Catalytic site flexibility facilitates the substrate and catalytic promiscuity of Vibrio dual lipase/transferase.

Although enzyme catalysis is typified by high specificity, enzymes can catalyze various substrates (substrate promiscuity) and/or different reaction types (catalytic promiscuity) using a single active site. This interesting phenomenon is widely distributed in enzyme catalysis, with both fundamental and applied importance. To date, the mechanistic understanding of enzyme promiscuity is very limited. Herein, we report the structural mechanism underlying the substrate and catalytic promiscuity of Vibrio dual lipase/transferase (VDLT). Crystal structures of the VDLT from Vibrio alginolyticus (ValDLT) and its fatty acid complexes were solved, revealing prominent structural flexibility. In particular, the "Ser-His-Asp" catalytic triad machinery of ValDLT contains an intrinsically flexible oxyanion hole. Analysis of ligand-bound structures and mutagenesis showed that the flexible oxyanion hole and other binding residues can undergo distinct conformational changes to facilitate substrate and catalytic promiscuity. Our study reveals a previously unknown flexible form of the famous catalytic triad machinery and proposes a "catalytic site tuning" mechanism to expand the mechanistic paradigm of enzyme promiscuity.

Lysine acetylation of Escherichia coli lactate dehydrogenase regulates enzyme activity and lactate synthesis.

As an evolutionarily conserved posttranslational modification, protein lysine acetylation plays important roles in many physiological and metabolic processes. However, there are few reports about the applications of lysine acetylation in metabolic regulations. Lactate is a main byproduct in microbial fermentation, and itself also an important bulk chemical with considerable commercial values in many fields. Lactate dehydrogenase (LdhA) is the key enzyme catalyzing lactate synthesis from pyruvate. Here, we reported that Escherichia coli LdhA can be acetylated and the acetylated lysine sites were identified by mass spectrometry. The effects and regulatory mechanisms of acetylated sites on LdhA activity were characterized. Finally, lysine acetylation was successfully used to regulate the lactate synthesis. LdhA (K9R) mutant overexpressed strain improved the lactate titer and glucose conversion efficiency by 1.74 folds than that of wild-type LdhA overexpressed strain. LdhA (K154Q-K248Q) mutant can inhibit lactate accumulation and improve 3HP production. Our study established a paradigm for lysine acetylation in lactate synthesis regulation and suggested that lysine acetylation may be a promising strategy to improve the target production and conversion efficiency in microbial synthesis. The application of lysine acetylation in regulating lactate synthesis also provides a reference for the treatment of lactate-related diseases.

Structural basis of staphylococcal Stl inhibition on a eukaryotic dUTPase.

dUTPases are key enzymes in all life kingdoms. A staphylococcal repressor protein (Stl) inhibited dUTPases from multiple species to various extents. Understanding the molecular basis underlying the inhibition differences is crucial to develop effective proteinaceous inhibitors of dUTPases. Herein, we report the complex structure of Stl N-terminal domain (StlN-ter) and Litopenaeus vannamei dUTPase domain (lvDUT65-210). Stl inhibited lvDUT65-210 through its N-terminal domain. The lvDUT65-210-StlN-ter complex structure revealed a heterohexamer encompassing three StlN-ter monomers bound to one lvDUT65-210 trimer, generating two types of Stl-dUTPase interfaces. Interface I is formed by Stl interaction with the lvDUT65-210 active-site region that is contributed by motifs I-IV from its two subunits; interface II results from Stl binding to the C-terminal motif V of the third lvDUT65-210 subunit. Structural comparison revealed both conserved features and obvious differences in Stl-dUTPase interaction patterns, giving clues about the inhibition differences of Stl on dUTPases. Noticeably, interface II is only observed in lvDUT65-210-StlN-ter. The Stl-interacting residues of lvDUT65-210 are conserved in other eukaryotic dUTPases, particularly human dUTPase. Altogether, our study presents the first structural model of Stl interaction with eukaryotic dUTPase, contributing to a more complete view of Stl inhibition and facilitating the development of proteinaceous inhibitor for eukaryotic dUTPases.

Kinematic ME-MAFA for Pseudolite Carrier-Phase Ambiguity Resolution in Precise Single-Point Positioning.

Precise single-point positioning using carrier-phase measurements can be provided by the synchronized pseudolite system. The primary task of carrier phase positioning is ambiguity resolution (AR) with rapidity and reliability. As the pseudolite system is usually operated in the dense multipath environment, cycle slips may lead the conventional least-squares ambiguity decorrelation adjustment (LAMBDA) method to incorrect AR. A new AR method based on the idea of the modified ambiguity function approach (MAFA), which is insensitive to the cycle slips, is studied in this paper. To improve the model strength of the MAFA and to eliminate the influence of constant multipath biases on the time-average model in static mode, the kinematic multi-epoch MAFA (kinematic ME-MAFA) algorithm is proposed. A heuristic method for predicting the 'float position' corresponding to every Voronoi cell of the next epoch, making use of Doppler-based velocity information, is implemented to improve the computational efficiency. If the success rate is very close to 1, it is possible to guarantee reliable centimeter-level accuracy positioning without further ambiguity validation. Therefore, a computing method of the success rate for the kinematic ME-MAFA is proposed. Both the numerical simulations and the kinematic experiment demonstrate the feasibility of the new AR algorithm according to its accuracy and reliability. The accuracy of the horizontal positioning solution is better than 1.7 centimeters in our pseudolite system.

Structural analysis of the CARB ß-lactamase from Vibrio parahaemolyticus facilitates application of the ß-lactam/ß-lactamase inhibitor therapy.

ß-Lactams are the most widely used antibiotics in treating bacterial infections. However, they are rarely applied in infections caused by Vibrio parahaemolyticus, as the bacterium is intrinsically resistant to penicillins by expressing ß-lactamase. Here we report structural characterization of the CARB ß-lactamase from V. parahaemolyticus (CARB-20). CARB-20 is a class A ß-lactamase, belonging to subclass A1 (containing 70STFKAL75, 130SDNTAANL137, 164RXEXXLN170, 231VGDKTG236, etc.), group LSBL2 (with the disulfide bridge C77-C123, motif 231IADRSGAG238 and R244). CARB-20 adopts a typical subclass A1 ß-lactamase fold consisting of two domains. Its active site is constituted by four conserved motifs, similar to that of known subclass A1 ß-lactamases. Analysis of the active site structure reveals its substrate preference for penicillin, ampicillin and carbenicillin but not for latterly developed cephalosporins. Meanwhile, ß-lactamase inhibitors such as clavulanate and sulbactam can well fit into the active site, supporting ß-lactams combined with ß-lactamase inhibitors as a potential approach for treating infection of V. parahaemolyticus. The residues around the active site show certain variations, which can be useful for specific inhibitor design. In the directed evolution experiment, CARB-20 exhibited plasticity in developing significant resistance to inhibitors by accumulated residue substitutions. Therefore, careful monitoring of enzyme mutations is necessary for successfully applying ß-lactam/ß-lactamase inhibitor combination therapy. Taken together, our results open up an avenue of inhibitor design targeting vibrio ß-lactamases, facilitating the application of ß-lactams in treating vibrio infections.

Structural analysis of a Vibrio phospholipase reveals an unusual Ser-His-chloride catalytic triad.

Phospholipases can disrupt host membranes and are important virulence factors in many pathogens. VvPlpA is a phospholipase A2 secreted by Vibrio vulnificus and essential for virulence. Its homologs, termed thermolabile hemolysins (TLHs), are widely distributed in Vibrio bacteria, but no structural information for this virulence factor class is available. Herein, we report the crystal structure of VvPlpA to 1.4-Å resolution, revealing that VvPlpA contains an N-terminal domain of unknown function and a C-terminal phospholipase domain and that these two domains are packed closely together. The phospholipase domain adopts a typical SGNH hydrolase fold, containing the four conserved catalytic residues Ser, Gly, Asn, and His. Interestingly, the structure also disclosed that the phospholipase domain accommodates a chloride ion near the catalytic His residue. The chloride is five-coordinated in a distorted bipyramid geometry, accepting hydrogen bonds from a water molecule and the amino groups of surrounding residues. This chloride substitutes for the most common Asp/Glu residue and forms an unusual Ser-His-chloride catalytic triad in VvPlpA. The chloride may orient the catalytic His and stabilize the charge on its imidazole ring during catalysis. Indeed, VvPlpA activity depended on chloride concentration, confirming the important role of chloride in catalysis. The VvPlpA structure also revealed a large hydrophobic substrate-binding pocket that is capable of accommodating a long-chain acyl group. Our results provide the first structure of the TLH family and uncover an unusual Ser-His-chloride catalytic triad, expanding our knowledge on the biological role of chloride.

Structural basis for specific calcium binding by the polycystic-kidney-disease domain of Vibrio anguillarum protease Epp.

Extracellular proteases are often produced as pre-pro-enzyme and then undergo multiple processing steps to mature into the active form. The protease Epp, a virulent factor of Vibrio anguillarum, belongs to this family. Its maturation might be regulated by Ca2+ via its polycystic kidney disease (PKD) domain, but the molecular mechanism is unknown. Herein, we report the crystal structure of the first PKD domain from V. anguillarum Epp (Epp-PKD1) and its specific Ca2+-binding capacity. Epp-PKD1 exists as a monomer, consisting of seven ß-strands which form two ß-sheets stacking with each other. One Ca2+ is bound by the residues Asn3, Gln4, Asp27, Asp29, Asp68 and a water molecule with a pentagonal bipyramidal geometry. Incubating the apo Epp-PKD1 with Ca2+ but not Mg2+, Mn2+, or Zn2+, enhances the thermal and chemical stability of Epp-PKD1, indicating its specific binding to Ca2+. Epp-PKD1 shares high similarity in both sequence and overall structure with that of Vibrio cholerae PrtV, a homologous protease of Epp, however, they differ in the oligomeric state and local structure at the Ca2+-binding site, suggesting maturation of PrtV and Epp might be differently regulated by Ca2+. Likely, proteases may take advantage of the structural diversity in PKD domains to tune their Ca2+-regulated maturation process.

The putative siderophore-interacting protein from Vibrio anguillarum: protein production, analysis, crystallization and X-ray crystallographic studies.

Siderophore-interacting proteins (SIPs) play an important role in iron acquisition in many bacteria. SIPs release iron from the internalized ferric siderophore complex by reducing ferric iron to ferrous iron, but how the iron is reduced is not well understood. Here, a sip gene was identified in the genome of Vibrio anguillarum 775. To further understand the catalytic mechanism of the protein, the SIP was overexpressed in Escherichia coli Rosetta (DE3) cells, purified and crystallized for X-ray diffraction analysis. The crystal diffracted to 1.113 Šresolution and belonged to space group P21, with unit-cell parameters a = 64.63, b = 58.47, c = 70.65 Å, ß = 114.19°.

Structural and enzymatic analysis of the cytochrome b5 reductase domain of Ulva prolifera nitrate reductase.

Rapid accumulations of unattached green macroalgae, referred to as blooms, constitute ecological disasters and occur in many coastal regions. Ulva are a major cause of blooms, owing to their high nitrogen utilization capacity, which requires nitrate reductase (NR) activity; however, molecular characterization of Ulva NR remains lacking. Herein we determined the crystal structure and performed an enzymatic analysis of the cytochrome b5 reductase domain of Ulva prolifera NR (UpCbRNR). The structural analysis revealed an N-terminal FAD-binding domain primarily consisting of six antiparallel ß strands, a C-terminal NADH-binding domain forming a Rossmann fold, and a three ß-stranded linker region connecting these two domains. The FAD cofactor was located in the cleft between the two domains and interacted primarily with the FAD-binding domain. UpCbRNR shares similarities in overall structure and cofactor interactions with homologs, and its catalytic ability is comparable to that of higher plant CbRNRs. Structure and sequence comparisons of homologs revealed two regions of sequence length variation potentially useful for phylogenetic analysis: one in the FAD-binding domain, specific to U. prolifera, and another in the linker region that may be used to differentiate between plant, fungi, and animal homologs. Our data will facilitate molecular-level understanding of nitrate assimilation in Ulva.

Malonyl-CoA pathway: a promising route for 3-hydroxypropionate biosynthesis.

3-Hydroxypropionate (3HP) is an attractive platform chemical, serving as a precursor to a variety of commodity chemicals like acrylate and acrylamide, as well as a monomer of a biodegradable plastic. To establish a sustainable way to produce these commercially important chemicals and materials, fermentative production of 3HP is widely investigated in recent years. It is reported that 3HP can be produced from several intermediates, such as glycerol, malonyl-CoA, and ß-alanine. Among all these biosynthetic routes, the malonyl-CoA pathway has some distinct advantages, including a broad feedstock spectrum, thermodynamic feasibility, and redox neutrality. To date, this pathway has been successfully constructed in various species including Escherichia coli, yeast and cyanobacteria, and optimized through carbon flux redirection, enzyme screening and engineering, and an increasing supply of energy and cofactors, resulting in significantly enhanced 3HP titer up to 40 g/L. These results show the feasibility of commercial manufacturing of 3HP and its derivatives in the future.

Functional balance between enzymes in malonyl-CoA pathway for 3-hydroxypropionate biosynthesis.

3-Hydroxypropionate (3HP) is an important platform chemical, and four 3HP biosynthetic routes were reported, in which the malonyl-CoA pathway has some expected advantages but presented the lowest 3HP yield. Here, we demonstrated that this low yield was caused by a serious functional imbalance between MCR-C and MCR-N proteins, responsible for the two-step reduction of malonyl-CoA to 3HP. Then we minimized the enzyme activity imbalance by directed evolution of rate-limiting enzyme MCR-C and fine tuning of MCR-N expression level. Combined with culture conditions optimization, our engineering approaches increased the 3HP titer 270-fold, from 0.15 g/L to 40.6 g/L, representing the highest 3HP production via malonyl-CoA pathway so far. This study not only significantly improved the 3HP productivity of recombinant Escherichia coli strain, but also proved the importance of metabolic balance in a multistep biosynthetic pathway, which should be always considered in any metabolic engineering study.

Development of genetically stable Escherichia coli strains for poly(3-hydroxypropionate) production.

Poly(3-hydroxypropionate) (P3HP) is a biodegradable and biocompatible thermoplastic. In our previous study, a pathway for P3HP production was constructed in recombinant Esecherichia coli. Seven exogenous genes in P3HP synthesis pathway were carried by two plasmid vectors. However, the P3HP production was severely suppressed by strain instability due to plasmid loss. In this paper, two strategies, chromosomal gene integration and plasmid addiction system (PAS) based on amino acid anabolism, were applied to construct a genetically stable strain. Finally, a combination of those two methods resulted in the best results. The resultant strain carried a portion of P3HP synthesis genes on chromosome and the others on plasmid, and also brought a tyrosine-auxotrophy based PAS. In aerobic fed-batch fermentation, this strain produced 25.7 g/L P3HP from glycerol, about 2.5-time higher than the previous strain with two plasmids. To the best of our knowledge, this is the highest P3HP production from inexpensive carbon sources.

Dissection of malonyl-coenzyme A reductase of Chloroflexus aurantiacus results in enzyme activity improvement.

The formation of fusion protein in biosynthetic pathways usually improves metabolic efficiency either channeling intermediates and/or colocalizing enzymes. In the metabolic engineering of biochemical pathways, generating unnatural protein fusions between sequential biosynthetic enzymes is a useful method to increase system efficiency and product yield. Here, we reported a special case. The malonyl-CoA reductase (MCR) of Chloroflexus aurantiacus catalyzes the conversion of malonyl-CoA to 3-hydroxypropionate (3HP), and is a key enzyme in microbial production of 3HP, an important platform chemical. Functional domain analysis revealed that the N-terminal region of MCR (MCR-N; amino acids 1-549) and the C-terminal region of MCR (MCR-C; amino acids 550-1219) were functionally distinct. The malonyl-CoA was reduced into free intermediate malonate semialdehyde with NADPH by MCR-C fragment, and further reduced to 3HP by MCR-N fragment. In this process, the initial reduction of malonyl-CoA was rate limiting. Site-directed mutagenesis demonstrated that the TGXXXG(A)X(1-2)G and YXXXK motifs were important for enzyme activities of both MCR-N and MCR-C fragments. Moreover, the enzyme activity increased when MCR was separated into two individual fragments. Kinetic analysis showed that MCR-C fragment had higher affinity for malonyl-CoA and 4-time higher K cat/K m value than MCR. Dissecting MCR into MCR-N and MCR-C fragments also had a positive effect on the 3HP production in a recombinant Escherichia coli strain. Our study showed the feasibility of protein dissection as a new strategy in biosynthetic systems.

Biosynthesis of poly(3-hydroxypropionate-co-3-hydroxybutyrate) with fully controllable structures from glycerol.

As the most representative biodegradable thermoplastic, poly(3-hydroxybutyrate) (P3HB) has a limited range of applications because of its poor thermal and physical properties. To improve its properties, a novel biosynthetic system was designed to produce poly(3-hydroxypropionate-co-3-hydroxybutyrate) (P(3HP-co-3HB)) with fully controllable structures from inexpensive carbon source. In this system, two parallel synthetic pathways controlled by independent regulatory systems were used to produce the 3HP and 3HB monomers, respectively. Through tuning the expression level of appropriate genes, P(3HP-co-3HB) copolyesters were synthesized with a wide range of 3HP fraction from 11.5 mol% to 94.6 mol%. Differential scanning calorimetry analysis demonstrated that the thermal properties of P(3HP-co-3HB) copolymer were totally dependent on its composition. The bioreactor cultivation was also performed and accumulated 9.8 g/L P(48.2 mol% 3HP-co-3HB) using glycerol as sole carbon source, which represented the highest production so far.

Biosynthesis of poly(3-hydroxypropionate) from glycerol by recombinant Escherichia coli.

Poly(3-hydroxypropionate) (P3HP) is a biodegradable and biocompatible thermoplastic. In this study, a P3HP biosynthetic pathway from glycerol was constructed in recombinant Escherichia coli. The genes for glycerol dehydratase and its reactivating factor (dhaB123 and gdrAB, from Klebsiella pneumoniae), propionaldehyde dehydrogenase (pduP, from Salmonella typhimurium), and polyhydroxyalkanoate synthase (phaC1, from Cupriavidus necator) were cloned and expressed in E. coli. After culture condition optimization, the final engineered strain accumulated 10.1 g/L P3HP (46.4% of the cell dry weight) using glycerol and glucose as cosubstrates in an aerobic fed-batch fermentation. To date, this is the highest P3HP production without addition of any expensive precursor.

Biosynthetic pathway for poly(3-hydroxypropionate) in recombinant Escherichia coli.

Poly(3-hydroxypropionate) (P3HP) is a biodegradable and biocompatible thermoplastic. In this study, we engineered a P3HP biosynthetic pathway in recombinant Escherichia coli. The genes for malonyl-CoA reductase (mcr, from Chloroflexus aurantiacus), propionyl-CoA synthetase (prpE, from E. coli), and polyhydroxyalkanoate synthase (phaC1, from Ralstonia eutropha) were cloned and expressed in E. coli. The E. coli genes accABCD encoding acetyl-CoA carboxylase were used to channel the carbon into the P3HP pathway. Using glucose as a sole carbon source, the cell yield and P3HP content were 1.32 g/L and 0.98% (wt/wt [cell dry weight]), respectively. Although the yield is relatively low, our study shows the feasibility of engineering a P3HP biosynthetic pathway using a structurally unrelated carbon source in bacteria.

Differential expression of aging biomarkers at different life stages of the annual fish Nothobranchius guentheri.

Short-lived vertebrates such as annual fishes serve as model organisms for gaining insights into the mechanisms of aging, but the study on the changes in age-related markers during the normal aging of the annual fish Nothobranchius guentheri, one of the earliest fish models for aging research, remains open. This study was undertaken to examine the changes in the expression of age-related markers at the different developmental stages of N. guentheri, and to evaluate its suitability as a model organism for aging studies. Here we clearly demonstrated that the expression of senescence-associated ß-galactosidase and accumulation of lipofuscin increased with age. Similarly, the protein oxidation and lipid peroxidation also increased with age. By contrast, the activities of catalase, glutathione peroxidase and superoxide dismutase decreased with age, while the activity of telomerase showed no apparent change with age. Taken together, these data suggest that the annual fish N. guentheri can be a suitable model for aging studies. It also provides a set of biomarkers that can be used to trace the process of normal tissue aging in N. guentheri.