to obtain catalytically active LipBL. Site-directed mutagenesis performed to map the active site of LipBL revealed that mutation of serine and lysine in the ß-lactamase motif (S(72)-M-T-K(75)) to alanine abolished the enzyme activity of LipBL, in contrast to mutation of the serine in the lipase consensus motif (S321A). Furthermore, mutagenesis was performed to understand the role of the G-X-S-X-G motif and other amino acids that are conserved among family VIII esterases. We describe how mutations in the conserved G-X-S-X-G motif altered the biochemical properties and substrate specificity of LipBL. Molecular modelling results indicate the location of the G-X-S(321)-X-G motif in a loop close to the catalytic centre of LipBL, presumably representing a substrate-binding site of LipBL.