Injuries and illness of athletes at the Tokyo 2020 Olympic and Paralympic summer games visiting outside facilities.

This study aimed to identify the reasons for transferring athletes to local medical facilities during the Olympic and Paralympic Games. Data on 567 injuries and other illnesses of athletes treated at the on-site clinics were collected from the Tokyo 2020 Organizing Committee. Of these, 84 athletes who required outpatient care during the Games were registered for this survey. During the Olympic and Paralympic Games, 66 (8.3/1 000) and 18 (7.2/1 000) athletes, respectively, consulted external medical facilities. In the Olympic Games, the reasons for these visits included 48 cases (72.7%) of injuries, 13 (19.7%) cases of illnesses, and 5 (7.6%) cases of heat stroke illness (HSI). Of these patients, 56 (84.9%) were treated as outpatients and 10 (15.1%) were hospitalized, while three of these patients required hospitalization for > 7 days. On the other hand, in the Paralympics Games, there were 7 (38.8%) cases of injuries, 9 (50.0%) other illnesses, 1 (5.6%) case of HSI, and 1 (5.6%) other cases, of which 11 (61.1%) were treated as outpatients and 7 (38.9%) were hospitalized, but none was hospitalized for > 7 days. Injuries accounted for 70% of the total cases at the 2021 Olympic Games, but only three (0.05%) were severe cases that required hospitalization for more than 1 week. In contrast, in the Paralympic Games, other illnesses accounted for approximately half of the total cases. This study provides details on the extent of injuries and other illnesses that were transferred to outside facilities, which has not been documented in previous games.

Multifunctional enzymes related to amino acid metabolism in bacteria.

In bacteria, D-amino acids are primarily synthesized from L-amino acids by amino acid racemases, but some bacteria use D-amino acid aminotransferases to synthesize D-amino acids. D-Amino acids are peptidoglycan components in the cell wall involved in several physiological processes, such as bacterial growth, biofilm dispersal, and peptidoglycan metabolism. Therefore, their metabolism and physiological roles have attracted increasing attention. Recently, we identified novel bacterial D-amino acid metabolic pathways, which involve amino acid racemases, with broad substrate specificity, as well as multifunctional enzymes with D-amino acid-metabolizing activity. Here, I review these multifunctional enzymes and their related D- and L-amino acid metabolic pathways in Escherichia coli and the hyperthermophile Thermotoga maritima.

Novel tetrahydrofolate-dependent d-serine dehydratase activity of serine hydroxymethyltransferases.

d-Serine plays vital physiological roles in the functional regulation of the mammalian brain, where it is produced from l-serine by serine racemase and degraded by d-amino acid oxidase. In the present study, we identified a new d-serine metabolizing activity of serine hydroxymethyltransferase (SHMT) in bacteria as well as mammals. SHMT is known to catalyze the conversion of l-serine and tetrahydrofolate (THF) to glycine and 5,10-methylenetetrahydrofolate, respectively. In addition, we found that human and Escherichia coli SHMTs have d-serine dehydratase activity, which degrades d-serine to pyruvate and ammonia. We characterized this enzymatic activity along with canonical SHMT activity. Intriguingly, SHMT required THF to catalyze d-serine dehydration and did not exhibit dehydratase activity toward l-serine. Furthermore, SHMT did not use d-serine as a substrate in the canonical hydroxymethyltransferase reaction. The d-serine dehydratase activities of two isozymes of human SHMT were inhibited in the presence of a high concentration of THF, whereas that of E. coli SHMT was increased. The pH and temperature profiles of d-serine dehydratase and serine hydroxymethyltransferase activities of these three SHMTs were partially distinct. The catalytic efficiency (kcat /Km ) of dehydratase activity was lower than that of hydroxymethyltransferase activity. Nevertheless, the d-serine dehydratase activity of SHMT was physiologically important because d-serine inhibited the growth of an SHMT deletion mutant of E. coli, ∆glyA, more than that of the wild-type strain. Collectively, these results suggest that SHMT is involved not only in l- but also in d-serine metabolism through the degradation of d-serine.

Drosophila Neuronal Glucose 6 Phosphatase is a modulator of Neuropeptide Release that regulates muscle glycogen stores via FMRFamide signaling.

Neuropeptides (NPs) and their cognate receptors are critical molecular effectors of diverse physiological processes and behaviors. We recently reported of a non-canonical function of the Drosophila Glucose-6-Phosphatase ( G6P ) gene in a subset of neurosecretory cells in the CNS that governs systemic glucose homeostasis in food deprived flies. Here, we show that G6P expressing neurons define 7 groups of neuropeptide secreting cells, 5 in the brain and 2 in the thoracic ganglia. Using the glucose homeostasis phenotype as a screening tool, we show that one such group, located in the thoracic ganglia and expressing FMRFamide ( FMRFa G6P ) neuropeptides, is necessary and sufficient to maintain systemic glucose homeostasis in starved flies. We further show that the receptor for FMRFamides (FMRFaR) is one key target of G6P dependent NP signaling and essential for the build-up of glycogen stores in the jump muscle. Lastly, measurements of the Golgi apparatus of FMRFa G6P neurons and neuropeptide released into the hemolymph suggests that G6P enhances FMRFa signaling by increasing the capacity of the neurosecretory system. We propose a general model in which the main role of G6P is to counteract glycolysis in peptidergic neurons for the purpose of optimizing the intracellular environment best suited for the expansion of the Golgi apparatus, boosting release of neuropeptides, which through the activation of specific neuropeptide receptors, enhances signaling in respective target tissues.

Incidence and factor analysis for the heat-related illness on the Tokyo 2020 Olympic and Paralympic Games.

Introduction: Among the 43 venues of Tokyo 2020 Olympic Games (OG) and 33 venues of Paralympic Games (PG) were held, the heat island effect was highly expected to cause heat-related illnesses in the outdoor venues with maximum temperatures exceeding 35°C. However, the actual number of heat-related illness cases during the competition was lower than that was initially expected, and it was unclear under what conditions or environment-related heat illnesses occurred among athletes. Object: To clarify the cause and factors contributing to the occurrence of heat-related illness among athletes participating in the Tokyo 2020 Olympic and Paralympic Games. Method: This retrospective descriptive study included 15 820 athletes from 206 countries. From 21 July 2021 to 8 August 2021 for the Olympics, and from 24 August 2021 to 5 September 2021 for the Paralympics. The number of heat-related illness cases at each venue, the incidence rate for each event, gender, home continent, as well as the type of competition, environmental factors (such as venue, time, location and wet-bulb globe temperature (WBGT)), treatment factor and the type of competition were analysed. Results: More number of heat-related illnesses among athletes occurred at the OG (n=110, 76.3%) than at the PG (n=36, 23.7%). A total of 100 cases (100%) at the OG and 31 cases (86.1%) at the PG occurred at the outdoors venues. In the OG, a total of 50 cases (57.9%) occurred during the competition of marathon running and race walking at Sapporo Odori Park. Six of those, were diagnosed with exertional heat illness and treated with cold water immersion (CWI) at OG and one case at PG. Another 20 cases occurred in athletics (track and field) competitions at Tokyo National Olympic Stadium. In total, 10 cases (10.0%) were diagnosed with severe heat illness in the OG and 3 cases (8.3%) in the PG. Ten cases were transferred to outside medical facilities for further treatment, but no case has been hospitalised due to severe condition. In the factor analysis, venue zone, outdoor game, high WBGT (<28°C) and endurance sports have been found to have a higher risk of moderate and severe heat-related illness (p<0.05). The incidence rate and severity could be attenuated by proper heat-related illness treatment (CWI, ice towel, cold IV transfusion and oral hydration) reduced the severity of the illness, providing summer hot environment sports. Conclusion: The Tokyo 2020 Olympic and Paralympic summer games were held. Contrary to expectations, we calculated that about 1 in 100 Olympic athletes suffered heat-related illness. We believe this was due to the risk reduction of heat-related illness, such as adequate prevention and proper treatment. Our experience in avoiding heat-related illness will provide valuable data for future Olympic summer Games.

Acute in-competition medical care at the Tokyo 2020 Olympics: a retrospective analysis.

OBJECTIVE: To analyse injuries and illnesses during the 2020 Tokyo Olympic Summer Games. METHODS: This retrospective descriptive study included 11 420 athletes from 206 National Olympic Committees and 312 883 non-athletes. Incidences of injuries and illnesses during the competition period from 21 July to 8 August 2021 were analysed. RESULTS: A total of 567 athletes (416 injuries, 51 non-heat-related illnesses and 100 heat-related illnesses) and 541 non-athletes (255 injuries, 161 non-heat-related illnesses and 125 heat-related illnesses) were treated at the competition venue clinic. Patient presentation and hospital transportation rates per 1000 athletes were 50 and 5.8, respectively. Marathons and race walking had the highest incidence of injury and illness overall (17.9%; n=66). The highest incidence of injury (per participant) was noted in boxing (13.8%; n=40), sport climbing (12.5%; n=5) and skateboarding (11.3%; n=9), excluding golf, with the highest incidence of minor injuries. Fewer infectious illnesses than previous Summer Olympics were reported among the participants. Of the 100 heat-related illnesses in athletes, 50 occurred in the marathon and race walking events. Only six individuals were transported to a hospital due to heat-related illness, and none required hospital admission. CONCLUSION: Injuries and heat-related illnesses were lower than expected at the 2020 Tokyo Olympic Summer Games. No catastrophic events occurred. Appropriate preparation including illness prevention protocols, and treatment and transport decisions at each venue by participating medical personnel may have contributed to these positive results.

Preparation of egg-koji for developing a novel food.

While chicken eggs contain many nutrients necessary for humans and there are various cooking methods, the nutritional components are used as they are, and there are no traditional foods that utilize microorganisms. Koji-mold, containing Aspergillus oryzae, A. sojae, and A. luchuensis, which has been used in various fermented foods since ancient times, grows on raw grain materials such as rice and barley to become koji. This can give flavors not found in the raw materials that can decompose and convert the nutritional components of the raw materials. Here, we succeeded for the first time in developing egg-koji that uses only eggs and koji-mold by selecting and combining cooked egg powder (CEP) and A. oryzae AO101 as the most suitable combination. To suppress the explosive growth of harmful bacteria, we improved the sterilization method, watering method, and amount of water. In addition, it was found that egg-koji has a characteristic enzyme activity balance, in which amylase is extremely low and protease at pH 6 was high compared to grain koji, such as rice and barley. Egg-koji might produce enzymes suitable for taking in nutrients when growing into CEP and would be expected to give a flavor that could not be achieved by cooking or additives.

YgeA is involved in L- and D-homoserine metabolism in Escherichia coli.

Noncanonical D-amino acids are involved in peptidoglycan and biofilm metabolism in bacteria. Previously, we identified amino acid racemases with broad substrate specificity, including YgeA from Escherichia coli, which strongly prefers homoserine as a substrate. In this study, we investigated the functions of this enzyme in vivo. When wild-type and ygeA-deficient E. coli strains were cultured in minimal medium containing D-homoserine, the D-homoserine level was significantly higher in the ygeA-deficient strain than in the wild-type strain, in which it was almost undetectable. Additionally, D-homoserine was detected in YgeA-expressed E. coli cells cultured in minimal medium containing L-homoserine. The growth of the ygeA-deficient strain was significantly impaired in minimal medium with or without supplemental D-homoserine, while L-methionine, L-threonine or L-isoleucine, which are produced via L-homoserine, restored the growth impairment. Furthermore, the wild-type strain formed biofilms significantly more efficiently than the ygeA-deficient strain. Addition of L- or D-homoserine significantly suppressed biofilm formation in the wild-type strain, whereas this addition had no significant effect in the ygeA-deficient strain. Together, these data suggest that YgeA acts as an amino acid racemase and plays a role in L- and D-homoserine metabolism in E. coli.

Characterization of human cystathionine γ-lyase enzyme activities toward d-amino acids.

Various d-amino acids play important physiological roles in mammals, but the pathways of their production remain unknown except for d-serine, which is generated by serine racemase. Previously, we found that Escherichia coli cystathionine ß-lyase possesses amino acid racemase activity in addition to ß-lyase activity. In the present work, we evaluated the enzymatic activities of human cystathionine γ-lyase, which shares a relatively high amino acid sequence identity with cystathionine ß-lyase. The enzyme did not show racemase activity toward various amino acids including alanine and lyase and dehydratase activities were highest toward l-cystathionine and l-homoserine, respectively. The enzyme also showed weak activity toward l-cysteine and l-serine but no activity toward d-amino acids. Intriguingly, the pH and temperature profiles of lyase activity were distinct from those of dehydratase activity. Catalytic efficiency was higher for lyase activity than for dehydratase activity.

Identification of a novel d-amino acid aminotransferase involved in d-glutamate biosynthetic pathways in the hyperthermophile Thermotoga maritima.

The hyperthermophilic bacterium Thermotoga maritima has an atypical peptidoglycan that contains d-lysine alongside the usual d-alanine and d-glutamate. We previously identified a lysine racemase involved in d-lysine biosynthesis, and this enzyme also possesses alanine racemase activity. However, T. maritima has neither alanine racemase nor glutamate racemase enzymes; hence, the precise biosynthetic pathways of d-alanine and d-glutamate remain unclear in T. maritima. In the present study, we identified and characterized a novel d-amino acid aminotransferase (TM0831) in T. maritima. TM0831 exhibited aminotransferase activity towards 23 d-amino acids, but did not display activity towards l-amino acids. It displayed high specific activities towards d-homoserine and d-glutamine as amino donors. The most preferred acceptor was 2-oxoglutarate, followed by glyoxylate. Additionally, TM0831 displayed racemase activity towards four amino acids including aspartate and glutamate. Catalytic efficiency (kcat /Km ) for aminotransferase activity was higher than for racemase activity, and pH profiles were distinct between these two activities. To evaluate the functions of TM0831, we constructed a TTHA1643 (encoding glutamate racemase)-deficient Thermus thermophilus strain (∆TTHA1643) and integrated the TM0831 gene into the genome of ∆TTHA1643. The growth of this TM0831-integrated strain was promoted compared with ∆TTHA1643 and was restored to almost the same level as that of the wild-type strain. These results suggest that TM0831 is involved in d-glutamate production. TM0831 is a novel d-amino acid aminotransferase with racemase activity that is involved in the production of d-amino acids in T. maritima.

Acetylornithine aminotransferase TM1785 performs multiple functions in the hyperthermophile Thermotoga maritima.

The hyperthermophilic bacterium Thermotoga maritima peptidoglycan contains unusual d-lysine alongside typical d-alanine and d-glutamate. We previously identified lysine racemase and threonine dehydratase, but knowledge of d-amino acid metabolism remains limited. Herein, we identified and characterized T. maritima acetylornithine aminotransferase TM1785. The enzyme was most active towards acetyl-l-ornithine, but also utilized l-glutamate, l-ornithine and acetyl-l-lysine as amino donors, and 2-oxoglutarate was the preferred amino acceptor. TM1785 also displayed racemase activity towards four amino acids and lyase activity towards l-cysteine, but no dehydratase activity towards l-serine, l-threonine or corresponding d-amino acids. Catalytic efficiency (kcat /Km ) was highest for aminotransferase activity and lowest for racemase activity. TM1785 is a novel acetylornithine aminotransferase associated with l-arginine biosynthesis that possesses two additional distinct activities.

Glyoxylate reductase/hydroxypyruvate reductase regulates the free d-aspartate level in mammalian cells.

Multiple d-amino acids are present in mammalian cells, and these compounds have distinctive physiological functions. Among the free d-amino acids identified in mammals, d-aspartate plays critical roles in the neuroendocrine and endocrine systems, as well as in the central nervous system. Mammalian cells have the molecular apparatus necessary to take up, degrade, synthesize, and release d-aspartate. In particular, d-aspartate is degraded by d-aspartate oxidase (DDO), a peroxisome-localized enzyme that catalyzes the oxidative deamination of d-aspartate to generate oxaloacetate, hydrogen peroxide, and ammonia. However, little is known about the molecular mechanisms underlying d-aspartate homeostasis in cells. In this study, we established a cell line that overexpresses cytoplasm-localized DDO; this cell line cannot survive in the presence of high concentrations of d-aspartate, presumably because high levels of toxic hydrogen peroxide are produced by metabolism of abundant d-aspartate by DDO in the cytoplasm, where hydrogen peroxide cannot be removed due to the absence of catalase. Next, we transfected these cells with a complementary DNA library derived from the human brain and screened for clones that affected d-aspartate metabolism and improved cell survival, even when the cells were challenged with high concentrations of d-aspartate. The screen identified a clone of glyoxylate reductase/hydroxypyruvate reductase (GRHPR). Moreover, the GRHPR metabolites glyoxylate and hydroxypyruvate inhibited the enzymatic activity of DDO. Furthermore, we evaluated the effects of GRHPR and peroxisome-localized DDO on d- and l-aspartate levels in cultured mammalian cells. Our findings show that GRHPR contributes to the homeostasis of these amino acids in mammalian cells.

Identification and biochemical characterization of threonine dehydratase from the hyperthermophile Thermotoga maritima.

The peptidoglycan of the hyperthermophile Thermotoga maritima contains an unusual component, D-lysine (D-Lys), in addition to the typical D-alanine (D-Ala) and D-glutamate (D-Glu). In a previous study, we identified a Lys racemase that is presumably associated with D-Lys biosynthesis. However, our understanding of D-amino acid metabolism in T. maritima and other bacteria remains limited, although D-amino acids in the peptidoglycan are crucial for preserving bacterial cell structure and resistance to environmental threats. Herein, we characterized enzymatic and structural properties of TM0356 that shares a high amino acid sequence identity with serine (Ser) racemase. The results revealed that TM0356 forms a tetramer with each subunit containing a pyridoxal 5'-phosphate as a cofactor. The enzyme did not exhibit racemase activity toward various amino acids including Ser, and dehydratase activity was highest toward L-threonine (L-Thr). It also acted on L-Ser and L-allo-Thr, but not on the corresponding D-amino acids. The catalytic mechanism did not follow typical Michaelis-Menten kinetics; it displayed a sigmoidal dependence on substrate concentration, with highest catalytic efficiency (kcat/K0.5) toward L-Thr. Interestingly, dehydratase activity was insensitive to allosteric regulators L-valine and L-isoleucine (L-Ile) at low concentrations, while these L-amino acids are inhibitors at high concentrations. Thus, TM0356 is a biosynthetic Thr dehydratase responsible for the conversion of L-Thr to α-ketobutyrate and ammonia, which is presumably involved in the first step of the biosynthesis of L-Ile.

D-Amino acid metabolism in bacteria.

Bacteria produce diverse d-amino acids, which are essential components of cell wall peptidoglycan. Incorporation of these d-amino acids into peptidoglycan contributes to bacterial adaptation to environmental changes and threats. d-Amino acids have been associated with bacterial growth, biofilm formation and dispersal and regulation of peptidoglycan metabolism. The diversity of d-amino acids in bacteria is primarily due to the activities of amino acid racemases that catalyse the interconversion of the d- and l-enantiomers of amino acids. Recent studies have revealed that bacteria possess multiple enzymes with amino acid racemase activities. Therefore, elucidating d-amino acid metabolism by these enzymes is critical to understand the biological significance and behaviour of d-amino acids in bacteria. In this review, we focus on the metabolic pathways of d-amino acids in six types of bacteria.

d-Serine and d-Alanine Regulate Adaptive Foraging Behavior in Caenorhabditis elegans via the NMDA Receptor.

d-Serine (d-Ser) is a coagonist for NMDA-type glutamate receptors and is thus important for higher brain function. d-Ser is synthesized by serine racemase and degraded by d-amino acid oxidase. However, the significance of these enzymes and the relevant functions of d-amino acids remain unclear. Here, we show that in the nematode Caenorhabditis elegans, the serine racemase homolog SERR-1 and d-amino acid oxidase DAAO-1 control an adaptive foraging behavior. Similar to many organisms, C. elegans immediately initiates local search for food when transferred to a new environment. With prolonged food deprivation, the worms exhibit a long-range dispersal behavior as the adaptive foraging strategy. We found that serr-1 deletion mutants did not display this behavior, whereas daao-1 deletion mutants immediately engaged in long-range dispersal after food removal. A quantitative analysis of d-amino acids indicated that d-Ser and d-alanine (d-Ala) are both synthesized and suppressed during food deprivation. A behavioral pharmacological analysis showed that the long-range dispersal behavior requires NMDA receptor desensitization. Long-term pretreatment with d-Ala, as well as with an NMDA receptor agonist, expanded the area searched by wild-type worms immediately after food removal, whereas pretreatment with d-Ser did not. We propose that d-Ser and d-Ala are endogenous regulators that cooperatively induce the long-range dispersal behavior in C. elegans through actions on the NMDA receptor.SIGNIFICANCE STATEMENT In mammals, d-serine (d-Ser) functions as an important neuromodulator of the NMDA-type glutamate receptor, which regulates higher brain functions. In Caenorhabditis elegans, previous studies failed to clearly define the physiological significance of d-Ser, d-alanine (d-Ala), and their metabolic enzymes. In this study, we found that these d-amino acids and their associated enzymes are active during food deprivation, leading to an adaptive foraging behavior. We also found that this behavior involved NMDA receptor desensitization.

A colorimetric assay method for measuring d-glutamate cyclase activity.

In mammals, metabolism of free d-glutamate is regulated by d-glutamate cyclase (DGLUCY), which reversibly converts d-glutamate to 5-oxo-d-proline and H2O. Metabolism of these d-amino acids by DGLUCY is thought to regulate cardiac function. In this study, we established a simple, accurate, and sensitive colorimetric assay method for measuring DGLUCY activity. To this end, we optimized experimental procedures for derivatizing 5-oxo-d-proline with 2-nitrophenylhydrazine hydrochloride. 5-Oxo-d-proline was derivatized with 2-nitrophenylhydrazine hydrochloride in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as a catalyst to generate the acid hydrazides, whose levels were then determined using a colorimetric method. Under optimized conditions, we examined the sensitivity and accuracy of the colorimetric method and compared our technique with other methods by high-performance liquid chromatography with ultraviolet-visible or fluorescence detection. Moreover, we assessed the suitability of this colorimetric method for measuring DGLUCY activity in biological samples. Our colorimetric method could determine DGLUCY activity with adequate validity and reliability. This method will help to elucidate the relationship among DGLUCY activity, the physiological and pathological roles of d-glutamate and 5-oxo-d-proline, and cardiac function.

Enzymatic properties and physiological function of glutamate racemase from Thermus thermophilus.

d-Amino acids are physiologically important components of peptidoglycan in the bacterial cell wall, maintaining cell structure and aiding adaptation to environmental changes through peptidoglycan remodelling. Therefore, the biosynthesis of d-amino acids is essential for bacteria to adapt to different environmental conditions. The peptidoglycan of the extremely thermophilic bacterium Thermus thermophilus contains d-alanine (d-Ala) and d-glutamate (d-Glu), but its d-amino acid metabolism remains poorly understood. Here, we investigated the enzyme activity and function of the product of the TTHA1643 gene, which is annotated to be a Glu racemase in the T. thermophilus HB8 genome. Among 21 amino acids tested, TTHA1643 showed highly specific activity toward Glu as the substrate. The catalytic efficiency (kcat/Km) of TTHA1643 toward d- and l-Glu was comparable; however, the kcat value was 18-fold higher for l-Glu than for d-Glu. Temperature and pH profiles showed that the racemase activity of TTHA1643 is high under physiological conditions for T. thermophilus growth. To assess physiological relevance, we constructed a TTHA1643-deficient strain (∆TTHA1643) by replacing the TTHA1643 gene with the thermostable hygromycin resistance gene. Growth of the ∆TTHA1643 strain in synthetic medium without d-Glu was clearly diminished relative to wild type, although the TTHA1643 deletion was not lethal, suggesting that alternative d-Glu biosynthetic pathways may exist. The deterioration in growth was restored by adding d-Glu to the culture medium, showing that d-Glu is required for normal growth of T. thermophilus. Collectively, our findings show that TTHA1643 is a Glu racemase and has the physiological function of d-Glu production in T. thermophilus.

Biochemical characterization of d-aspartate oxidase from Caenorhabditis elegans: its potential use in the determination of free d-glutamate in biological samples.

d-Aspartate oxidase (DDO) is a flavin adenine dinucleotide (FAD)-containing flavoprotein that stereospecifically acts on acidic d-amino acids (i.e., free d-aspartate and d-glutamate). Mammalian DDO, which exhibits higher activity toward d-aspartate than d-glutamate, is presumed to regulate levels of d-aspartate in the body and is not thought to degrade d-glutamate in vivo. By contrast, three DDO isoforms are present in the nematode Caenorhabditis elegans, DDO-1, DDO-2, and DDO-3, all of which exhibit substantial activity toward d-glutamate as well as d-aspartate. In this study, we optimized the Escherichia coli culture conditions for production of recombinant C. elegans DDO-1, purified the protein, and showed that it is a flavoprotein with a noncovalently but tightly attached FAD. Furthermore, C. elegans DDO-1, but not mammalian (rat) DDO, efficiently and selectively degraded d-glutamate in addition to d-aspartate, even in the presence of various other amino acids. Thus, C. elegans DDO-1 might be a useful tool for determining these acidic d-amino acids in biological samples.

Involvement of penicillin-binding proteins in the metabolism of a bacterial peptidoglycan containing a non-canonical D-amino acid.

Bacteria produce various D-amino acids, including non-canonical D-amino acids, to adapt to environmental changes and overcome a variety of threats. These D-amino acids are largely utilized as components of peptidoglycan, and they promote peptidoglycan remodeling and biofilm disassembly. The biosynthesis, maturation, and recycling of peptidoglycan are catalyzed by penicillin-binding proteins (PBPs). However, although non-canonical D-amino acids are known to be incorporated into peptidoglycan, the maturation and recycling of peptidoglycan containing such residues remain uncharacterized. Therefore, we investigated whether PBP4 and PBP5, low molecular mass (LMM) PBPs from Escherichia coli and Bacillus subtilis, are involved in these events of peptidoglycan metabolism. Enzyme assays using p-nitroaniline (pNA)-derivatized D-amino acids and peptidoglycan-mimicking peptides revealed that PBP4 and PBP5 from both species have peptidase activity toward substrates containing D-Asn, D-His, or D-Trp. These D-amino acids slowed the growth of dacA- or dacB-deficient E. coli (∆dacA or ∆dacB) relative to the wild-type strain. Additionally, these D-amino acids affected biofilm formation by the ∆dacB strain. Collectively, PBP4 and PBP5 are involved in the cleavage of peptidoglycan containing non-canonical D-amino acids, and these properties affect growth and biofilm formation.