Homozygous missense variant in POPDC3 causes recessive limb-girdle muscular dystrophy type 26.

BACKGROUND: Limb-girdle muscular dystrophy (LGMD) comprises a heterogeneous group of diseases, affecting different muscles, predominantly skeletal muscles and cardiac muscles of the body. LGMD is classified into two main subtypes A and B, which are further subclassified into eight dominant and thirty recessive subtypes. Three genes, namely POPDC1, POPDC2 and POPDC3, encode popeye domain-containing protein (POPDC), and the variants of POPDC1 and POPDC3 genes have been associated with LGMD. METHODS: In the present study, we performed whole-exome sequencing (WES) analysis on a single-family to investigate the hallmark features of LGMD. The results of WES were further confirmed by Sanger sequencing and 3D protein modeling was also conducted. RESULTS: WES data analysis and Sanger sequencing revealed a homozygous missense variant (c.460A>G; p.Lys154Glu) at a highly conserved amino acid position in the POPDC3. Mutations in the POPDC3 gene have been previously associated with recessive limb-girdle muscular dystrophy type 26. 3D protein modeling further suggested that the identified variant might affect the POPDC3 structure and proper function. CONCLUSIONS: The present study confirms the role of POPDC3 in LGMD, and will facilitate genetic counseling of the family to mitigate the risks of the carrier or affects on future pregnancies.

Role of Lactobacillus plantarum 299v versus Placebo in symptomatic improvement of irritable bowel syndrome patients.

Objectives: To find the efficacy of lactobacillus plantarum 299v compared to placebo in symptomatic improvement of irritable bowel syndrome patients. METHODS: The randomised control trial was conducted at the Lady Reading Hospital, Peshawar, Pakistan, from July 20, 2014, to January 20, 2015, and comprised irritable bowel syndrome patients who were randomised into intervention group A treated with lactobacillus plantarum 299v and control group B treated with placebo. Symptoms, like abdominal pain, bloating and complete rectal emptying, were noted and compared between the groups. Data was analysed using SPSS 19. RESULTS: Of the 190 patients assessed, 120(63%) were included; 60(50%) in each of the two groups. After loss to follow-up, the study was completed by 55(91.7%) in group A and 53(88.3%) in group B. The mean age in group A was 37.53±9.02 and it was 34.40±11.23 in group B (p=0.652). The male-to-female ratio and irritable bowel syndrome type in both the groups was not significantly different (p>0.055). There was no significant difference in terms of relieving abdominal pain, bloating and rectal emptying between the groups (p>0.05). CONCLUSIONS: No significant efficacy was established in favour of treating in irritable bowel syndrome with lactobacillus plantarum 299v. Trials Registration: IRCT20200504047303N1.

Comparative in vitro activity of selected antibacterial agents against Escherichia coli isolated from hospitalized patients suffering UTI.

Escherichia coli is the most studied among those bacteria causing urinary tract infections. This study was aimed to find out antibacterial activity and minimum inhibitory concentration of selected antibacterial agents against E. coli isolates of hospitalized UTI patients. The specimens were inoculated on Eosin Methylene Blue medium. E. coli isolates were identified via colonial morphology, biochemical testing and API-20 kit. The susceptibility pattern of antibacterial agents was determined applying disc diffusion method (Kirby-Bauer) and dilution tube method. Among all, 38.82% (n=158/407) specimens were positive for E. coli, while the rest showed either no growth or exhibited colonies other than E. coli. while observing the susceptibility pattern, Imipenem was found the most effective (73.42%) antibacterial agent, followed by nitrofurantoin (52.53%), cefpirome (44.94%) and tazobactam/ piperacillin (44.94%), whereas the E. coli isolates were highly resistant to sulfamethoxazole/trimethoprim (71.52%), followed by Amoxicillin-clavulanic acid (67.72%), nalidixic acid (66.46%) and Tobramycin (62.03%), when tested by disc diffusion method. The isolates were susceptible to cefpirome (39.87%) and tobramycin (39.87%) and resistant to sulfamethoxazole/trimethoprim (75.32%), followed by levofloxacin (61.39%), when tested by tube dilution method. The study concluded high degree of resistance against Sulfamethoxazole/trimethoprim, in contrast, cephalosporin and Imipenem exhibited good potency which can be recommended for UTI.

REPORT - CYP2D6*4 null allele frequency in sixteen Pakistani ethnic groups.

CYP2D6 belongs to a family of Cytochrome P450 and is involved in metabolism of a number of commonly prescribed drugs. This study was designed to identify *4 allelic frequency of CYP2D6 in Pakistani population. The ethno-geographic variations in the CYP2D6 alleles are responsible for varied expression of this enzyme and thus influence the metabolic rate and efficacy of prescribed drugs. In total, 976 volunteers belonging to 16 different ethnic groups of Pakistan were screened for CYP2D6*4 polymorphism. The *4 allele was detected in all the ethnic groups with varied frequency ranging from 3.73%-13.64% and an overall average of 7.22% in different ethnic groups of the population. Maximum frequency was detected in northern Pakistani population including Meo (13.64%), Punjabi (11.96%) and Pathan (10.42%). Low frequency (<4%) of*4 polymorphism was observed in Kalash and Makrani groups, whereas an intermediate frequency (5-9%) was observed in all the other ethnic groups. The data indicates that despite ethnic diversity poor metabolizers in Pakistani population are expected to carry CYP2D6*4 allele at a relatively higher frequency than most other Asian populations. (Word count = 186).

Evaluation of chromosomal abnormalities and Y chromosome microdeletion in infertile males of 10 families.

This study was designed to investigate the hormonal, seminal changes and chromosomal aberrations in cases of male infertility. A total of ten infertile families from Khyber Pakhtunkhwa of Pakistan were included in the study. The families were clinically evaluated by standard criteria; diagnosis of azoospermic and oligospermic males was confirmed. Seminal, hormonal, ultra sonographic and histopathological examinations were carried out for all the affected participants of the study. Karyotyping was performed on peripheral blood lymphocytes according to standard methods. Hormones were altered in six families. Ultrasonographic abnormal finding was observed in six families. Karyotyping analysis revealed numerical aberration in family G (0X) and family I (XXY). The remainingfamilies had no structural or numerical aberration. Y chromosome microdeletion analysis revealed AZFc deletion in both the affected participants of the family C. The remaining families were found normal for microdeletion. The occurrence of chromosomal anomalies and Y chromosome microdeletions among infertile males strongly suggests the need to include these two tests in routine investigations of male in fertility cases.

Analysis of NT-proBNP and uric acid due to left ventricle hypertrophy in the patients of aortic valve disease.

OBJECTIVE: To evaluate the concentration of N terminal proBNP (NT-proBNP) and partially the serum uric acid in the severe condition of aortic valve dysfunction for assessment of left ventricle hypertrophy. METHODS: The study was conducted in the signal transduction lab department of biochemistry Quaid-I-Azam University, Islamabad from September 2013 to February 2017. NT-proBNP and serum uric acid were measured in one hundred patients of aortic valve dysfunction. The patients were divided into three main groups: 1) Aortic stenosis, 2) Aortic regurgitation, and 3) Aortic stenosis with Aortic regurgitation. The results were compared between disease and controls groups. RESULTS: High level of plasma NT-proBNP was detected in all the three disease groups of aortic valve (stenosis, p<0.001), (regurgitation, p<0.001) and (stenosis with regurgitation, p<0.001). In addition, non-significantly increased level of serum uric acid was also observed in left ventricle hypertrophy in all the three respective disease groups of aortic valve. CONCLUSION: Increased secretion of NT-proBNP during cardiac remodeling can be related to the severity of left ventricle hypertrophy due to aortic valve abnormality in all the disease groups of severe stenosis, severe regurgitation, and combine disease condition of severe stenosis and severe regurgitation. However, non-significant increase in uric acid concentration is also identified which may be due to one of the factors involved in left ventricle hypertrophy in all the three disease groups of aortic valve. The interaction of uric acid with NT-proBNP during cardiac remolding due to aortic valve dysfunction is still not clear.

Potential Implications for Designing Drugs Against the Brown Spider Venom Phospholipase-D.

Loxoscelism refers to the clinical symptoms that develop after brown spider bites. Brown spider venoms contain several phospholipase-D isoforms, which are the main toxins responsible for both the cutaneous and systemic effects of loxoscelism. Understanding of the phospholipase-D catalytic mechanism is crucial for the development of specific treatment that could reverse the toxic effects caused by the spider bite. Based on enzymatic, biological, structural, and thermodynamic tests, we show some features suitable for designing drugs against loxoscelism. Firstly, through molecular docking and molecular dynamics predictions, we found three different molecules (Suramin, Vu0155056, and Vu0359595) that were able to bind the enzyme's catalytic site and interact with catalytically important residues (His12 or His47) and with the Mg2+ co-factor. The binding promoted a decrease in the recombinant brown spider venom phospholipase-D (LiRecDT1) enzymatic activity. Furthermore, the presence of the inhibitors reduced the hemolytic, dermonecrotic, and inflammatory activities of the venom toxin in biological assays. Altogether, these results indicate the mode of action of three different LiRecDT1 inhibitors, which were able to prevent the venom toxic effects. This strengthen the idea of the importance of designing a specific drug to treat the serious clinical symptoms caused by the brown spider bite, a public health problem in several parts of the world, and until now without specific treatment. J. Cell. Biochem. 118: 726-738, 2017. © 2016 Wiley Periodicals, Inc.

Tyrosine binding and promiscuity in the arginine repressor from the pathogenic bacterium Corynebacterium pseudotuberculosis.

The arginine repressor (ArgR) regulates arginine biosynthesis in a number of microorganisms and consists of two domains interlinked by a short peptide; the N-terminal domain is involved in DNA binding and the C-terminal domain binds arginine and forms a hexamer made-up of a dimer of trimers. The crystal structure of the C-terminal domain of ArgR from the pathogenic Corynebacterium pseudotuberculosis determined at 1.9 Å resolution contains a tightly bound tyrosine at the arginine-binding site indicating hitherto unobserved promiscuity. Structural analysis of the binding pocket displays clear molecular adaptations to accommodate tyrosine binding suggesting the possible existence of an alternative regulatory process in this pathogenic bacterium.

Crystal structure of mature 2S albumin from Moringa oleifera seeds.

2S albumins, the seed storage proteins, are the primary sources of carbon and nitrogen and are involved in plant defense. The mature form of Moringa oleifera (M. oleifera), a chitin binding protein isoform 3-1 (mMo-CBP3-1) a thermostable antifungal, antibacterial, flocculating 2S albumin is widely used for the treatment of water and is potentially interesting for the development of both antifungal drugs and transgenic crops. The crystal structure of mMo-CBP3-1 determined at 1.7 Å resolution demonstrated that it is comprised of two proteolytically processed α-helical chains, stabilized by four disulfide bridges that is stable, resistant to pH changes and has a melting temperature (TM) of approximately 98 °C. The surface arginines and the polyglutamine motif are the key structural factors for the observed flocculating, antibacterial and antifungal activities. This represents the first crystal structure of a 2S albumin and the model of the pro-protein indicates the structural changes that occur upon formation of mMo-CBP3-1 and determines the structural motif and charge distribution patterns for the diverse observed activities.

Crystal structure of Staphylococcus aureus exfoliative toxin D-like protein: Structural basis for the high specificity of exfoliative toxins.

Exfoliative toxins are serine proteases secreted by Staphylococcus aureus that are associated with toxin-mediated staphylococcal syndromes. To date, four different serotypes of exfoliative toxins have been identified and 3 of them (ETA, ETB, and ETD) are linked to human infection. Among these toxins, only the ETD structure remained unknown, limiting our understanding of the structural determinants for the functional differentiation between these toxins. We recently identified an ETD-like protein associated to S. aureus strains involved in mild mastitis in sheep. The crystal structure of this ETD-like protein was determined at 1.95 Å resolution and the structural analysis provide insights into the oligomerization, stability and specificity and enabled a comprehensive structural comparison with ETA and ETB. Despite the highly conserved molecular architecture, significant differences in the composition of the loops and in both the N- and C-terminal α-helices seem to define ETD-like specificity. Molecular dynamics simulations indicate that these regions defining ET specificity present different degrees of flexibility and may undergo conformational changes upon substrate recognition and binding. DLS and AUC experiments indicated that the ETD-like is monomeric in solution whereas it is present as a dimer in the asymmetric unit indicating that oligomerization is not related to functional differentiation among these toxins. Differential scanning calorimetry and circular dichroism assays demonstrated an endothermic transition centered at 52 °C, and an exothermic aggregation in temperatures up to 64 °C. All these together provide insights about the mode of action of a toxin often secreted in syndromes that are not associated with either ETA or ETB.

Comparative genomics of spike, envelope, and nucleocapsid protein of severe acute respiratory syndrome coronavirus 2.

Background: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) upsurge sprang up in Wuhan, China, in late December 2019. Objectives: Due to the exceptionally high mutation frequency, comparative genomics of viruses isolated throughout time and in various geographical locations are crucial. To better understand how SARS-CoV-2 heterogeneity has changed around the globe, this research was conducted. Methods: Nucleotide and protein sequences of SARS-CoV-2, SARS-CoV, and bat SARS-like CoV were extracted from the NCBI Virus database. The Wuhan SARS-CoV-2 variant was used as a reference. Molecular Evolutionary Genetics Study performed the phylogenetic analysis, while the Genome Detective Coronavirus Typing Tool performed the mutational analysis. Results: The evolutionary research has revealed that bats are the primary host for coronavirus evolution and the origin of the formation of SARS-CoV and SARS-CoV-2. Numerous mutations have been discovered in the spike, envelope, and nucleocapsid protein. Conclusions: The current research findings may have an implication that facilitates the development of prospective immunization candidates/small pharmacological compounds targeting COVID-19.

Structural and functional analyses of the echinomycin resistance conferring protein Ecm16 from Streptomyces lasalocidi.

Echinomycin is a natural product DNA bisintercalator antibiotic. The echinomycin biosynthetic gene cluster in Streptomyces lasalocidi includes a gene encoding the self-resistance protein Ecm16. Here, we present the 2.0 Å resolution crystal structure of Ecm16 bound to adenosine diphosphate. The structure of Ecm16 closely resembles that of UvrA, the DNA damage sensor component of the prokaryotic nucleotide excision repair system, but Ecm16 lacks the UvrB-binding domain and its associated zinc-binding module found in UvrA. Mutagenesis study revealed that the insertion domain of Ecm16 is required for DNA binding. Furthermore, the specific amino acid sequence of the insertion domain allows Ecm16 to distinguish echinomycin-bound DNA from normal DNA and link substrate binding to ATP hydrolysis activity. Expression of ecm16 in the heterologous host Brevibacillus choshinensis conferred resistance against echinomycin and other quinomycin antibiotics, including thiocoraline, quinaldopeptin, and sandramycin. Our study provides new insight into how the producers of DNA bisintercalator antibiotics fend off the toxic compounds that they produce.

Pediatric Acute Lymphoblastic Leukemia: Clinical Characteristics, Treatment Outcomes, and Prognostic Factors: 10 Years' Experience From a Low- and Middle-Income Country.

PURPOSE: Acute lymphoblastic leukemia (ALL) represents around 70% of pediatric leukemia. In high-income countries, the 5-year survival is above 90%, but survival in low- and middle-income countries is inferior. This study documents the treatment outcome and prognostic factors of pediatric ALL in Pakistan. MATERIALS AND METHODS: In this prospective cohort study, all newly diagnosed patients with ALL/lymphoblastic lymphoma from age 1 to 16 years enrolled between January 1, 2012, and December 31, 2021, were included. The treatment was based on the standard arm of the UKALL2011 protocol. RESULTS: Data from 945 patients with ALL, including 597 males (63.2%), were analyzed. The mean age at diagnosis was 5.73 ± 3.51 years. Pallor was the commonest presentation in 95.2% followed by fever in 84.2% of patients. The mean WBC count was 56.6 ± 103.4 × 109/L. Neutropenic fever followed by myopathy was the most common complication during induction. In univariate analysis, the high WBC count (P ≤ 0.001), intensive chemotherapy (P ≤ 0.001), malnutrition (P = .007), poor response to induction chemotherapy (P = .001), delayed presentation (P = .004), and use of steroids before chemotherapy (P = .023) significantly adversely affected overall survival (OS). The delayed presentation was the most significant prognostic factor in the multivariate analysis (P ≤ .002). After a median follow-up of 54.64 ± 33.80 months, the 5-year OS and disease-free survival (DFS) were 69.9% and 67.8%, respectively. CONCLUSION: In this largest cohort of childhood ALL from Pakistan, a high WBC count, malnutrition, delayed presentation, previous steroids use, intensive chemotherapy, and poor response to the induction chemotherapy were associated with decreased OS and DFS rates.

Crystallization and preliminary X-ray diffraction analysis of an L-amino-acid oxidase from Bothrops jararacussu venom.

Snake-venom L-amino-acid oxidases (SV-LAAOs) trigger a wide range of local and systematic effects, including inhibition of platelet aggregation, cytotoxicity, haemolysis, apoptosis and haemorrhage. These effects mainly arise from the uncontrolled release of the hydrogen peroxide that is produced by the redox reaction involving L-amino acids catalyzed by these flavoenzymes. Taking their clinical relevance into account, few SV-LAAOs have been structurally characterized and the structural determinants responsible for their broad direct and indirect pharmacological activities remain unclear. In this work, an LAAO from Bothrops jararacussu venom (BJu-LAAO) was purified and crystallized. The BJu-LAAO crystals belonged to space group P2(1), with unit-cell parameters a = 66.38, b = 72.19, c = 101.53 Å, ß = 90.9°. The asymmetric unit contained two molecules and the structure was determined and partially refined at 3.0 Å resolution.

Purification, crystallization and preliminary X-ray diffraction analysis of crotamine, a myotoxic polypeptide from the Brazilian snake Crotalus durissus terrificus.

Crotamine, a highly basic myotoxic polypeptide (molecular mass 4881 Da) isolated from the venom of the Brazilian rattlesnake Crotalus durissus terrificus, causes skeletal muscle contraction and spasms, affects the functioning of voltage-sensitive sodium channels by inducing sodium influx and possesses antitumour activity, suggesting potential pharmaceutical applications. Crotamine was purified from C. durissus terrificus venom; the crystals diffracted to 1.9 Å resolution and belonged to the orthorhombic space group I2(1)2(1)2(1) or I222, with unit-cell parameters a = 67.75, b = 74.4, c = 81.01 Å. The self-rotation function indicated that the asymmetric unit contained three molecules. However, structure determination by molecular replacement using NMR-determined coordinates was unsuccessful and a search for potential derivatives has been initiated.

Purification, crystallization and preliminary X-ray diffraction analysis of a class P-III metalloproteinase (BmMP-III) from the venom of Bothrops moojeni.

Snake-venom metalloproteinases (SVMPs) comprise a family of haemostatically active toxins which can cause haemorrhage, coagulopathy, inhibition of platelet aggregation and inflammatory response. These effects are attributed to the proteolytic action of SVMPs on extracellular matrix components, plasma proteins and cell-surface proteins. SVMPs are classified into four classes (P-I to P-IV) based on their domain structures. In order to understand the multiple roles played by the domains of P-III SVMPs, a P-III SVMP (BmMP-III) from the venom of Bothrops moojeni was purified, characterized and crystallized. The crystals belonged to space group I4(1)22, with unit-cell parameters a = b = 108.16, c = 196.09 Å. Initially, flash-cooled crystals diffracted poorly to a resolution of about 10 Å. However, a significant improvement in the diffraction resolution was observed upon annealing and a complete data set was collected to 3.3 Šresolution. The asymmetric unit contained one molecule and the structure was determined and partially refined to an R factor of 34%. Structural comparisons indicated that the cysteine-rich domain can adopt different conformations in relation to the catalytic domain, which may modulate the enzyme activity.

Three-Dimensional Structure Characterization and Inhibition Study of Exfoliative Toxin D From Staphylococcus aureus.

The Staphylococcus aureus exfoliative toxins (ETs) are the main toxins that produce staphylococcal scalded skin syndrome (SSSS), an abscess skin disorder. The victims of the disease are usually newborns and kids, as well as grown-up people. Five ETs namely, exfoliative toxins A, B, C, D, and E have been identified in S. aureus. The three-dimensional (3D) structure of exfoliative toxins A, B, C and E is known, while that of exfoliative toxin D (ETD) is still unknown. In this work, we have predicted the 3D structure of ETD using protein modeling techniques (software used for 3D structure modeling comprising the MODELLER 9v19 program, SWISS-Model, and I-TESSER). The validation of the build model was done using PROCHECK (Ramachandran plot), ERRAT2, and Verify 3D programs. The results from 3D modeling show that the build model was of good quality as indicated by a GMQE score of 0.88 and by 91.1% amino acid residues in the most favored region of the Ramachandran plot, the ERRAT2 quality factor of 90.1%, and a verify3D score of >0.2 for 99.59% of amino acid residues. The 3D structure analysis indicates that the overall structure of ETD is similar to the chymotrypsin-like serine protease fold. The structure is composed of 13 ß-strands and seven α-helices that fold into two well-defined six-strand ß-barrels whose axes are roughly perpendicular to each other. The active site residues include histidine-97, aspartic acid-147, and serine-221. This represents the first structure report of ETD. Structural comparison with the other ETs shows some differences, particularly in the loop region, which also change the overall surface charge of these toxins. This may convey variable substrate specificity to these toxins. The inhibition of these toxins by natural (2S albumin and flocculating proteins from Moringa oleifera seeds) and synthetic inhibitors (suramin) was also carried out in this study. The results from docking indicate that the inhibitors bind near the C-terminal domain which may restrict the movement of this domain and may halt the access of the substrate to the active site of this enzyme. Molecular dynamic simulation was performed to see the effect of inhibitor binding to the enzyme. This work will further elucidate the structure-function relationship of this enzyme. The inhibition of this enzyme will lead to a new treatment for SSSS.

Biodiesel from Dodonaea Plant Oil: Synthesis and Characterization-A Promising Nonedible Oil Source for Bioenergy Industry.

In this work, Dodonaea oil was studied as a potential biodiesel source. Dodonaea (Dodonaea viscosa Jacq.) is an evergreen shrubby plant that thrives in tropical and subtropical conditions. The plant produces high-grade biodiesel in terms of both quantity and quality despite its naturally high fat content. In the transesterification followed by esterification reaction, varied ratios of oil to methanol, constant temperature (60°), reaction duration (1 h), and different catalyst concentrations (0.25-0.75% (w/w) were utilized. A maximum biodiesel yield of 90% was achieved. For fuel characteristic analysis, the prepared biodiesel was specified and compared to ASTM criteria. The chemical composition was verified using analytical techniques such as FT-IR and NMR spectroscopy. As a result of the foregoing, Dodonaea is considered a possible bioenergy source, particularly in the transport sector.

C-Reactive Protein and High-Sensitive Cardiac Troponins Correlate with Oxidative Stress in Valvular Heart Disease Patients.

Background: Valvular heart disease (VHD) is a major contributor to loss of physical function and longevity. Oxidative stress is one of the key causative factors involved in heart disease including VHD. Here, we aimed to illuminate the role and relation of oxidative stress to the VHD risk markers in the human population. Materials and Methods: 150 VHD patients and 103 healthy individuals as control were selected for the study and were divided into three groups: the aortic valve, mitral valve, and combined disease based on valvular calcification. Results: Our results demonstrated enhanced oxidative stress in the VHD condition, as we found elevated levels of reactive oxygen species (ROS) at the serum, supported by an increased level of thiobarbituric acid reactive substances (TBARs) in the cardiac valvular tissues of the VHD patients. In contrast, we experienced declined antioxidants including Super Oxide Dismutase (SOD), catalase (CAT), and peroxidase (POD) activities. Concurrently, increasing levels of C-reactive protein (CRP), high-sensitivity cardiac troponin I (hs-cTnI), and high-sensitivity cardiac troponin T (hs-cTnT) were detected in the aortic, mitral, and combined disease condition, suggesting a key association of oxidative stress to VHD conditions. Furthermore, regression analysis validated a key association between the impairment of the redox system (ROS and antioxidant enzyme activities) and VHD condition. Conclusion: Taken together, dysregulated oxidative stress contributes to the progression of VHD via positively correlating with CRP, hs-TnI, and hs-TnT level.

Structure of a novel class II phospholipase D: catalytic cleft is modified by a disulphide bridge.

Phospholipases D (PLDs) are principally responsible for the local and systemic effects of Loxosceles envenomation including dermonecrosis and hemolysis. Despite their clinical relevance in loxoscelism, to date, only the SMase I from Loxosceles laeta, a class I member, has been structurally characterized. The crystal structure of a class II member from Loxosceles intermedia venom has been determined at 1.7Å resolution. Structural comparison to the class I member showed that the presence of an additional disulphide bridge which links the catalytic loop to the flexible loop significantly changes the volume and shape of the catalytic cleft. An examination of the crystal structures of PLD homologues in the presence of low molecular weight compounds at their active sites suggests the existence of a ligand-dependent rotamer conformation of the highly conserved residue Trp230 (equivalent to Trp192 in the glycerophosphodiester phosphodiesterase from Thermus thermophofilus, PDB code: 1VD6) indicating its role in substrate binding in both enzymes. Sequence and structural analyses suggest that the reduced sphingomyelinase activity observed in some class IIb PLDs is probably due to point mutations which lead to a different substrate preference.