A Plurizyme with Transaminase and Hydrolase Activity Catalyzes Cascade Reactions.

Engineering dual-function single polypeptide catalysts with two abiotic or biotic catalytic entities (or combinations of both) supporting cascade reactions is becoming an important area of enzyme engineering and catalysis. Herein we present the development of a PluriZyme, TR2 E2 , with efficient native transaminase (kcat : 69.49±1.77 min-1 ) and artificial esterase (kcat : 3908-0.41 min-1 ) activities integrated into a single scaffold, and evaluate its utility in a cascade reaction. TR2 E2 (pHopt : 8.0-9.5; Topt : 60-65 °C) efficiently converts methyl 3-oxo-4-(2,4,5-trifluorophenyl)butanoate into 3-(R)-amino-4-(2,4,5-trifluorophenyl)butanoic acid, a crucial intermediate for the synthesis of antidiabetic drugs. The reaction proceeds through the conversion of the ß-keto ester into the ß-keto acid at the hydrolytic site and subsequently into the ß-amino acid (e.e. >99 %) at the transaminase site. The catalytic power of the TR2 E2 PluriZyme was proven with a set of ß-keto esters, demonstrating the potential of such designs to address bioinspired cascade reactions.

Crystal structures of a novel family IV esterase in free and substrate-bound form.

Bacterial lipolytic enzymes of family IV are homologs of the mammalian hormone-sensitive lipases (HSL) and have been successfully used for various biotechnological applications. The broad substrate specificity and ability for enantio-, regio-, and stereoselective hydrolysis are remarkable features of enzymes from this class. Many crystal structures are available for esterases and lipases, but structures of enzyme-substrate or enzyme-inhibitor complexes are less frequent although important to understand the molecular basis of enzyme-substrate interaction and to rationalize biochemical enzyme characteristics. Here, we report on the structures of a novel family IV esterase isolated from a metagenomic screen, which shows a broad substrate specificity. We solved the crystal structures in the apo form and with a bound substrate analogue at 1.35 and 1.81 Å resolution, respectively. This enzyme named PtEst1 hydrolyzed more than 60 out 96 structurally different ester substrates thus being substrate promiscuous. Its broad substrate specificity is in accord with a large active site cavity, which is covered by an α-helical cap domain. The substrate analogue methyl 4-methylumbelliferyl hexylphosphonate was rapidly hydrolyzed by the enzyme leading to a complete inactivation caused by covalent binding of phosphinic acid to the catalytic serine. Interestingly, the alcohol leaving group 4-methylumbelliferone was found remaining in the active site cavity, and additionally, a complete inhibitor molecule was found at the cap domain next to the entrance of the substrate tunnel. This unique situation allowed gaining valuable insights into the role of the cap domain for enzyme-substrate interaction of esterases belonging to family IV. DATABASE: Structural data of PtEst1 are available in the worldwide protein data bank (https://www.rcsb.org) under the accession codes: 6Z68 (apo-PtEst1) and 6Z69 (PtEst1-inhibitor complex).

A Novel Polyester Hydrolase From the Marine Bacterium Pseudomonas aestusnigri - Structural and Functional Insights.

Biodegradation of synthetic polymers, in particular polyethylene terephthalate (PET), is of great importance, since environmental pollution with PET and other plastics has become a severe global problem. Here, we report on the polyester degrading ability of a novel carboxylic ester hydrolase identified in the genome of the marine hydrocarbonoclastic bacterium Pseudomonas aestusnigri VGXO14 T . The enzyme, designated PE-H, belongs to the type IIa family of PET hydrolytic enzymes as indicated by amino acid sequence homology. It was produced in Escherichia coli, purified and its crystal structure was solved at 1.09 Å resolution representing the first structure of a type IIa PET hydrolytic enzyme. The structure shows a typical α/ß-hydrolase fold and high structural homology to known polyester hydrolases. PET hydrolysis was detected at 30°C with amorphous PET film (PETa), but not with PET film from a commercial PET bottle (PETb). A rational mutagenesis study to improve the PET degrading potential of PE-H yielded variant PE-H (Y250S) which showed improved activity, ultimately also allowing the hydrolysis of PETb. The crystal structure of this variant solved at 1.35 Å resolution allowed to rationalize the improvement of enzymatic activity. A PET oligomer binding model was proposed by molecular docking computations. Our results indicate a significant potential of the marine bacterium P. aestusnigri for PET degradation.

Organic-Solvent-Tolerant Carboxylic Ester Hydrolases for Organic Synthesis.

Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by carboxylic ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of carboxylic ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant esterase from Pseudomonas aestusnigri In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard ester hydrolase CalB from Candida antarcticaIMPORTANCE Major challenges hampering biotechnological applications of esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant carboxylic ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.

Agar plate-based screening methods for the identification of polyester hydrolysis by Pseudomonas species.

Hydrolases acting on polyesters like cutin, polycaprolactone or polyethylene terephthalate (PET) are of interest for several biotechnological applications like waste treatment, biocatalysis and sustainable polymer modifications. Recent studies suggest that a large variety of such enzymes are still to be identified and explored in a variety of microorganisms, including bacteria of the genus Pseudomonas. For activity-based screening, methods have been established using agar plates which contain nanoparticles of polycaprolactone or PET prepared by solvent precipitation and evaporation. In this protocol article, we describe a straightforward agar plate-based method using emulsifiable artificial polyesters as substrates, namely Impranil® DLN and liquid polycaprolactone diol (PLD). Thereby, the currently quite narrow set of screening substrates is expanded. We also suggest optional pre-screening with short-chain and middle-chain-length triglycerides as substrates to identify enzymes with lipolytic activity to be further tested for polyesterase activity. We applied these assays to experimentally demonstrate polyesterase activity in bacteria from the P. pertucinogena lineage originating from contaminated soils and diverse marine habitats.

The biotechnological potential of marine bacteria in the novel lineage of Pseudomonas pertucinogena.

Marine habitats represent a prolific source for molecules of biotechnological interest. In particular, marine bacteria have attracted attention and were successfully exploited for industrial applications. Recently, a group of Pseudomonas species isolated from extreme habitats or living in association with algae or sponges were clustered in the newly established Pseudomonas pertucinogena lineage. Remarkably for the predominantly terrestrial genus Pseudomonas, more than half (9) of currently 16 species within this lineage were isolated from marine or saline habitats. Unlike other Pseudomonas species, they seem to have in common a highly specialized metabolism. Furthermore, the marine members apparently possess the capacity to produce biomolecules of biotechnological interest (e.g. dehalogenases, polyester hydrolases, transaminases). Here, we summarize the knowledge regarding the enzymatic endowment of the marine Pseudomonas pertucinogena bacteria and report on a genomic analysis focusing on the presence of genes encoding esterases, dehalogenases, transaminases and secondary metabolites including carbon storage compounds.

Marine Biosurfactants: Biosynthesis, Structural Diversity and Biotechnological Applications.

Biosurfactants are amphiphilic secondary metabolites produced by microorganisms. Marine bacteria have recently emerged as a rich source for these natural products which exhibit surface-active properties, making them useful for diverse applications such as detergents, wetting and foaming agents, solubilisers, emulsifiers and dispersants. Although precise structural data are often lacking, the already available information deduced from biochemical analyses and genome sequences of marine microbes indicates a high structural diversity including a broad spectrum of fatty acid derivatives, lipoamino acids, lipopeptides and glycolipids. This review aims to summarise biosyntheses and structures with an emphasis on low molecular weight biosurfactants produced by marine microorganisms and describes various biotechnological applications with special emphasis on their role in the bioremediation of oil-contaminated environments. Furthermore, novel exploitation strategies are suggested in an attempt to extend the existing biosurfactant portfolio.

Bioprospecting Reveals Class III ω-Transaminases Converting Bulky Ketones and Environmentally Relevant Polyamines.

Amination of bulky ketones, particularly in (R) configuration, is an attractive chemical conversion; however, known ω-transaminases (ω-TAs) show insufficient levels of performance. By applying two screening methods, we discovered 10 amine transaminases from the class III ω-TA family that were 38% to 76% identical to homologues. We present examples of such enzymes preferring bulky ketones over keto acids and aldehydes with stringent (S) selectivity. We also report representatives from the class III ω-TAs capable of converting (R) and (S) amines and bulky ketones and one that can convert amines with longer alkyl substituents. The preference for bulky ketones was associated with the presence of a hairpin region proximal to the conserved Arg414 and residues conforming and close to it. The outward orientation of Arg414 additionally favored the conversion of (R) amines. This configuration was also found to favor the utilization of putrescine as an amine donor, so that class III ω-TAs with Arg414 in outward orientation may participate in vivo in the catabolism of putrescine. The positioning of the conserved Ser231 also contributes to the preference for amines with longer alkyl substituents. Optimal temperatures for activity ranged from 45 to 65°C, and a few enzymes retained ≥50% of their activity in water-soluble solvents (up to 50% [vol/vol]). Hence, our results will pave the way to design, in the future, new class III ω-TAs converting bulky ketones and (R) amines for the production of high-value products and to screen for those converting putrescine.IMPORTANCE Amine transaminases of the class III ω-TAs are key enzymes for modification of chemical building blocks, but finding those capable of converting bulky ketones and (R) amines is still challenging. Here, by an extensive analysis of the substrate spectra of 10 class III ω-TAs, we identified a number of residues playing a role in determining the access and positioning of bulky ketones, bulky amines, and (R)- and (S) amines, as well as of environmentally relevant polyamines, particularly putrescine. The results presented can significantly expand future opportunities for designing (R)-specific class III ω-TAs to convert valuable bulky ketones and amines, as well as for deepening the knowledge into the polyamine catabolic pathways.

Determinants and Prediction of Esterase Substrate Promiscuity Patterns.

Esterases receive special attention because of their wide distribution in biological systems and environments and their importance for physiology and chemical synthesis. The prediction of esterases' substrate promiscuity level from sequence data and the molecular reasons why certain such enzymes are more promiscuous than others remain to be elucidated. This limits the surveillance of the sequence space for esterases potentially leading to new versatile biocatalysts and new insights into their role in cellular function. Here, we performed an extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases, when tested with 96 diverse esters. We determined the primary factors shaping their substrate range by analyzing substrate range patterns in combination with structural analysis and protein-ligand simulations. We found a structural parameter that helps rank (classify) the promiscuity level of esterases from sequence data at 94% accuracy. This parameter, the active site effective volume, exemplifies the topology of the catalytic environment by measuring the active site cavity volume corrected by the relative solvent accessible surface area (SASA) of the catalytic triad. Sequences encoding esterases with active site effective volumes (cavity volume/SASA) above a threshold show greater substrate spectra, which can be further extended in combination with phylogenetic data. This measure provides also a valuable tool for interrogating substrates capable of being converted. This measure, found to be transferred to phosphatases of the haloalkanoic acid dehalogenase superfamily and possibly other enzymatic systems, represents a powerful tool for low-cost bioprospecting for esterases with broad substrate ranges, in large scale sequence data sets.

First Insights into the Genome Sequence of Pseudomonas oleovorans DSM 1045.

The Gram-negative proteobacterium Pseudomonas oleovorans DSM 1045 is considered a promising source for enzymes of biotechnological interest, e.g., hydrolases and transaminases. Here, we present a draft sequence of its 4.86-Mb genome, enabling the identification of novel biocatalysts.

Rim Fracture of a Tibial Base-Plate Due to Retained Cement as a Cause of Catastrophic Failure of Unicompartmental Knee Arthroplasty: A Case Report.

CASE: We report a case of catastrophic implant failure due to fatigue fracture of a well-functioning unicompartmental knee arthroplasty at 7 years in an active 55-year-old man, who presented with sudden onset of atraumatic knee pain and effusion. The patient underwent revision to total knee arthroplasty. Intraoperative findings included a metallic fragment fractured off the rim of the tibial base-plate and cement retained in the tibial tray. CONCLUSION: Retained cement in the tibial tray resulted in increased tibial tray contact stresses and fatigue fracture of the rim of the tray, creating a free metallic loose body that produced symptoms.

Is Scheduled Intravenous Acetaminophen Effective in the Pain Management Protocol of Geriatric Hip Fractures?

BACKGROUND: Hip fractures have significant effects on the geriatric population and the health care system. Prior studies have demonstrated both the safety of intravenous (IV) acetaminophen and its efficacy in decreasing perioperative narcotic consumption. The purpose of this study is to evaluate the effect of scheduled IV acetaminophen for perioperative pain control on length of hospital stay, pain level, narcotic use, rate of missed physical therapy (PT) sessions, adverse effects, and discharge disposition in geriatric patients with hip fractures. METHODS: A retrospective review was performed of all patients 65 years and older admitted to a level I trauma center, who received operative treatment for a hip fracture over a 2-year period. Demographic data, in-hospital variables, and outcome measures were analyzed. Three hundred thirty-six consecutive fractures in 332 patients met inclusion criteria. These patients were divided into 2 cohorts. Group 1 (169 fractures) consisted of patients treated before the initiation of a standardized IV acetaminophen perioperative pain control protocol, and group 2 (167 fractures) consisted of those treated after the protocol was initiated. RESULTS: Group 2 had a statistically significant shorter mean length of hospital stay (4.4 vs 3.8 days), lower mean pain score (4.2 vs 2.8), lower mean narcotic usage (41.3 vs 28.3 mg), lower rate of PT sessions missed (21.8% vs 10.4%), and higher likelihood of discharge home (7% vs 19%; P ≤ .001). Use of IV acetaminophen was also consistently and independently predictive of the same variables (P < .01). CONCLUSION: The utilization of scheduled IV acetaminophen as part of a standardized pain management protocol for geriatric hip fractures resulted in shortened length of hospital stay, decreased pain levels and narcotic use, fewer missed PT sessions, and higher rate of discharge to home. LEVEL OF EVIDENCE: Therapeutic level III.

Factors used by program directors to select hand surgery fellows.

PURPOSE: To identify factors and attributes hand surgery fellowship program directors consider important in selecting applicants for interview and ranking. METHODS: A web-based questionnaire was sent to all hand fellowship program directors in the United States. The questionnaire was designed to identify the most important criteria in granting an interview, sources of letters of recommendation, the interview process, and factors used to rank a candidate. Each criterion was ranked in importance on a 1 to 5 Likert scale, with 1 being not important and 5 being critical. All responses were anonymous. The most important criterion for each section of the survey was determined by comparing the average Likert scores. RESULTS: Fifty-two of 76 program directors responded (68%). The criteria with the highest mean Likert scores for offering an applicant an interview were, in order, quality letters of recommendation from hand surgeons, completion of an orthopedic surgery residency, comments regarding the applicant's technical competence, applicant having an MD degree (as opposed to a DO degree), and residency program reputation. The letters of recommendation with the highest value were from the division chief of hand surgery and another hand surgeon in the division/department. The most important features of the interview were maturity of applicant, ability of applicant to articulate thoughts, ability to listen well, self-confidence, and relevant questions asked. The most important factors in ranking a candidate were applicant integrity, commitment to hard work, quality of letters of recommendation, quality of the interview, and ability to work well with other members of the hand surgery team. CONCLUSIONS: There are identifiable factors considered important by hand surgery fellowship directors when selecting and ranking an applicant. This information may be valuable to medical students and residents contemplating careers in hand surgery.

Short, locked humeral nailing via Neviaser portal: an anatomic study.

OBJECTIVE: Supraspinatus tendon trauma may contribute to residual shoulder pain after nail fixation for proximal humeral fractures. Some have proposed a more medial starting point for humeral nail insertion to avoid cuff tendon footprint damage. We hypothesized that percutaneous nail insertion via Neviaser portal would not only be possible, but would avoid tendon trauma, while sacrificing articular cartilage. MATERIALS AND METHODS: Under c-arm guidance and in percutaneous fashion, we nailed 16 consecutive complete (head and neck, etc, intact) specimen right proximal humeri with locked short humeral nails (Aequalis) via Neviaser portal. Each shoulder was dissected to study the damage to the rotator cuff and long head of the biceps tendons as well as to the articular surfaces. We measured the humeral-thoracic abduction arc before the damaged articular surface contacted the superior glenoid. There were 5 male specimens and 11 female specimens with a mean age of 83 years at the time of death. RESULTS: We successfully inserted 15 of 16 humeral nails through this percutaneous approach. No supraspinatus tendon or long head of the biceps tendon was damaged. All nails passed entirely through supraspinatus muscle belly. Thirteen of 15 starting points were entirely on articular surface. Mean arc of abduction before superior glenoid contact was 76 degrees (range, 50 degrees-130 degrees). Mean distance from the edge of the articular surface to the most lateral part of the nail insertion was 11 mm (0-25 mm). CONCLUSIONS: Short, locked humeral nail insertion is possible in percutaneous fashion via Neviaser portal without tendon injury. However, successful insertion comes at the cost of articular cartilage damage.

Osteolytic lesion of the tibial diaphysis after cementless TKA.

Biomaterial wear debris is a known contributing factor in aseptic loosening of total joint prostheses, particularly when cementless tibial trays are used in total knee arthroplasty (TKA). Local inflammatory response can lead to osteolysis and aseptic loosening of implants. The resulting lesions require careful clinical evaluation. This article presents a case of a 76-year old man with a remote history of prostate cancer and cigarette smoking who presented with acute onset left knee and tibia pain 15 years after TKA. Radiographs showed an osteolytic lesion in the distal tibial diaphysis and magnetic resonance imaging revealed a cystic lesion with evidence concerning for pathologic mid-shaft fracture. Biopsy of the lesion confirmed a foreign body reaction and revision TKA was performed. The patient was seen at 3-year follow-up without complication. The existing literature presents cases reporting osteolytic lesions of the distal femur and proximal tibial metaphysis due to polyethylene wear debris and foreign body reaction following TKA. We are unaware of case reports involving osteolysis of this etiology extending into the distal tibial diaphysis. We conclude that polyethylene wear debris with foreign body reaction should be considered in the differential diagnosis of an osteolytic lesion extending into the tibial diaphysis following TKA.