Serum brain-derived neurotrophic factor and neurocognitive function in children with type 1 diabetes.

BACKGROUND/PURPOSE: This study aimed to clarify whether brain-derived neurotrophic factor (BDNF) is a biomarker for cognitive dysfunction in children with type 1 diabetes. METHODS: We conducted a cross-sectional case-control study of children aged between 6 and 18 years with type 1 diabetes and healthy volunteers. Serum BDNF level was measured in all of the studied children, and they all underwent intelligence tests with the Wechsler Intelligence Scale for Children, Fourth Edition (WISC-IV). We further compared the cognitive function and BDNF levels in the diabetic children with positive glutamic acid decarboxylase 65 antibody (GAD65-Ab) and those with negative GAD65-Ab. RESULTS: Forty-five children with type 1 diabetes (mean age 14.0 ± 2.6 years, 42% male) and 50 normal controls (mean age 13.2 ± 2.3 years, 54% male) were recruited. The serum BDNF level was significantly lower in the diabetes group than in the controls (15.92 ± 7.2 vs. 18.5 ± 5.1 ng/mL, respectively, t = -2.03, p = 0.045) and much lower in the subgroup with GAD65-Ab positive type 1 diabetes. The average Full-Scale IQ, verbal comprehension, perceptual reasoning and working memory scores in the diabetes group were significantly lower than in the controls (all p < 0.05). Among the children with type 1 diabetes, poor glycemic control was related to lower general cognitive abilities (r = -0.34, p < 0.02), lower verbal comprehension (r = -0.305, p < 0.05), and lower perceptual reasoning scores (r = -0.346, p = 0.02). CONCLUSION: The children with type 1 diabetes had a lower serum BDNF level and poorer neurocognitive function than normal healthy children, especially those with GAD65-Ab positive diabetes. Poor glycemic control was correlated with worse cognitive performance.

Evaluating the Antioxidants, Whitening and Antiaging Properties of Rice Protein Hydrolysates.

Plant-derived protein hydrolysates have potential applications in nutrition. Rice protein hydrolysates (RPHs), an excellent source of proteins, have attracted attention for the development of cosmeceuticals. However, few studies have reported the potential application of RPH in analysis, and this study examined their antioxidant activities and the inhibitory activities of skin aging enzymes. The results indicated that the total phenolic and flavonoid concentrations were 2.06 ± 0.13 mg gallic acid equivalent/g RPHs and 25.96 ± 0.52 µg quercetin equivalent/g RPHs, respectively. RPHs demonstrated dose-dependent activity for scavenging free radicals from 1,1-diphenyl-2-picrylhydrazyl [half-maximal inhibitory concentration (IC50) = 42.58 ± 2.1 mg/g RPHs] and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (IC50 = 2.11 ± 0.88 mg/g RPHs), dose-dependent reduction capacity (6.95 ± 1.40 mg vitamin C equivalent/g RPHs) and oxygen radical absorbance capacity (473 µmol Trolox equivalent/g RPHs). The concentrations of the RPH solution required to achieve 50% inhibition of hyaluronidase and tyrosinase activities were determined to be 8.91 and 107.6 mg/mL, respectively. This study demonstrated that RPHs have antioxidant, antihyaluronidase, and antityrosinase activities for future cosmetic applications.

Endoplasmic Reticulum Protein TXNDC5 Augments Myocardial Fibrosis by Facilitating Extracellular Matrix Protein Folding and Redox-Sensitive Cardiac Fibroblast Activation.

RATIONALE: Cardiac fibrosis plays a critical role in the pathogenesis of heart failure. Excessive accumulation of extracellular matrix (ECM) resulting from cardiac fibrosis impairs cardiac contractile function and increases arrhythmogenicity. Current treatment options for cardiac fibrosis, however, are limited, and there is a clear need to identify novel mediators of cardiac fibrosis to facilitate the development of better therapeutics. Exploiting coexpression gene network analysis on RNA sequencing data from failing human heart, we identified TXNDC5 (thioredoxin domain containing 5), a cardiac fibroblast (CF)-enriched endoplasmic reticulum protein, as a potential novel mediator of cardiac fibrosis, and we completed experiments to test this hypothesis directly. OBJECTIVE: The objective of this study was to determine the functional role of TXNDC5 in the pathogenesis of cardiac fibrosis. METHODS AND RESULTS: RNA sequencing and Western blot analyses revealed that TXNDC5 mRNA and protein were highly upregulated in failing human left ventricles and in hypertrophied/failing mouse left ventricle. In addition, cardiac TXNDC5 mRNA expression levels were positively correlated with those of transcripts encoding transforming growth factor ß1 and ECM proteins in vivo. TXNDC5 mRNA and protein were increased in human CF (hCF) under transforming growth factor ß1 stimulation in vitro. Knockdown of TXNDC5 attenuated transforming growth factor ß1-induced hCF activation and ECM protein upregulation independent of SMAD3 (SMAD family member 3), whereas increasing expression of TXNDC5 triggered hCF activation and proliferation and increased ECM protein production. Further experiments showed that TXNDC5, a protein disulfide isomerase, facilitated ECM protein folding and that depletion of TXNDC5 led to ECM protein misfolding and degradation in CF. In addition, TXNDC5 promotes hCF activation and proliferation by enhancing c-Jun N-terminal kinase activity via increased reactive oxygen species, derived from NAD(P)H oxidase 4. Transforming growth factor ß1-induced TXNDC5 upregulation in hCF was dependent on endoplasmic reticulum stress and activating transcription factor 6-mediated transcriptional control. Targeted disruption of Txndc5 in mice (Txndc5-/-) revealed protective effects against isoproterenol-induced cardiac hypertrophy, reduced fibrosis (by ≈70%), and markedly improved left ventricle function; post-isoproterenol left ventricular ejection fraction was 59.1±1.5 versus 40.1±2.5 (P<0.001) in Txndc5-/- versus wild-type mice, respectively. CONCLUSIONS: The endoplasmic reticulum protein TXNDC5 promotes cardiac fibrosis by facilitating ECM protein folding and CF activation via redox-sensitive c-Jun N-terminal kinase signaling. Loss of TXNDC5 protects against ß agonist-induced cardiac fibrosis and contractile dysfunction. Targeting TXNDC5, therefore, could be a powerful new therapeutic approach to mitigate excessive cardiac fibrosis, thereby improving cardiac function and outcomes in patients with heart failure.

Discovery of 2-aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrugs for delivering nucleoside HCV NS5B polymerase inhibitors.

Our HCV research program investigated novel 2'-dihalogenated nucleoside HCV polymerase inhibitors and identified compound 1, a 5'-phosphoramidate prodrug of 2'-deoxy-2'-α-bromo-ß-chloro uridine. Although 1 had a favorable in vitro activity profile in HCV replicons, oral dosing in dog resulted in low levels of the active 5'-triphosphate (TP) in liver. Metabolism studies using human hepatocytes provided a simple assay for screening alternative phosphoramidate prodrug analogs. Compounds that produced high TP concentrations in hepatocytes were tested in dog liver biopsy studies. This method identified 2-aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrug 14, which provided 100-fold higher TP concentrations in dog liver in comparison to 1 (4 and 24 h after 5 mg/kg oral dose).

Synthesis and evaluation of 2'-dihalo ribonucleotide prodrugs with activity against hepatitis C virus.

Hepatitis C virus (HCV) nucleoside inhibitors have been a key focus of nearly 2 decades of HCV drug research due to a high barrier to drug resistance and pan-genotypic activity profile provided by molecules in this drug class. Our investigations focused on several potent 2'-halogenated uridine-based HCV polymerase inhibitors, resulting in the discovery of novel 2'-deoxy-2'-dihalo-uridine analogs that are potent inhibitors in replicon assays for all genotypes. Further studies to improve in vivo performance of these nucleoside inhibitors identified aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrugs 18a and 18c, which provide high levels of the active triphosphate in dog liver. AIBEE prodrug 18c was compared with sofosbuvir (1) by co-dosing both compounds by oral administration in dog (5 mg/kg each) and measuring liver concentrations of the active triphosphate metabolite at both 4 and 24 h post dosing. In this study, 18c provided liver triphosphate concentrations that were 6-fold higher than sofosbuvir (1) at both biopsy time points, suggesting that 18c could be a highly effective agent for treating HCV infected patients in the clinic.

Prevalence of preschool children developmental disabilities in northeastern Taiwan - Screening with Taipei City Developmental Screening Checklist for Preschoolers, 2nd Version.

BACKGROUND/PURPOSE: The prevalence of developmental disabilities in Taiwan remains unclear, especially in young children under the age 3. We aimed to study the prevalence of developmental disabilities and verify a useful developmental screening tool in a community setting in Taiwan. METHODS: We conducted a prospective cross-sectional study in northeastern Taiwan from July 2008 to December 2009 in children aged 4 months to 6 years old from well-child visits. We devised a screening program using Taipei City Developmental Screening Checklist for Preschoolers, 2nd Version (Taipei-II), a validated parent-report milestone checklist tailored to the Taiwanese culture and language to assess the prevalence of developmental disabilities in Taiwan. Information about the children's medical conditions and their family were recorded. RESULTS: A total of 3214 children were recruited, of whom 365 had developmental disabilities, with an overall prevalence of 11.36%. Speech and language delay/disorders were the most common developmental problems followed by motor delays, with prevalence rates of 4.79% and 2.33%, respectively. Low economic status, prematurity and/or small for gestational age and a history of perinatal hypoxia or underlying medical disorders were the main risk factors correlated with developmental delays. However, foreign-born mother and aboriginal families were not important factors for poor developmental outcomes. CONCLUSION: The prevalence rate of developmental disabilities in northeastern Taiwan was 11.36%. Low economic status, prematurity and/or small for gestational age and a history of underlying medical disorders were the main risk factors correlated with developmental disabilities. Taipei II is an easy-to-use and effective developmental surveillance tool for Taiwanese children.

Application of Sonography in the Diagnosis and Follow-Up of Trapped Temporal Horn of Lateral Ventricle: Two Case Reports.

Trapped temporal horn of lateral ventricle (TTHLV) is a rare condition of isolated focal hydrocephalus. We report two cases with different presentations, etiologies, and surgical managements. The first case involved an extremely preterm male baby with a history of ventriculitis and intraventricular hemorrhage; he received external ventricle drainage twice due to obstructive hydrocephalus. TTHLV was detected by sonography. He received a ventriculoperitoneal shunt involving two catheters to bypass the adhesion site. There was no ventricular dilatation during 2 years of follow-up. The second case involved a term baby with an enlarged head; brain sonography revealed left focal hydrocephalus with TTHLV and mild midline shift. Neuroendoscopic cystoventriculostomy with fenestration from the left trigone to the frontal horn was performed and serial follow-up brain sonography for 3 months showed decreased ventricle size. The suitable surgical techniques for the management of TTHLV should be adjusted according to the patients' condition to obtain more favorable outcomes. Brain sonography can be a useful tool for the diagnosis and for following up the surgical outcomes in infants with TTHLV.

Posterior Fossa Hemorrhage in a Term Neonate with Hemophilia A.

Posterior fossa hemorrhage is rare in term baby and difficult to assess. The clinical signs are nonspecific and usually delay the diagnosis. We present a 5-day-old male neonate of posterior fossa hemorrhage with the initial presentations of fever and seizure and early deduced by cranial ultrasonography findings as hyperechoic, asymmetric, ill-defined density and complicated with hydrocephalus. Magnetic resonance imaging of the head verified the diagnosis. Hemophilia A was confirmed thereafter by serology.

Rare Brain Tumor in a Neonate.

Neonatal brain tumor is rare and its outcome is generally poor. We reported a 17-day-old neonate presented as enlarged head girth. The pathological finding showed an embryonal tumor with multilayered rosettes.

Effects of water quality on dissolution of yerba mate extract powders.

Yerba mate tea is known as one of the most popular nonalcoholic beverages favoured by South Americans due to its nutrition facts and medicinal properties. The processing of yerba mate tea is found to affect the properties of its final forms. This study presents an investigation into the effects of water sources on the dissolution of yerba mate extract powders. Comparisons were conducted between yerba mate teas prepared by dissolving yerba mate extract powders into tap water and deionized water. Topics to be explored in this work are the major compositions and antioxidant activities, including total phenol content, reducing power, DPPH scavenging activity, and ABTS(+)• scavenging capacity. It is indicated that there is little difference for antioxidant activities and major constituents of yerba mate teas between both water sources. However, a deeper color is seen in the tap water case, resulting from the reaction between tannic acid and ions. This research finding can be treated as a way to benefit the yerba mate tea processing for applications.

Discovery of Small Molecule Interleukin 17A Inhibitors with Novel Binding Mode and Stoichiometry: Optimization of DNA-Encoded Chemical Library Hits to In Vivo Active Compounds.

Dysregulation of IL17A drives numerous inflammatory and autoimmune disorders with inhibition of IL17A using antibodies proven as an effective treatment. Oral anti-IL17 therapies are an attractive alternative option, and several preclinical small molecule IL17 inhibitors have previously been described. Herein, we report the discovery of a novel class of small molecule IL17A inhibitors, identified via a DNA-encoded chemical library screen, and their subsequent optimization to provide in vivo efficacious inhibitors. These new protein-protein interaction (PPI) inhibitors bind in a previously undescribed mode in the IL17A protein with two copies binding symmetrically to the central cavities of the IL17A homodimer.

Association of attention-deficit/hyperactivity disorder with diabetes: a population-based study.

BACKGROUND: Cognitive impairment has been documented in adult diabetes but is unclear in pediatric diabetes. No study had been conducted to explore the relationship between attention-deficit/hyperactivity disorder (ADHD) and diabetes. Using a population-based data set, we aimed to examine the association between ADHD and a prior diagnosis of diabetes mellitus (DM) in Taiwan. METHODS: A total of 4,302 patients with ADHD were selected as cases and 21,510 randomly selected subjects as controls. We used conditional logistic regression to calculate the odds ratio (OR) for having previously received a diagnosis of DM between subjects with and without ADHD. RESULTS: In this study, 116 of the 25,812 sampled subjects (0.5%) had received a diagnosis of DM prior to their index date. Subjects with ADHD had a higher proportion of prior DM diagnoses than controls (0.9% vs. 0.4%, P < 0.001). After adjusting for age, sex, index year, geographic location, and obesity, ADHD was significantly associated with a prior diagnosis of type 2 DM (OR = 2.75, 95% confidence interval (CI) = 1.82-4.16). However, no significant association was observed between ADHD and type 1 DM. CONCLUSION: The findings suggest that ADHD was associated with a previous diagnosis of type 2 DM.

Genome-Wide Sequencing Modalities for Children with Unexplained Global Developmental Delay and Intellectual Disabilities-A Narrative Review.

Unexplained global developmental delay (GDD) and intellectual disabilities (ID) together affect nearly 2% of the pediatric population. Establishing an etiologic diagnosis is crucial for disease management, prognostic evaluation, and provision of physical and psychological support for both the patient and the family. Advancements in genome sequencing have allowed rapid accumulation of gene-disorder associations and have accelerated the search for an etiologic diagnosis for unexplained GDD/ID. We reviewed recent studies that utilized genome-wide analysis technologies, and we discussed their diagnostic yield, strengths, and limitations. Overall, exome sequencing (ES) and genome sequencing (GS) outperformed chromosomal microarrays and targeted panel sequencing. GS provides coverage for both ES and chromosomal microarray regions, providing the maximal diagnostic potential, and the cost of ES and reanalysis of ES-negative results is currently still lower than that of GS alone. Therefore, singleton or trio ES is the more cost-effective option for the initial investigation of individuals with GDD/ID in clinical practice compared to a staged approach or GS alone. Based on these updated evidence, we proposed an evaluation algorithm with ES as the first-tier evaluation for unexplained GDD/ID.

The effect of age at onset of type 1 diabetes mellitus on epilepsy vulnerability.

AIMS: To examine whether type 1 diabetes age onset correlates with epilepsy incidence. METHODS: We used type 1 diabetes longitudinal data with onset age ≤ 40 years enrolled in Taiwan National Health Insurance program to examine type 1 diabetes onset age effect on epilepsy occurrence. RESULTS: In 6,165 type 1 diabetes patients, onset age groups included 3,571 patients (58%) ≤ 18 years (childhood-onset) and 2,594 patients (42%) > 18 years (adulthood-onset). After 8.6 years median follow-up following type 1 diabetes onset, epilepsy incidence rate in adulthood-onset group was 2.26-fold higher than that in childhood-onset group. Epilepsy incidence rate ratio was lowest in those with onset age 6-12 years in comparison to that in patients with onset age ≤ 6 years, but was highest in onset age of 30-40 years. Longer follow-up duration correlates with higher epilepsy risk in adulthood-onset group. Multiple logistic regression analysis showed that onset age 30-40 years, male, more than one diabetic ketoacidosis episode, and unprovoked seizure events were independent risk factors for epilepsy following type 1 diabetes onset. CONCLUSIONS: There is age-related vulnerability to epilepsy following type 1 diabetes onset. Adulthood-onset type 1 diabetes is an independent risk factor for epilepsy susceptibility after type 1 diabetes.

Effects of molecular weight fraction on antioxidation capacity of rice protein hydrolysates.

Rice protein was used as a starting material to provide rice protein hydrolysates (RPH) through enzyme-assisted extraction. RPH was further fractionated using ultrafiltration membrane (UF) and classified by molecular weight (MW; MW < 1 kDa, MW 1-10 kDa, and MW > 10 kDa). Peptides with MW < 1 kDa possessed superior antioxidant properties (p < 0.05). Therefore, UF demonstrated great efficacy in selectively separating antioxidant peptides. A Pearson correlation analysis revealed that the total phenolic concentration was correlated with oxygen radical absorbance capacity (ORAC; r = 0.999, p < 0.05). Amino acid contents had negative correlations with the scavenging activity (specifically, IC50) of 1,1-diphenyl-2-picrylhydrazyl and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radicals (r = - 0.986 to - 1.000). Reducing power was related to aromatic amino acid contents (r = 0.997, p < 0.05). In this study, enzymatic hydrolysis was discovered to be an effective method of extracting and isolating natural antioxidant proteins from broken rice, thus preserving the nutritional quality of rice and making those proteins more accessible in future applications.

The Epidemiological Analysis of COVID-19 Outbreaks in Nursing Homes during the Period of Omicron Variant Predominance.

BACKGROUND: The main purpose of this study was to evaluate the epidemic trend and risk factors associated with COVID-19 outbreaks in nursing homes during the period of Omicron variant predominance. METHODS: The study analyzed the risk factors associated with SARS-CoV-2 infection and death among the 327 residents and 129 healthcare workers (HCWs) in three hospital-affiliated nursing homes through a multivariate Cox regression model. RESULTS: The rates of receiving a COVID-19 booster dose were 70.3% for the residents and 93.0% for the healthcare workers (HCWs), respectively. A number of asymptomatic individuals, including 54 (16.5%) residents and 15 (11.6%) HCWs, were detected through mass screening surveillance tests. The COVID-19 infection rates during the outbreaks were 41.6% among residents and 48.1% among HCWs, respectively. The case fatality rate among residents was 10.3%. None of the HCWs were hospitalized or died. The multivariate Cox regression model showed that the risk of COVID-19 infection increased in males (HR 2.46; 95% CI 1.47-4.11; p = 0.001), Barthel index ≥ 61 (HR 1.93; 95% CI 1.18-3.17; p = 0.009), and dementia (HR 1.61; 95% CI 1.14-2.27; p = 0.007). The risk of COVID-19 death increased with pneumonia (HR 11.03; 95% CI 3.02-40.31; p < 0.001), hospitalization (HR 7.18; 95% CI 1.97-26.25; p = 0.003), and admission to an intensive care unit (HR 8.67; 95% CI 2.79-26.89; p < 0.001). CONCLUSIONS: This study highlighted the high infection rates with a substantial proportion of asymptomatic infections for both residents and HCWs, as well as a high case fatality rate for the residents among nursing homes during the Omicron epidemic period. We suggest implementing mass screening through regular surveillance testing as an effective strategy for early detection of COVID-19 and for preventing transmission during an epidemic period. Pneumonia is the primary risk associated with COVID-19 death. Early detection and prompt treatment of pneumonia for vulnerable residents in nursing homes are crucial to protect them from potential mortality.

Psychosocial stress in children with Tourette syndrome and chronic tic disorder.

BACKGROUND: This study aimed to understand the longitudinal relationship between psychosocial stress with tic exacerbation in children with Tourette syndrome (TS) and chronic tic disorder. METHODS: Consecutive ratings of tic severity as well as child and parental reports of psychosocial stress were obtained for 373 children (296 males, 77 females; mean age 9y 5mo; SD 3y 3mo) with TS and chronic tic disorder between January 2018 and December 2020. The Yale Global Tic Severity Scale (YGTSS) global severity score, total tic score, and impairment rating were calculated. The stressful events and YGTSS measurements were used and treated as time-varying variables in the analyses. Models that controlled for non-independence among the repeated observations using a random intercept and random slope model were employed. Each participant was treated as a random factor in the modelling. RESULTS: Family-related stress, personal relationship stress and school-related stress were independently associated with increasing YGTSS global severity, total tic score, and impairment rating over time. An increased number of stressful events were associated with increased severity of tics. CONCLUSION: Family, personal relationships, and school-related stress were consistently associated with the exacerbation of tics. Managing these stressful events is important in the treatment of TS and chronic tic disorder.

Development of erlotinib derivatives as CIP2A-ablating agents independent of EGFR activity.

Cancerous inhibitor of PP2A (CIP2A) is a novel human oncoprotein that inhibits PP2A, contributing to tumor aggressiveness in various cancers. Several studies have shown that downregulation of CIP2A by small molecules reduces PP2A-dependent phosphorylation of Akt and induces cell death. Here, a series of mono- and di-substituted quinazoline and pyrimidine derivatives based on the skeleton of erlotinib (an EGFR inhibitor) were synthesized and their bioactivities against hepatocellular carcinoma were evaluated. The di-substituted quinazoline and pyrimidine derivatives were more potent inhibitors of cancer-cell proliferation than the mono-substituted derivatives. In particular, compound 1 with chloride at position 2 of quinazoline was as potent as erlotinib in inducing cell death but no inhibition for EGFR activity. Further assays confirmed a correlation between cell death, and CIP2A and Akt inhibition by these derivatives. Among all the derivatives, compounds 19 and 22 showed the most potent antiproliferative activities and the strongest inhibition of CIP2A and p-Akt expression.

Identification and characterization of a novel class of extracellular poly(3-hydroxybutyrate) depolymerase from Bacillus sp. strain NRRL B-14911.

The catalytic, linker, and denatured poly(3-hydroxybutyrate) (dPHB)-binding domains of bacterial extracellular PHB depolymerases (PhaZs) are classified into several different types. We now report a novel class of extracellular PHB depolymerase from Bacillus sp. strain NRRL B-14911. Its catalytic domain belongs to type 1, whereas its putative linker region neither possesses the sequence features of the three known types of linker domains nor exhibits significant amino acid sequence similarity to them. Instead, this putative linker region can be divided into two distinct linker domains of novel types: LD1 and LD2. LD1 shows significant amino acid sequence similarity to certain regions of a large group of PHB depolymerase-unrelated proteins. LD2 and its homologs are present in a small group of PhaZs. The remaining C-terminal portion of this PhaZ can be further divided into two distinct domains: SBD1 and SBD2. Each domain showed strong binding to dPHB, and there is no significant sequence similarity between them. Each domain neither possesses the sequence features of the two known types of dPHB-binding domains nor shows significant amino acid sequence similarity to them. These unique features indicate the presence of two novel and distinct types of dPHB-binding domains. Homologs of these novel domains also are present in the extracellular PhaZ of Bacillus megaterium and the putative extracellular PhaZs of Bacillus pseudofirmus and Bacillus sp. strain SG-1. The Bacillus sp. NRRL B-14911 PhaZ appears to be a representative of a novel class of extracellular PHB depolymerases.

Structure, assembly, and mechanism of a PLP-dependent dodecameric L-aspartate beta-decarboxylase.

The type-I PLP enzyme l-aspartate beta-decarboxylase converts aspartate to alanine and CO(2). Similar to the homodimeric aminotransferases, its protein subunit comprises a large and a small domain, of 410 and 120 residues, respectively. The crystal structure reveals a dodecamer made of six identical dimers arranged in a truncated tetrahedron whose assembly involves tetramer and hexamer as intermediates. The additional helical motifs I and II participate in the oligomer formation. Triple mutations of S67R/Y68R/M69R or S67E/Y68E/M69E in motif I produced an inactive dimer. The PLP is bound covalently to Lys315 in the active site, while its phosphate group interacts with a neighboring Tyr134. Removal of the bulky side chain of Arg37, which overhangs the PLP group, improved the substrate affinity. Mutations in flexible regions produced the more active K17A and the completely inactive R487A. The structure also suggests that substrate binding triggers conformational changes essential for catalyzing the reaction.