Ping-Yi Li

Structural basis for dimerization and catalysis of a novel esterase from the GTSAG motif subfamily of the bacterial hormone-sensitive lipase family

Background: Catalytic mechanisms of GTSAG motif subfamily enzymes of the bacterial hormone-sensitive lipases (HSLs) family are largely unknown. Results: E25, a GTSAG motif subfamily esterase, adopts a novel dimerization pattern. Dimerization keeps the catalytic Asp282 orientation for E25 catalysis. Conclusion: Dimerization and some catalytic profiles of E25 are distinctive from other HSLs. Significance: Our study sheds light on protein folding and evolution of HSLs. Hormone-sensitive lipases (HSLs) are widely distributed in microorganisms, plants, and animals. Microbial HSLs are classified into two subfamilies, an unnamed new subfamily and the GDSAG motif subfamily. Due to the lack of structural information, the detailed catalytic mechanism of the new subfamily is not yet clarified. Based on sequence analysis, we propose to name the new subfamily as the GTSAG motif subfamily. We identified a novel HSL esterase E25, a member of the GTSAG motif subfamily, by functional metagenomic screening, and resolved its structure at 2.05 Å. E25 is mesophilic (optimum temperature at 50 °C), salt-tolerant, slightly alkaline (optimum pH at 8.5) for its activity, and capable of hydrolyzing short chain monoesters (C2–C10). E25 tends to form dimers both in the crystal and in solution. An E25 monomer contains an N-terminal CAP domain, and a classical α/β hydrolase-fold domain. Residues Ser186, Asp282, and His312 comprise the catalytic triad. Structural and mutational analyses indicated that E25 adopts a dimerization pattern distinct from other HSLs. E25 dimer is mainly stabilized by an N-terminal loop intersection from the CAP domains and hydrogen bonds and salt bridges involving seven highly conserved hydrophilic residues from the catalytic domains. Further analysis indicated that E25 also has some catalytic profiles different from other HSLs. Dimerization is essential for E25 to exert its catalytic activity by keeping the accurate orientation of the catalytic Asp282 within the catalytic triad. Our results reveal the structural basis for dimerization and catalysis of an esterase from the GTSAG motif subfamily of the HSL family.

Directly LD-pumped passively Q-switched YVO4-Nd:YVO4 laser at 1.34 μm with a V3+: YAG saturable absorber

The performance of a directly LD-pumped passively Q-switched YVO4-Nd:YVO4 laser at 1.34 μm with a V3+:YAG saturable absorber was demonstrated for the first time to the best of our knowledge. The average output power of 580 mW was obtained at the pump power of 8.8 W, corresponding to the optical conversion efficiency of 6.6% and slope efficiency of 11%. The minimum pulse width was 280 ns with the pulse repetition rate of 230 kHz, which was attained with a T = 5.6% output coupler at the pump power of 8.8 W.

Genetic structure of three fosmid‐fragments encoding 16S rRNA genes of the Miscellaneous Crenarchaeotic Group (MCG): implications for physiology and evolution of marine sedimentary archaea

Archaea of the Miscellaneous Crenarchaeotic Group (MCG) exist widely in soil, freshwater and marine sediments of both surface and subsurface. However, current knowledge about this group is limited to its phylogenetic diversity. An archaeal 16S library was constructed from a sediment sample from the South China Sea, which was dominated by MCG and Marine Group I (MG-I). A metagenomic library was constructed from the same sediment sample, and three MCG fosmids (E6-3G, E37-7F and E48-1C) containing 16S rRNA genes were screened. Annotation showed that the three genomic fragments encode a variety of open reading frames (ORFs) that are potentially homologous to important functional genes related to lipid biosynthesis, energy metabolism, and resistance to oxidants. No colinear regions were found between MCG fosmids and reported archaeal genomic fragments or genomes, suggesting that the MCG archaea are quite different from the sequenced archaea in gene arrangement. Analyses of both the phylogenies of 16S rRNA genes and several informational processing genes and nucleotide frequencies showed that MCG archaea are distinct from MG-I plus relatives. In addition, tetranucleotide frequency analysis in combination with phylogenetic analysis suggested that some fragments in the MCG fosmids are probably derived from non-MCG or non-archaeal genomes.

Interdomain hydrophobic interactions modulate the thermostability of microbial esterases from the hormone-sensitive lipase family.

Background: The effect of interdomain interactions on the thermostability of microbial hormone-sensitive lipases (HSLs) remains unclear. Results: The absence of interdomain hydrophobic interactions between loop 1 and α7 leads to the thermolability of E40, a thermolabile HSL esterase. Conclusion: Interdomain hydrophobic interactions are a key element for the thermostability of microbial HSLs. Significance: Our study is helpful for protein engineering of thermolabile HSLs. Microbial hormone-sensitive lipases (HSLs) contain a CAP domain and a catalytic domain. However, it remains unclear how the CAP domain interacts with the catalytic domain to maintain the stability of microbial HSLs. Here, we isolated an HSL esterase, E40, from a marine sedimental metagenomic library. E40 exhibited the maximal activity at 45 °C and was quite thermolabile, with a half-life of only 2 min at 40 °C, which may be an adaptation of E40 to the permanently cold sediment environment. The structure of E40 was solved to study its thermolability. Structural analysis showed that E40 lacks the interdomain hydrophobic interactions between loop 1 of the CAP domain and α7 of the catalytic domain compared with its thermostable homologs. Mutational analysis showed that the introduction of hydrophobic residues Trp202 and Phe203 in α7 significantly improved E40 stability and that a further introduction of hydrophobic residues in loop 1 made E40 more thermostable because of the formation of interdomain hydrophobic interactions. Altogether, the results indicate that the absence of interdomain hydrophobic interactions between loop 1 and α7 leads to the thermolability of E40. In addition, a comparative analysis of the structures of E40 and other thermolabile and thermostable HSLs suggests that the interdomain hydrophobic interactions between loop 1 and α7 are a key element for the thermostability of microbial HSLs. Therefore, this study not only illustrates the structural element leading to the thermolability of E40 but also reveals a structural determinant for HSL thermostability.

High efficiency Nd:YAG ceramic eye-safe laser operating at 1442.8 nm

We report on a diode-pumped Nd:YAG ceramic laser operating at 1442.8 nm for the first time. In our experiment, two different Nd:YAG ceramics with the Nd-doped concentrations of 1.0 and 0.6 at. % and a Nd:YAG with the Nd-doped concentration of 1.0 at. % were used as the laser gain mediums, respectively. At a pump power of 20.7 W, a maximum output power of up to 3.96 W with optical-to-optical efficiency of up to 19.1% was obtained by using the 1.0 at. % Nd-doped ceramic as the laser gain medium. To the best of our knowledge, this is the highest output power of a LD-pumped 1.44 μm Nd:YAG ceramic laser and the highest optical-to-optical efficiency of a LD-pumped 1.44 μm Nd-doped crystal laser.

Gene Cloning, Expression and Characterization of a Novel Xylanase from the Marine Bacterium, Glaciecola mesophila KMM241

Marine xylanases are rather less studied compared to terrestrial xylanases. In this study, a new xylanase gene, xynB, was cloned from the marine bacterium, Glaciecola mesophila KMM241, and expressed in Escherichia coli. xynB encodes a multi-domain xylanase XynB of glycoside hydrolase (GH) family 8. The recombinant XynB comprises an N-terminal domain (NTD) with unknown function and a catalytic domain, which is structurally novel among the characterized xylanases of GH family 8. XynB has the highest identity (38%) to rXyn8 among the characterized xylanases. The recombinant XynB showed maximal activity at pH 6–7 and 35 °C. It is thermolabile and salt-tolerant. XynB is an endo-xylanase that demands at least five sugar moieties for effective cleavage and to hydrolyze xylohexaose and xylopentaose into xylotetraose, xylotriose and xylobiose. NTD was expressed in Escherichia coli to analyze its function. The recombinant NTD exhibited a high binding ability to insoluble xylan and avicel and little binding ability to chitosan and chitin. Since the NTD shows no obvious homology to any known carbohydrate-binding module (CBM) sequence in public databases, XynB may contain a new type of CBM.

Characterization of a cryptic plasmid pSM429 and its application for heterologous expression in psychrophilic Pseudoalteromonas

BackgroundPseudoalteromonas is an important genus widespread in marine environment, and a lot of psychrophilic Pseudoalteromonas strains thrive in deep sea and polar sea. By now, there are only a few genetic systems for Pseudoalteromonas reported and no commercial Pseudoalteromonas genetic system is available, which impedes the study of Pseudoalteromonas, especially for psychrophilic strains. The aim of this study is to develop a heterologous expression system for psychrophilic Pseudoalteromonas.ResultsA cryptic plasmid pSM429 isolated from psychrophilic Pseudoalteromonas sp. BSi20429 from the Arctic sea ice, was sequenced and characterized. The plasmid pSM429 is 3874 bp in length, with a G+C content of 28%. Four putative open reading frames (ORFs) were identified on pSM429. Based on homology, the ORF4 was predicted to encode a replication initiation (Rep) protein. A shuttle vector (Escherichia coli, Pseudoalteromonas), pWD, was constructed by ligating pSM429 and pUC19 and inserting a chloramphenicol acetyl transferase (CAT) cassette conferring chloramphenicol resistance. To determine the minimal replicon of pSM429 and to check the functionality of identified ORFs, various pWD derivatives were constructed. All derivatives except the two smallest ones were shown to allow replication in Pseudoalteromonas sp. SM20429, a plasmid-cured strain of Pseudoalteromonas sp. BSi20429, suggesting that the orf4 and its flanking intergenic regions are essential for plasmid replication. Although not essential, the sequence including some repeats between orf1 and orf2 plays important roles in segregational stability of the plasmid. With the aid of pWD-derived plasmid pWD2, the erythromycin resistance gene and the cd gene encoding the catalytic domain of a cold-adapted cellulase were successfully expressed in Pseudoalteromonas sp. SM20429.ConclusionsPlasmid pSM429 was isolated and characterized, and the regions essential for plasmid replication and stability were determined, helping the development of pSM429-based shuttle vectors. The shuttle vectors pWD and its derivatives could be used as cloning vectors for Pseudoalteromonas, offering new perspectives in the genetic manipulation of Pseudoalteromonas strains. With the aid of pWD-derived vector and its host, the erythromycin resistance gene and the cd gene of a cold-adapted protein were successfully expressed, indicating that the potential use of this system for recombinant protein production, especially for cold-adapted proteins.

Characterization of a New Cold-Adapted and Salt-Activated Polysaccharide Lyase Family 7 Alginate Lyase from Pseudoalteromonas sp. SM0524

Marine bacterial alginate lyases play a role in marine alginate degradation and carbon cycling. Although a large number of alginate lyases have been characterized, reports on alginate lyases with special characteristics are still rather less. Here, a gene alyPM encoding an alginate lyase of polysaccharide lyase family 7 (PL7) was cloned from marine Pseudoalteromonas sp. SM0524 and expressed in Escherichia coli. AlyPM shows 41% sequence identity to characterized alginate lyases, indicating that AlyPM is a new PL7 enzyme. The optimal pH for AlyPM activity was 8.5. AlyPM showed the highest activity at 30°C and remained 19% of the highest activity at 5°C. AlyPM was unstable at temperatures above 30°C and had a low Tm of 37°C. These data indicate that AlyPM is a cold-adapted enzyme. Moreover, AlyPM is a salt-activated enzyme. AlyPM activity in 0.5–1.2 M NaCl was sixfolds higher than that in 0 M NaCl, probably caused by a significant increase in substrate affinity, because the Km of AlyPM in 0.5 M NaCl decreased more than 20-folds than that in 0 M NaCl. AlyPM preferably degraded polymannuronate and mainly released dimers and trimers. These data indicate that AlyPM is a new PL7 endo-alginate lyase with special characteristics.

Identification and Characterization of a Novel Salt-Tolerant Esterase from the Deep-Sea Sediment of the South China Sea

Marine esterases play an important role in marine organic carbon degradation and cycling. Halotolerant esterases from the sea may have good potentials in industrial processes requiring high salts. Although a large number of marine esterases have been characterized, reports on halotolerant esterases are only a few. Here, a fosmid library containing 7,200 clones was constructed from a deep-sea sediment sample from the South China Sea. A gene H8 encoding an esterase was identified from this library by functional screening and expressed in Escherichia coli. Phylogenetic analysis showed that H8 is a new member of family V of bacterial lipolytic enzymes. H8 could effectively hydrolyze short-chain monoesters (C4–C10), with the highest activity toward p-nitrophenyl hexanoate. The optimal temperature and pH for H8 activity were 35°C and pH 10.0, respectively. H8 had high salt tolerance, remaining stable in 4.5 M NaCl, which suggests that H8 is well adapted to the marine saline environment and that H8 may have industrial potentials. Unlike reported halophilic/halotolerant enzymes with high acidic/basic residue ratios and low pI values, H8 contains a large number of basic residues, leading to its high basic/acidic residue ratio and high predicted pI (9.09). Moreover, more than 10 homologous sequences with similar basic/acidic residue ratios and predicted pI values were found in database, suggesting that H8 and its homologs represent a new group of halotolerant esterases. We also investigated the role of basic residues in H8 halotolerance by site-directed mutation. Mutation of Arg195, Arg203 or Arg236 to acidic Glu significantly decreased the activity and/or stability of H8 under high salts, suggesting that these basic residues play a role in the salt tolerance of H8. These results shed light on marine bacterial esterases and halotolerant enzymes.

Novel Molecular Insights into the Catalytic Mechanism of Marine Bacterial Alginate Lyase AlyGC from Polysaccharide Lyase Family 6

Alginate lyases that degrade alginate via a β-elimination reaction fall into seven polysaccharide lyase (PL) families. Although the structures and catalytic mechanisms of alginate lyases in the other PL families have been clarified, those in family PL6 have yet to be revealed. Here, the crystal structure of AlyGC, a PL6 alginate lyase from marine bacterium Glaciecola chathamensis S18K6T, was solved, and its catalytic mechanism was illustrated. AlyGC is a homodimeric enzyme and adopts a structure distinct from other alginate lyases. Each monomer contains a catalytic N-terminal domain and a functionally unknown C-terminal domain. A combined structural and mutational analysis using the structures of AlyGC and of an inactive mutant R241A in complex with an alginate tetrasaccharide indicates that conformational changes occur in AlyGC when a substrate is bound and that the two active centers in AlyGC may not bind substrates simultaneously. The C-terminal domain is shown to be essential for the dimerization and the catalytic activity of AlyGC. Residues Tyr130, Arg187, His242, Arg265, and Tyr304 in the active center are also important for the activity of AlyGC. In catalysis, Lys220 and Arg241 function as the Brønsted base and acid, respectively, and a Ca2+ in the active center neutralizes the negative charge of the C5 carboxyl group of the substrate. Finally, based on our data, we propose a metal ion-assisted catalytic mechanism of AlyGC for alginate cleavage with a state change mode, which provides a better understanding for polysaccharide lyases and alginate degradation.