Beyond Glycolysis: Aldolase A is a Novel Effector in Reelin Mediated Dendritic Development.

Reelin, a secreted glycoprotein, plays a crucial role in guiding neocortical neuronal migration, dendritic outgrowth and arborization, and synaptic plasticity in the adult brain. Reelin primarily operates through the canonical lipoprotein receptors apolipoprotein E receptor 2 (Apoer2) and very low-density lipoprotein receptor (Vldlr). Reelin also engages with non-canonical receptors and unidentified co-receptors; however, the effects of which are less understood. Using high-throughput tandem mass tag LC-MS/MS-based proteomics and gene set enrichment analysis, we identified both shared and unique intracellular pathways activated by Reelin through its canonical and non-canonical signaling in primary murine neurons during dendritic growth and arborization. We observed pathway crosstalk related to regulation of cytoskeleton, neuron projection development, protein transport, and actin filament-based process. We also found enriched gene sets exclusively by the non-canonical Reelin pathway including protein translation, mRNA metabolic process and ribonucleoprotein complex biogenesis suggesting Reelin fine-tunes neuronal structure through distinct signaling pathways. A key discovery is the identification of aldolase A, a glycolytic enzyme and actin binding protein, as a novel effector of Reelin signaling. Reelin induced de novo translation and mobilization of aldolase A from the actin cytoskeleton. We demonstrated that aldolase A is necessary for Reelin-mediated dendrite growth and arborization in primary murine neurons and mouse brain cortical neurons. Interestingly, the function of aldolase A in dendrite development is independent of its known role in glycolysis. Altogether, our findings provide new insights into the Reelin-dependent signaling pathways and effector proteins that are crucial for actin remodeling and dendritic development. Significance: Reelin is an extracellular glycoprotein and exerts its function primarily by binding to the canonical lipoprotein receptors Apoer2 and Vldlr. Reelin is best known for its role in neuronal migration during prenatal brain development. Reelin also signals through a non-canonical pathway outside of Apoer2/Vldlr; however, these receptors and signal transduction pathways are less defined. Here, we examined Reelin's role during dendritic outgrowth in primary murine neurons and identified shared and distinct pathways activated by canonical and non-canonical Reelin signaling. We also found aldolase A as a novel effector of Reelin signaling, that functions independently of its known metabolic role, highlighting Reelin's influence on actin dynamics and neuronal structure and growth.

Tight control of the APP-Mint1 interaction in regulating amyloid production.

Generation of amyloid-ß (Aß) peptides through the proteolytic processing of the amyloid precursor protein (APP) is a pathogenic event in Alzheimer's disease (AD). APP is a transmembrane protein and endocytosis of APP mediated by the YENPTY motif is a key step in Aß generation. Mints, a family of cytosolic adaptor proteins, directly bind to the YENPTY motif of APP and facilitate APP trafficking and processing. Here, we generated and examined two Mint1 mutants, Tyr633Ala of Mint1 (Mint1Y633A) that enhanced APP binding, and Tyr549Ala and Phe610Ala mutant (Mint1Y549A/F610A), that reduced APP binding. We investigated how perturbing the APP-Mint1 interaction through these Mint1 mutants alter APP and Mint1 cellular dynamics and Mint1's interaction with its other binding partners. We found that Mint1Y633A increased binding affinity specifically for APP and presenilin1 (catalytic subunit of γ-secretase), that subsequently enhanced APP endocytosis in primary murine neurons. Conversely, Mint1Y549A/F610A exhibited reduced APP affinity and Aß secretion. The effect of Mint1Y549A/F610A on Aß release was greater compared to knocking down all three Mint proteins supporting the APP-Mint1 interaction is a critical factor in Aß production. Altogether, this study highlights the potential of targeting the APP-Mint1 interaction as a therapeutic strategy for AD.

A 30-Year Worldwide Research Productivity of Scientific Publication in Full-Endoscopic Decompression Spine Surgery: Quantitative and Qualitative Analysis.

OBJECTIVE: The ever-growing number of articles related to full-endoscopic spine surgery published in the last few decades presents a challenge which is perplexing and time-consuming in identifying the current research status. The study aims to identify and analyze the most cited works related to full-endoscopic decompression spine surgery, compare the articles published by different publishers and area, and show the current publication status of full-endoscopic research. METHODS: Using Bibliometrix, CiteSpace, and VOSviewer, we analyzed the bibliometric data selected from the Web of Science database between 1992 and 2022. Spine has the highest H-index with the most-cited journal in the field of full-endoscopic decompression spine surgery. China ranked as the most productive country, whereas the most cited with high H-index papers came from South Korea. For the author analysis, Yeung AT, Ruetten S, Hoogland T, Ahn Y, Choi G, and Mayer HM were the most impactful authors in the global and local citations. The most productive organization is Wooridul Spine Hospital. CONCLUSION: The bibliometric study showed a growing trend of research on full-endoscopic decompression spine surgery over the past 30 years. It has demonstrated that there is a significant increase in the number of authors, institutions, and internationally collaborated countries. However, the quality of studies is still low, and the lack of high-quality clinical evidence and the trend of general journal submissions has somewhat affected the quality of endoscopy journals in recent years.

Barriers to research opportunities among osteopathic medical students.

CONTEXT: Despite the increase of importance placed on research, both by residency program directors and the medical field at large, osteopathic medical students (OMS) have significantly fewer research experiences than United States (U.S.) allopathic medical students and non-U.S. international medical graduates. However, few studies have addressed this long-standing discrepancy, and none directly have focused on osteopathic medical students to assess their unique needs. The literature would benefit from identifying the barriers osteopathic medical students encounter when participating in research and understanding the currently available resources. OBJECTIVES: To assess the barriers that OMS face when seeking research opportunities, identify resources currently available to osteopathic medical students at their respective schools, and investigate factors that contribute to an osteopathic medical student's desire to pursue research opportunities. Additionally, to investigate osteopathic medical students' confidence in research methodology. METHODS: A survey was created by the investigators and administered to participants over a three-month period via a GoogleForm. Research participants were surveyed for demographic information, as well as their involvement in research projects in the past, mentor availability, institutional resources, motivation to participate in research, individual barriers to participation, and confidence in their ability to do independent research. Responses were de-identified and analyzed using Microsoft Excel functions to count data and calculate percentages, as well as Pearson's chi square analysis. RESULTS: After relevant exclusion, 668 responses were included. Of the students surveyed, 85.9% (574) indicated they currently and/or in the past were involved in research. More than half of the respondents that are not currently involved in research are interested in pursuing it (86.9%; 344). The primary barriers students reported facing include lack of time (57.8%; 386), feeling overwhelmed and unsure how to start (53.4%; 357), and lack of access to research (53%; 354). 34.7% (232) of students stated they either did not have resources from their school or were unsure whether these resources were available. The two most cited motivations to pursue research included boosting their residency application and/or interest in the area of study. Male gender and current research were associated with reported confidence in research ( [4, n=662]=10.6, p<0.05). CONCLUSIONS: Findings from this study provide a synopsis of the barriers to research opportunities among osteopathic medical students. Notably, ⅓ of OMSs reported an absence or unawareness of available research resources at their osteopathic medical schools.

A Systematic Review of the Risks of Motor Vehicle Crashes Associated with Psychiatric Disorders.

OBJECTIVE: Psychiatric disorders and their treatments have the potential to adversely impact driving skills. However, it is unclear to what extent this poses a public health risk by increasing the risk of motor vehicle crashes (MVCs). The aim of this systematic review was to synthesize and critically appraise evidence on the risk of MVC for drivers with psychiatric disorders. METHOD: We conducted a systematic review of the MVC risk associated with psychiatric disorders using seven databases in November 2019. Two reviewers examined each study and extracted data. The National Heart, Lung, and Blood Institute Quality Assessment tools were used to assess each study's quality of evidence. RESULTS: We identified 24 studies that met the inclusion criteria, including eight cohort, 10 case-control, and six cross-sectional designs. Quality assessment ratings were "Good" for four studies, "Fair" for 10, and "Poor" for 10. Self-report or questionnaires were used in place of objective measures of either MVC, psychiatric disorder, or both in 12 studies, and only seven adjusted for driving exposure. Fifteen studies reported an increased risk of MVC associated with psychiatric disorders, and nine did not. There was no category of disorder that was consistently associated with increased MVC risk. CONCLUSION: The available evidence is mixed, not of high quality, and does not support a blanket restriction on drivers with psychiatric disorder. An individualized approach, as recommended by international guidelines, should continue. Further research should include objective assessments of psychiatric disorders and MVC risk and adjust for driving exposure.

Recommendations on the post-acute management of the osteoporotic fracture - Patients with "very-high" Re-fracture risk.

Osteoporosis is a systemic skeletal disease where there is low bone mass and deterioration of bone microarchitecture, leading to an increased risk of a fragility fracture. The aim of this clinical guideline from Fragility Fracture Network Hong Kong SAR, is to provide evidence-based recommendations on the post-acute treatment of the osteoporotic fracture patient that presents for clinical care at the Fracture Liaison Service (FLS). It is now well established that the incidence of a second fracture is especially high after the first 2 years of the initial osteoporotic fracture. Therefore, the recent osteoporotic fracture should be categorized as "very-high" re-fracture risk. Due to the significant number of silent vertebral fractures in the elderly population, it is also recommended that vertebral fracture assessment (VFA) should be incorporated into FLS. This would have diagnostic and treatment implications for the osteoporotic fracture patient. The use of a potent anti-osteoporotic agent, and preferably an anabolic followed by an anti-resorptive agent should be considered, as larger improvements in BMD is strongly associated with a reduction in fractures. Managing other risk factors including falls and sarcopenia are imperative during rehabilitation and prevention of another fracture. Although of low incidence, one should remain vigilant of the atypical femoral fracture. The aging population is increasing worldwide, and it is expected that the treatment of osteoporotic fractures will be routine. The recommendations are anticipated to aid in the daily clinical practice for clinicians. The Translational potential of this article: Fragility fractures have become a common encounter in clinical practise in the hospital setting. This article provides recommendations on the post-acute management of fragility fracture patients at the FLS.

First Case of Splenic Abscess Parvimonas micra and Bacteremia Porphyromonas gingivalis Coinfection.

We report a case of a 76-year-old Caucasian male with bacteremia caused by Porphyromonas gingivalis and splenic abscess caused by Parvimonas micra. This patient presented with nonspecific symptoms: fever, chills, body aches, and shortness of breath. He was treated with IV piperacillin-tazobactam that was later switched to ampicillin sodium/sulbactam sodium during his hospital course and underwent a splenectomy. He ultimately expired due to acute respiratory failure and cardiac arrest, secondary to post-surgical complications. To our knowledge, this is the first case of P. micra and P. gingivalis coinfection.

Clinical Discordance in Monozygotic Twins With Autism Spectrum Disorder.

There is a significant concordance of autism spectrum disorder in monozygotic (MZ) twins, where behavioral manifestations are heavily influenced by genetic factors. We describe a case of male monozygotic twins with autism spectrum disorder (ASD), raised in the same household, that present with different clinical manifestations. One of the twins presents with intermittent frank syncopal episodes, sinus bradycardia, and elevated alkaline phosphatase (ALP), while the other has symptoms of attention-deficit/hyperactivity disorder (ADHD), normal cardiological findings, and normal ALP level. The clinical discordance in this pair of monozygotic twins may be due to any of the following: 1) neuroanatomic cerebellar differences, 2) variable expression of genotype, and 3) inconsistent neurotransmitter regulation.

Spontaneous Miscarriage Management Experience: A Systematic Review.

BACKGROUND: The estimated frequency of spontaneous miscarriage is about a quarter of all clinically identified pregnancies in the United States. Women typically go to the emergency department (ED) or outpatient clinic when they experience symptoms, including but not limited to vaginal bleeding, abdominal pain, and contractions. The care that is provided varies from place to place. METHODS: Researchers searched articles from 2010 to 2021 for reports mentioning treatment for spontaneous abortion. Search terms included "miscarriage aftercare" and "spontaneous abortion care," seeking articles addressing the psychological effects of miscarriage and reporting patient experiences in different clinical settings. Data were independently reviewed, graded for evidence quality, and assessed for risk bias using the AMSTAR checklist. RESULTS: The search strategy yielded 2,275 articles, six of which met the inclusion criteria. Conservative, medical, and surgical management were provided, with surgical management being more common among women with higher education and socioeconomic status. All qualitative studies reported dissatisfaction with care provided in the emergency department, partially due to a lack of emotional support. Structured bereavement intervention was beneficial for women experiencing early pregnancy loss and led to fewer reports of despair. The quantitative studies referenced interventions that aided patients in coping with pregnancy loss and identified several factors influencing the type of treatment received as well as the patient's ability to cope with feeling depressed following a miscarriage. CONCLUSION: Psychological management is not regularly addressed in the emergency department, and protocols including bereavement education for healthcare providers as well as patient involvement in management would improve the overall patient experience with spontaneous miscarriage care.

Human APOER2 Isoforms Have Differential Cleavage Events and Synaptic Properties.

Human apolipoprotein E receptor 2 (APOER2) is a type I transmembrane protein with a large extracellular domain (ECD) and a short cytoplasmic tail. APOER2-ECD contains several ligand-binding domains (LBDs) that are organized into exons with aligning phase junctions, which allows for in-frame exon cassette splicing events. We have identified 25 human APOER2 isoforms from cerebral cortex using gene-specific APOER2 primers, where the majority are exon-skipping events within the N-terminal LBD regions compared with six identified in the heart. APOER2 undergoes proteolytic cleavage in response to ligand binding that releases a C-terminal fragment (CTF) and transcriptionally active intracellular domain (ICD). We tested whether the diversity of human brain-specific APOER2 variants affects APOER2 cleavage. We found isoforms with differing numbers of ligand-binding repeats generated different amounts of CTFs compared with full-length APOER2 (APOER2-FL). Specifically, APOER2 isoforms lacking exons 5-8 (Δex5-8) and lacking exons 4-6 (Δex4-6) generated the highest and lowest amounts of CTF generation, respectively, in response to APOE peptide compared with APOER2-FL. The differential CTF generation of Δex5-8 and Δex4-6 coincides with the proteolytic release of the ICD, which mediates transcriptional activation facilitated by the Mint1 adaptor protein. Functionally, we demonstrated loss of mouse Apoer2 decreased miniature event frequency in excitatory synapses, which may be because of a decrease in the total number of synapses and/or VAMP2 positive neurons. Lentiviral infection with human APOER2-FL or Δex4-6 isoform in Apoer2 knockout neurons restored the miniature event frequency but not Δex5-8 isoform. These results suggest that human APOER2 isoforms have differential cleavage events and synaptic properties.SIGNIFICANCE STATEMENT Humans and mice share virtually the same number of protein-coding genes. However, humans have greater complexity of any higher eukaryotic organisms by encoding multiple protein forms through alternative splicing modifications. Alternative splicing allows pre-mRNAs transcribed from genes to be spliced in different arrangements, producing structurally and functionally distinct protein variants that increase proteomic diversity and are particularly prevalent in the human brain. Here, we identified 25 distinct human APOER2 splice variants from the cerebral cortex using gene-specific APOER2 primers, where the majority are exon-skipping events that exclude N-terminal ligand-binding regions of APOER2. We show that some of the APOER2 variants have differential proteolytic properties in response to APOE ligand and exhibit distinct synaptic properties.

Single molecule, long-read Apoer2 sequencing identifies conserved and species-specific splicing patterns.

Apolipoprotein E receptor 2 (Apoer2) is a synaptic receptor in the brain that binds disease-relevant ligand Apolipoprotein E (Apoe) and is highly alternatively spliced. We examined alternative splicing (AS) of conserved Apoer2 exons across vertebrate species and identified gain of exons in mammals encoding functional domains such as the cytoplasmic and furin inserts, and loss of an exon in primates encoding the eighth LDLa repeat, likely altering receptor surface levels and ligand-binding specificity. We utilized single molecule, long-read RNA sequencing to profile full-length Apoer2 isoforms and identified 68 and 48 unique full-length Apoer2 transcripts in the mouse and human cerebral cortex, respectively. Furthermore, we identified two exons encoding protein functional domains, the third EGF-precursor like repeat and glycosylation domain, that are tandemly skipped specifically in mouse. Our study provides new insight into Apoer2 isoform complexity in the vertebrate brain and highlights species-specific differences in splicing decisions that support functional diversity.

In-silico analysis of closed-loop vasopressor control of phenylephrine versus norepinephrine.

We have previously demonstrated in in-silico, pre-clinical animal models, and finally human clinical studies the ability of a novel closed-loop vasopressor titration system to manage norepinephrine infusion rates to keep mean arterial blood pressure in a very tight range, reduce hypotension time and severity, and reduce overtreatment. We hypothesized that the same controller could, with modification for pharmacologic differences, suitably titrate a lower-potency longer duration of action agent like phenylephrine. Using the same physiologic simulation model as was used previously for in-silico testing of our controller for norepinephrine, we first updated the model to include a new vasopressor agent modeled after phenylephrine. A series of simulation tests patterned after our previous norepinephrine study was then conducted, this time using phenylephrine for management, in order to both test the system with the new agent and allow for comparisons between the two. Hundreds of simulation trials were conducted across a range of patient and environmental variances. The controller performance was characterized based on time in target, time above and below target, coefficient of variation, and using Varvel's criteria. The controller kept the simulated patients' MAP in target for 94% of management time in the simple scenarios and more than 85% of time in the most challenging scenarios. Varvel criteria were all under 1% error for expected pharmacologic responses and were consistent with those established for norepinephrine in our previous studies. The controller was able to acceptably titrate phenylephrine in this simulated patient model consistent with performance previously seen for norepinephrine after adjusting for the anticipated differences between the two agents.

FOXR1 regulates stress response pathways and is necessary for proper brain development.

The forkhead box (Fox) family of transcription factors are highly conserved and play essential roles in a wide range of cellular and developmental processes. We report an individual with severe neurological symptoms including postnatal microcephaly, progressive brain atrophy and global developmental delay associated with a de novo missense variant (M280L) in the FOXR1 gene. At the protein level, M280L impaired FOXR1 expression and induced a nuclear aggregate phenotype due to protein misfolding and proteolysis. RNAseq and pathway analysis showed that FOXR1 acts as a transcriptional activator and repressor with central roles in heat shock response, chaperone cofactor-dependent protein refolding and cellular response to stress pathways. Indeed, FOXR1 expression is increased in response to cellular stress, a process in which it directly controls HSPA6, HSPA1A and DHRS2 transcripts. The M280L mutant compromises FOXR1's ability to respond to stress, in part due to impaired regulation of downstream target genes that are involved in the stress response pathway. Quantitative PCR of mouse embryo tissues show Foxr1 expression in the embryonic brain. Using CRISPR/Cas9 gene editing, we found that deletion of mouse Foxr1 leads to a severe survival deficit while surviving newborn Foxr1 knockout mice have reduced body weight. Further examination of newborn Foxr1 knockout brains revealed a decrease in cortical thickness and enlarged ventricles compared to littermate wild-type mice, suggesting that loss of Foxr1 leads to atypical brain development. Combined, these results suggest FOXR1 plays a role in cellular stress response pathways and is necessary for normal brain development.

Hypotonia-cystinuria 2p21 deletion syndrome: Intrafamilial variability of clinical expression.

Two siblings presented similarly with congenital hypotonia, lactic acidosis, and failure to thrive. Later in childhood, the brother developed cystinuria and nephrolithiasis whereas the older sister suffered from cystinuria and chronic neurobehavioral disturbances. Biopsied muscle studies demonstrated deficient cytochrome c oxidase activities consistent with a mitochondrial disease. Whole exome sequencing (WES), however, revealed a homozygous 2p21 deletion involving two contiquous genes, SLC3A1 (deletion of exons 2-10) and PREPL (deletion of exons 2-14). The molecular findings were consistent with the hypotonia-cystinuria 2p21 deletion syndrome, presenting similarly in infancy with mitochondrial dysfunction but diverging later in childhood and displaying intrafamilial phenotypic variability.

The methyltransferase METTL9 mediates pervasive 1-methylhistidine modification in mammalian proteomes.

Post-translational methylation plays a crucial role in regulating and optimizing protein function. Protein histidine methylation, occurring as the two isomers 1- and 3-methylhistidine (1MH and 3MH), was first reported five decades ago, but remains largely unexplored. Here we report that METTL9 is a broad-specificity methyltransferase that mediates the formation of the majority of 1MH present in mouse and human proteomes. METTL9-catalyzed methylation requires a His-x-His (HxH) motif, where "x" is preferably a small amino acid, allowing METTL9 to methylate a number of HxH-containing proteins, including the immunomodulatory protein S100A9 and the NDUFB3 subunit of mitochondrial respiratory Complex I. Notably, METTL9-mediated methylation enhances respiration via Complex I, and the presence of 1MH in an HxH-containing peptide reduced its zinc binding affinity. Our results establish METTL9-mediated 1MH as a pervasive protein modification, thus setting the stage for further functional studies on protein histidine methylation.

Targeting the APP-Mint2 Protein-Protein Interaction with a Peptide-Based Inhibitor Reduces Amyloid-ß Formation.

There is an urgent need for novel therapeutic approaches to treat Alzheimer's disease (AD) with the ability to both alleviate the clinical symptoms and halt the progression of the disease. AD is characterized by the accumulation of amyloid-ß (Aß) peptides which are generated through the sequential proteolytic cleavage of the amyloid precursor protein (APP). Previous studies reported that Mint2, a neuronal adaptor protein binding both APP and the γ-secretase complex, affects APP processing and formation of pathogenic Aß. However, there have been contradicting results concerning whether Mint2 has a facilitative or suppressive effect on Aß generation. Herein, we deciphered the APP-Mint2 protein-protein interaction (PPI) via extensive probing of both backbone H-bond and side-chain interactions. We also developed a proteolytically stable, high-affinity peptide targeting the APP-Mint2 interaction. We found that both an APP binding-deficient Mint2 variant and a cell-permeable PPI inhibitor significantly reduced Aß42 levels in a neuronal in vitro model of AD. Together, these findings demonstrate a facilitative role of Mint2 in Aß formation, and the combination of genetic and pharmacological approaches suggests that targeting Mint2 is a promising therapeutic strategy to reduce pathogenic Aß levels.

Plasma glycemic measures and fecundability in a Singapore preconception cohort study.

OBJECTIVE: To examine the association between plasma glycemia in women attempting to conceive and fecundability, as measured by time to pregnancy. DESIGN: Prospective preconception population-based study. SETTING: Hospital. PATIENT(S): Asian preconception women, 18-45 years old, attempting conception for ≤12 cycles at study entry. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): We ascertained time to pregnancy within a year of glycemic assessment in menstrual cycles. We estimated fecundability ratios (FRs) and 95% confidence intervals using discrete-time proportional hazards models, adjusting for age, ethnicity, education, body mass index, and cycle regularity and accounting for left truncation and right censoring. RESULT(S): We studied a population sample of 766 women from the Singapore Preconception Study of Long-Term Maternal and Child Outcomes prospective cohort. Compared with women with normoglycemia, women with dysglycemia (prediabetes and diabetes, defined by the American Diabetes Association) had a lower FR (0.56). Compared with the respective lowest quintiles, women in the highest quintile of fasting glucose (≥5.1 mmol/L) had an FR of 0.60, while women in the highest 2-hour postload glucose quintile (≥6.9 mmol/L) had an FR of 0.66. Overall, the FRs decreased generally across the range of fasting and 2-hour plasma glucose. Glycated hemoglobin was not associated with fecundability. CONCLUSION(S): Increasing preconception plasma glucose is associated with reduced fecundability, even within the normal range of glucose concentrations. CLINICAL TRIAL REGISTRATION NUMBER: NCT03531658.

Surface texture limits transfer of S. aureus, T4 bacteriophage, influenza B virus and human coronavirus.

Spread of pathogens on contaminated surfaces plays a key role in disease transmission. Surface technologies that control pathogen transfer can help control fomite transmission and are of great interest to public health. Here, we report a novel bead transfer method for evaluating fomite transmission in common laboratory settings. We show that this method meets several important criteria for quantitative test methods, including reasonableness, relevancy, resemblance, responsiveness, and repeatability, and therefore may be adaptable for standardization. In addition, this method can be applied to a wide variety of pathogens including bacteria, phage, and human viruses. Using the bead transfer method, we demonstrate that an engineered micropattern limits transfer of Staphylococcus aureus by 97.8% and T4 bacteriophage by 93.0% on silicone surfaces. Furthermore, the micropattern significantly reduces transfer of influenza B virus and human coronavirus on silicone and polypropylene surfaces. Our results highlight the potential of using surface texture as a valuable new strategy in combating infectious diseases.

Phylogenetics-based identification and characterization of a superior 2,3-butanediol dehydrogenase for Zymomonas mobilis expression.

BACKGROUND: Zymomonas mobilis has recently been shown to be capable of producing the valuable platform biochemical, 2,3-butanediol (2,3-BDO). Despite this capability, the production of high titers of 2,3-BDO is restricted by several physiological parameters. One such bottleneck involves the conversion of acetoin to 2,3-BDO, a step catalyzed by 2,3-butanediol dehydrogenase (Bdh). Several Bdh enzymes have been successfully expressed in Z. mobilis, although a highly active enzyme is yet to be identified for expression in this host. Here, we report the application of a phylogenetic approach to identify and characterize a superior Bdh, followed by validation of its structural attributes using a mutagenesis approach. RESULTS: Of the 11 distinct bdh genes that were expressed in Z. mobilis, crude extracts expressing Serratia marcescens Bdh (SmBdh) were found to have the highest activity (8.89 µmol/min/mg), when compared to other Bdh enzymes (0.34-2.87 µmol/min/mg). The SmBdh crystal structure was determined through crystallization with cofactor (NAD+) and substrate (acetoin) molecules bound in the active site. Active SmBdh was shown to be a tetramer with the active site populated by a Gln247 residue contributed by the diagonally opposite subunit. SmBdh showed a more extensive supporting hydrogen-bond network in comparison to the other well-studied Bdh enzymes, which enables improved substrate positioning and substrate specificity. This protein also contains a short α6 helix, which provides more efficient entry and exit of molecules from the active site, thereby contributing to enhanced substrate turnover. Extending the α6 helix to mimic the lower activity Enterobacter cloacae (EcBdh) enzyme resulted in reduction of SmBdh function to nearly 3% of the total activity. In great contrast, reduction of the corresponding α6 helix of the EcBdh to mimic the SmBdh structure resulted in ~ 70% increase in its activity. CONCLUSIONS: This study has demonstrated that SmBdh is superior to other Bdhs for expression in Z. mobilis for 2,3-BDO production. SmBdh possesses unique structural features that confer biochemical advantage to this protein. While coordinated active site formation is a unique structural characteristic of this tetrameric complex, the smaller α6 helix and extended hydrogen network contribute towards improved activity and substrate promiscuity of the enzyme.