Sudipta Bhattacharyya

Crystal structure and fluorescence studies reveal the role of helical dimeric interface of staphylococcal fabg1 in positive cooperativity for NADPH

Crystal structure of Staphylococcal β‐ketoacyl‐ACP reductase 1 (SaFabG1) complexed with NADPH is determined at 2.5 Å resolution. The enzyme is essential in FAS‐II pathway and utilizes NADPH to reduce β‐ketoacyl‐ACP to (S)‐β‐hydroxyacyl‐ACP. Unlike the tetrameric FabGs, dimeric SaFabG1 shows positive homotropic cooperativity towards NADPH. Analysis of FabG:NADPH binary crystal structure endorses that NADPH interacts directly with the helices α4 and α5 those are present on a dimerization interface. A steady shift in tryptophan (of α4 helix) emission peak upon steady increment of NADPH concentration reveals that the dimeric interface is formed by α4‐α4′ and α5‐α5′ helices. This dimeric interface imparts positive homotropic cooperativity towards NADPH. PEG, a substrate mimicking molecule is also found near the active site of the enzyme. Proteins 2012;

Structural elucidation of the binding site and mode of inhibition of Li+ and Mg2+ in inositol monophosphatase

Mg2+‐dependent, Li+‐sensitive phosphatases are a widely distributed family of enzymes with significant importance throughout the biological kingdom. Inositol monophosphatase (IMPase) is an important target of Li+‐based therapeutic agents in manic depressive disorders. However, despite decades of intense research efforts, the precise mechanism of Li+‐induced inhibition of IMPase remains obscured. Here we describe a structural investigation of the Li+ binding site in staphylococcal IMPase I (SaIMPase I) using X–ray crystallography. The biochemical study indicated common or overlapping binding sites for Mg2+ and Li+ in the active site of SaIMPase I. The crystal structure of the SaIMPase I ternary product complex shows the presence of a phosphate and three Mg2+ ions (namely Mg1, Mg2 and Mg3) in the active site. As Li+ is virtually invisible in X–ray crystallography, competitive displacement of Mg2+ ions from the SaIMPase I ternary product complex as a function of increasing LiCl concentration was used to identify the Li+ binding site. In this approach, the disappearing electron density of Mg2+ ions due to Li+ ion binding was traced, and the Mg2+ ion present at the Mg2 binding site was found to be replaced. Moreover, based on a detailed comparative investigation of the phosphate orientation and coordination states of Mg2+ binding sites in enzyme–substrate and enzyme–product complexes, inhibition mechanisms for Li+ and Mg2+ are proposed.

Crystal structure of Staphylococcal dual specific inositol monophosphatase/NADP(H) phosphatase (SAS2203) delineates the molecular basis of substrate specificity.

Inositol monophosphatase (IMPase) family of proteins are Mg(2+) activated Li(+) inhibited class of ubiquitous enzymes with promiscuous substrate specificity. Herein, the molecular basis of IMPase substrate specificity is delineated by comparative crystal structural analysis of a Staphylococcal dual specific IMPase/NADP(H) phosphatase (SaIMPase - I) with other IMPases of different substrate compatibility, empowered by in silico docking and Escherichia coli SuhB mutagenesis analysis. Unlike its eubacterial and eukaryotic NADP(H) non-hydrolyzing counterparts, the composite structure of SaIMPase - I active site pocket exhibits high structural resemblance with archaeal NADP(H) hydrolyzing dual specific IMPase/FBPase. The large and shallow SaIMPase - I active site cleft efficiently accommodate large incoming substrates like NADP(H), and therefore, justifies the eminent NADP(H) phosphatase activity of SaIMPase - I. Compared to other NADP(H) non-hydrolyzing IMPases, the profound difference in active site topology as well as the unique NADP(H) recognition capability of SaIMPase - I stems from the differential length and orientation of a distant helix α4 (in human and bovine α5) and its preceding loop. We identified the length of α4 and its preceding loop as the most crucial factor that regulates IMPase substrate specificity by employing a size exclusion mechanism. Hence, in SaIMPase - I, the substrate promiscuity is a gain of function by trimming the length of α4 and its preceding loop, compared to other NADP(H) non-hydrolyzing IMPases. This study thus provides a biochemical - structural framework revealing the length and orientation of α4 and its preceding loop as the predisposing factor for the determination of IMPase substrate specificity.

Macroscopic amyloid fiber formation by staphylococcal biofilm associated SuhB protein.

Staphylococcus aureus is a commensal and opportunistic pathogen that causes lethal infections. Biofilm forming ability of S. aureus enhances its virulence since biofilm provides the bacteria protective shield against antibiotics and host immunity. Polysaccharide independent biofilm formation by several virulent S. aureus strains have been identified recently, where protein components substitute polysaccharide intercellular adhesin (PIA) involved in bacterial cell attachment. The suhB gene has been reported to be essential in staphylococcal PIA-independent biofilm formation. Overexpression of staphylococcal SuhB (SasuhB) in E. coli produces extracellular macroscopic fibers made of recombinant SaSuhB protein. The amyloidic nature of the fiber is evaluated by high resolution electron microscopy, X-ray fiber diffraction and amyloid specific dyes, such as Congo red and thioflavin-T binding assay. The fibers appear to be sticky in nature and bind a large number of bacterial cells. The results suggest the possible role of SaSuhB-fibers as a structural component as well as an adhesin in biofilm matrix.

A structurally novel hemopexin fold protein of rice plays role in chlorophyll degradation

Proteins containing hemopexin fold domain are suggested to have diverse functions in various living organisms. In order to investigate the structure and function of this type of protein in rice plant (Oryza sativa), the gene encoding a hemopexin fold protein (OsHFP) was cloned, analyzed in silico and characterized. Molecular modeling revealed that the OsHFP is closely related to other hemopexin fold proteins, but is unique with a cylindrical central tunnel as well as extended N- and C-terminal domains. The recombinant OsHFP was found to bind hemin, the oxidized form of heme in vitro. The expression of the single copy OsHFP gene was detected in rice flower buds. Heterologous expression of OsHFP in green leaf tissues resulted in chlorophyll degradation; however, stable expression of OsHFP was observed in transgenic hairy roots, a non-green tissue. The possible role of OsHFP in regulating programmed cell death in anther green tissues of rice is proposed.

Cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of an inositol monophosphatase family protein (SAS2203) from Staphylococcus aureus MSSA476

The gene product of the sas2203 ORF of Staphylococcus aureus MSSA476 encodes a 30 kDa molecular-weight protein with a high sequence resemblance (29% identity) to tetrameric inositol monophosphatase from Thermotoga maritima. The protein was cloned, expressed, purified to homogeneity and crystallized. Crystals appeared in several conditions and good diffraction-quality crystals were obtained from 0.2 M Li(2)SO(4), 20% PEG 3350, 0.1 M HEPES pH 7.0 using the sitting-drop vapour-diffusion method. A complete diffraction data set was collected to 2.6 Å resolution using a Rigaku MicroMax-007 HF Cu Kα X-ray generator and a Rigaku R-AXIS IV(++) detector. The diffraction data were consistent with the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 49.98, b = 68.35, c = 143.79 Å, α = β = γ = 90°, and the crystal contained two molecules in the asymmetric unit.

Structural elucidation of the NADP(H) phosphatase activity of staphylococcal dual-specific IMPase/NADP(H) phosphatase.

NADP(H)/NAD(H) homeostasis has long been identified to play a pivotal role in the mitigation of reactive oxygen stress (ROS) in the intracellular milieu and is therefore critical for the progression and pathogenesis of many diseases. NAD(H) kinases and NADP(H) phosphatases are two key players in this pathway. Despite structural evidence demonstrating the existence and mode of action of NAD(H) kinases, the specific annotation and the mode of action of NADP(H) phosphatases remains obscure. Here, structural evidence supporting the alternative role of inositol monophosphatase (IMPase) as an NADP(H) phosphatase is reported. Crystal structures of staphylococcal dual-specific IMPase/NADP(H) phosphatase (SaIMPase-I) in complex with the substrates D-myo-inositol-1-phosphate and NADP(+) have been solved. The structure of the SaIMPase-I-Ca(2+)-NADP(+) ternary complex reveals the catalytic mode of action of NADP(H) phosphatase. Moreover, structures of SaIMPase-I-Ca(2+)-substrate complexes have reinforced the earlier proposal that the length of the active-site-distant helix α4 and its preceding loop are the predisposing factors for the promiscuous substrate specificity of SaIMPase-I. Altogether, the evidence presented suggests that IMPase-family enzymes with a shorter α4 helix could be potential candidates for previously unreported NADP(H) phosphatase activity.

Crystallization and preliminary X-ray diffraction analysis of the high molecular weight ketoacyl reductase FabG4 complexed with NADH. Corrigendum

A corrigendum to the article by Dutta et al. [(2012) Acta Cryst. F68, 786–789].

Cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of an atypical two-cysteine peroxiredoxin (SAOUHSC_01822) from Staphylococcus aureus NCTC 8325

An atypical two-cysteine peroxidase, SAOUHSC_01822, from the virulent Staphylococcus aureus strain NCTC 8325 plays a major role in the response of the bacterium to oxidative stress. The protein was cloned, expressed, purified to homogeneity and crystallized. The protein was crystallized from 2 M ammonium sulfate, 0.1 M Na HEPES pH 7, 2%(v/v) PEG 400. A complete diffraction data set was collected to 2.3 angstrom resolution using a Rigaku MicroMax HF007 Cu K alpha X-ray generator and a Rigaku R-AXIS IV(+)(+) detector. The crystals belonged to space group P2(1), with unit-cell parameters a = 43.50, b = 149.35, c = 73.73 angstrom, beta = 104.4 degrees, and contained four molecules in the asymmetric unit.