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Biliverdin IX-alpha (BV), a tetrapyrrole, is found ubiquitously in most living organisms. It functions as a metabolite, pigment, and signaling compound. While BV is known to bind to diverse protein families such as heme-metabolizing enzymes and phytochromes, not many BV-bound lipocalins (ubiquitous, small lipid-binding proteins) have been studied. The molecular basis of binding and conformational selectivity of BV in lipocalins remains unexplained. Sandercyanin (SFP)-BV complex is a blue lipocalin protein present in the mucus of the Canadian walleye (

The pacific beetle cockroach,Diploptera punctata, is a viviparous cockroach that produces a milk-like substance to support the growing embryo with a brood sac. The structure of thein vivogrown crystals present in the gut of the embryo showed that the milk-derived crystals are heterogenous and are made of three proteins (called Lili-Mips). Multiple fatty acids could be modeled into the active site, and we hypothesized that each of the three isoforms of the protein bound to a different fatty acid. We previously reported that the recombinantly expressed Lili-Mip2 has a structure similar to the structure of the protein determined fromin vivocrystals, and this single isoform also binds to several fatty acids. In this study, we aimed to probe the specificity and affinity of fatty acid binding and test the stability of different isoforms. We show that all the isoforms can bind to different fatty acids with very similar affinities, and the local abundance of a fatty acid determined bound fatty acid ratios. Lili-Mips’ thermostability is pH dependent, where stability is highest at acidic pH and declines as the pH increases to physiological levels near 7.0. The measurement of the pH in the gut lumen and the gut cells suggests that the pH in the gut is acidic and the pH inside the gut cells is closer to neutral pH. We propose that the protein has evolved to be highly stable in the acidic gut lumen and, when absorbed inside the gut cells, becomes less stable to enable the breakdown of the glycosylated lipo-protein complex to provide essential metabolites for survival and development of the embryo. The different orientations of Phe-98 and Phe-100 control the binding pocket volume and allow the binding of different chain-length fatty acids to bind with similar affinities.

In bacteria that live in hosts whose terminal sugar is a sialic acid, Glucosamine 6-phosphate deaminase (NagB) catalyzes the last step in the conversion of sialic acid into Fructose-6-phosphate, which enters the glycolytic pathway. The enzyme exists as a hexamer in Gram-negative bacteria and is shown to be allosterically regulated. In Gram-positive bacteria, it exists as a monomer and lacks allosteric regulation. Our identification of a dimeric Gram-negative bacterial NagB motivated us to characterize the structural basis of the various oligomeric forms. We characterized the crystal structures of NagB from two Gram-negative pathogens, Haemophilus influenzae (Hi) and Pasturella multocida (Pm). The Hi-NagB is active as a hexamer, while Pm-NagB is active as a dimer. We confirm that this is not a crystallographic artifact by cryo-electron microscopy. Both Hi-NagB and Pm-NagB contain the C-terminal helix, and the residues in the interface involved in oligomerization are conserved. The hexamer is described as a dimer of trimers. In the Pm-NagB dimer, the dimeric interface is conserved. This would suggest that the three possible oligomeric forms of NagB are a monomer, a dimer, and a trimer of dimers. Computational modeling and MD simulations indicate that the residues at the trimeric interface have less stabilizing energy of oligomer formation than those in the dimer interface. We propose that Pm-NagB is the evolutionary link between the monomer and the hexamer forms.

Enzymes catalyze reactions by binding and orienting substrates with dynamic interactions. Horse liver alcohol dehydrogenase catalyzes hydrogen transfer with quantum-mechanical tunneling that involves fast motions in the active site. The structures and B factors of ternary complexes of the enzyme with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol or NAD+ and 2,2,2-trifluoroethanol were determined to 1.1–1.3 Å resolution below the `glassy transition' in order to extract information about the temperature-dependent harmonic motions, which are reflected in the crystallographic B factors. The refinement statistics and structures are essentially the same for each structure at all temperatures. The B factors were corrected for a small amount of radiation decay. The overall B factors for the complexes are similar (13–16 Å2) over the range 25–100 K, but increase somewhat at 150 K. Applying TLS refinement to remove the contribution of pseudo-rigid-body displacements of coenzyme binding and catalytic domains provided residual B factors of 7–10 Å2 for the overall complexes and of 5–10 Å2 for C4N of NAD+ and the methylene carbon of the alcohols. These residual B factors have a very small dependence on temperature and include local harmonic motions and apparently contributions from other sources. Structures at 100 K show complexes that are poised for hydrogen transfer, which involves atomic displacements of ∼0.3 Å and is compatible with the motions estimated from the residual B factors and molecular-dynamics simulations. At 298 K local conformational changes are also involved in catalysis, as enzymes with substitutions of amino acids in the substrate-binding site have similar positions of NAD+ and pentafluorobenzyl alcohol and similar residual B factors, but differ by tenfold in the rate constants for hydride transfer.

Several pathogenic bacteria import and catabolize sialic acids as a source of carbon and nitrogen. Within the sialic acid catabolic pathway, the enzyme N-acetylmannosamine kinase (NanK) catalyzes the phosphorylation of N-acetylmannosamine to N-acetylmannosamine-6-phosphate. This kinase belongs to the ROK superfamily of enzymes, which generally contain a conserved zinc-finger (ZnF) motif that is important for their structure and function. Previous structural studies have shown that the ZnF motif is absent in NanK of Fusobacterium nucleatum (Fn-NanK), a Gram-negative bacterium that causes the gum disease gingivitis. However, the effect in loss of the ZnF motif on the kinase activity is unknown. Using kinetic and thermodynamic studies, we have studied the functional properties of Fn-NanK to its substrates ManNAc and ATP, compared its activity with other ZnF motif-containing NanK enzymes from closely related Gram-negative pathogenic bacteria Haemophilus influenzae (Hi-NanK), Pasteurella multocida (Pm-NanK), and Vibrio cholerae (Vc-NanK). Our studies show a 10-fold decrease in substrate binding affinity between Fn-NanK (apparent K-M approximate to 700 mu M) and ZnF motif-containing NanKs (apparent K-M 60 approximate to mu M). To understand the structural features that combat the loss of the ZnF motif in Fn-NanK, we solved the crystal structures of functionally homologous ZnF motif-containing NanKs from P. multocida and H. influenzae. Here, we report Pm-NanK:unliganded, Pm-NanK:AMPPNP, Pm-NanK:ManNAc, Hi-NanK:ManNAc, and Hi-NanK:ManNAc-6P:ADP crystal structures. Structural comparisons of Fn-NanK with Hi-NanK, Pm-NanK, and hMNK (human N-acetylmannosamine kinase domain of UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase, GNE) show that even though there is less sequence identity, they have high degree of structural similarity. Furthermore, our structural analyses highlight that the ZnF motif of Fn-NanK is substituted by a set of hydrophobic residues, which forms a hydrophobic cluster that helps the proper orientation of ManNac in the active site. In summary, ZnF-containing and ZnF-lacking NanK enzymes from different Gram-negative pathogenic bacteria are functionally very similar but differ in their metal requirement. Our structural studies unveil the structural modifications in Fn-NanK that compensate the loss of the ZnF motif in comparison to other NanK enzymes.

N,N‐dimethyl formamide (DMF) is an extensively used organic solvent but is also a potent pollutant. Certain bacterial species from genera such as Paracoccus, Pseudomonas, and Alcaligenes have evolved to use DMF as a sole carbon and nitrogen source for growth via degradation by a dimethylformamidase (DMFase). We show that DMFase from Paracoccus sp. strain DMF is a halophilic and thermostable enzyme comprising a multimeric complex of the α2β2 or (α2β2)2 type. One of the three domains of the large subunit and the small subunit are hitherto undescribed protein folds of unknown evolutionary origin. The active site consists of a mononuclear iron coordinated by two Tyr side‐chain phenolates and one carboxylate from Glu. The Fe3+ ion in the active site catalyzes the hydrolytic cleavage of the amide bond in DMF. Kinetic characterization reveals that the enzyme shows cooperativity between subunits, and mutagenesis and structural data provide clues to the catalytic mechanism., Break it down again: Certain bacteria can break down N,N‐dimethyl formamide (DMF) through the action of a dimethylformamidase (DMFase). The structure and mechanism of a DMFase from a bacterium of the genus Paracoccus are presented. The enzyme comprises a multimeric α2β2 or (α2β2)2 complex, and the active site consists of FeIII coordinated by two Tyr side‐chain phenolates and one carboxylate from Glu in an unusual square pyramidal geometry.

In environments where glucose is limited, some pathogenic bacteria metabolize host-derived sialic acid as a nutrient source. N-Acetylmannosamine kinase (NanK) is the second enzyme of the bacterial sialic acid import and degradation pathway and adds phosphate to N-acetylmannosamine using ATP to prime the molecule for future pathway reactions. Sequence alignments reveal that Gram-positive NanK enzymes belong to the Repressor, ORF, Kinase (ROK) family, but many lack the canonical Zn-binding motif expected for this function, and the sugar-binding EXGH motif is altered to EXGY. As a result, it is unclear how they perform this important reaction. Here, we study the Staphylococcus aureus NanK (SaNanK), which is the first characterization of a Gram-positive NanK. We report the kinetic activity of SaNanK along with the ligand-free, N-acetylmannosamine-bound and substrate analog GlcNAc-bound crystal structures (2.33, 2.20, and 2.20 Å resolution, respectively). These demonstrate, in combination with small-angle X-ray scattering, that SaNanK is a dimer that adopts a closed conformation upon substrate binding. Analysis of the EXGY motif reveals that the tyrosine binds to the N-acetyl group to select for the "boat" conformation of N-acetylmannosamine. Moreover, SaNanK has a stacked arginine pair coordinated by negative residues critical for thermal stability and catalysis. These combined elements serve to constrain the active site and orient the substrate in lieu of Zn binding, representing a significant departure from canonical NanK binding. This characterization provides insight into differences in the ROK family and highlights a novel area for antimicrobial discovery to fight Gram-positive and S. aureus infections.

Biliverdin IXα (BV) binds to several prokaryotic and eukaryotic proteins. How nature exploits the versatility of BV's properties is not fully understood. Unlike free BV, the Sandercyanin fluorescent protein bound to BV (SFP-BV) shows enhanced red fluorescence (675 nm) on excitation in the UV region (380 nm). Site-directed mutagenesis showed that the BV complex of two SFP variants, F55A and E79A, resulted in the loss of red fluorescence. Crystal structures of the complexes of these proteins with BV show the absence of stacking interactions of the F55 phenyl ring with BV. BV changes from ZZZssa conformation in the wild-type to ZZZsss conformation in the variants. In the nonfluorescent mutants, the lowest excited state is destabilized, resulting in nonradiative decay.

O-Glycans on cell surfaces play important roles in cell–cell, cell–matrix and receptor–ligand interaction. Therefore, glycan-based interactions are important for tissue regeneration and homeostasis. Free-living flatworm Schmidtea mediterranea, because of its robust regenerative potential, is of great interest in the field of stem cell biology and tissue regeneration. Nevertheless, information on the composition and structure of O-glycans in planaria is unknown. Using mass spectrometry and in silico approaches, we characterized the glycome and the related transcriptome of mucin-type O-glycans of planarian S. mediterranea. Mucin-type O-glycans were composed of multiple isomeric, methylated, and unusually extended mono- and disubstituted O-N-acetylgalactosamine structures. Extensions made of hexoses and 3-O-methyl hexoses were the glycoforms observed. From glycotranscriptomic analysis, 60 genes belonging to five distinct enzyme classes were identified to be involved in mucin-type O-glycan biosynthesis. These genes shared homology with those in other invertebrate systems. Although a majority of the genes involved in mucin-type O-glycan biosynthesis were highly expressed during organogenesis and in differentiated cells, a few select genes in each enzyme class were specifically enriched during early embryogenesis. Our results indicate a unique temporal and spatial role for mucin-type O-glycans during embryogenesis and organogenesis and in adulthood. In summary, this is the first report on O-glycans in planaria. This study expands the structural and biosynthetic possibilities in cellular glycosylation in the invertebrate glycome and provides a framework towards understanding the biological role of mucin-type O-glycans in tissue regeneration using planarians.

Planaria is an ideal system to study factors involved in regeneration and tissue homeostasis. Little is known about the role of metabolites and small molecules in stem cell maintenance and lineage specification in planarians. Using liquid chromatography and mass spectrometry (LC-MS)-based quantitative metabolomics, we determined the relative levels of metabolites in stem cells, progenitors, and differentiated cells of the planarian Schmidtea mediterranea. Tryptophan and its metabolic product serotonin are significantly enriched in stem cells and progenitor population. Serotonin biosynthesis in these cells is brought about by a noncanonical enzyme, phenylalanine hydroxylase. Knockdown of Smed-pah leads to complete disappearance of eyes in regenerating planaria, while exogenous supply of serotonin and its precursor rescues the eyeless phenotype. Our results demonstrate a key role for serotonin in eye regeneration.

CMP-N-acetylneuraminate synthetase (CMAS) is a key enzyme in the sialic acid incorporation pathway and plays a crucial role in the virulence and survival of several pathogenic bacteria. Here, the structural and functional properties of CMAS from the pathogenic bacterium Vibrio cholerae are reported. Upon CDP binding, a partial domain closure is observed that was previously unreported in homologous structures. Kinetic studies reveal that the enzyme shows substrate promiscuity and can activate both Neu5Ac and Neu5Gc., Several pathogenic bacteria utilize sialic acid, including host-derived N-acetylneuraminic acid (Neu5Ac), in at least two ways: they use it as a nutrient source and as a host-evasion strategy by coating themselves with Neu5Ac. Given the significant role of sialic acid in pathogenesis and host-gut colonization by various pathogenic bacteria, including Neisseria meningitidis, Haemophilus influenzae, Pasteurella multocida and Vibrio cholerae, several enzymes of the sialic acid catabolic, biosynthetic and incorporation pathways are considered to be potential drug targets. In this work, findings on the structural and functional characterization of CMP-N-acetylneuraminate synthetase (CMAS), a key enzyme in the incorporation pathway, from Vibrio cholerae are reported. CMAS catalyzes the synthesis of CMP-sialic acid by utilizing CTP and sialic acid. Crystal structures of the apo and the CDP-bound forms of the enzyme were determined, which allowed the identification of the metal cofactor Mg2+ in the active site interacting with CDP and the invariant Asp215 residue. While open and closed structural forms of the enzyme from eukaryotic and other bacterial species have already been characterized, a partially closed structure of V. cholerae CMAS (VcCMAS) observed upon CDP binding, representing an intermediate state, is reported here. The kinetic data suggest that VcCMAS is capable of activating the two most common sialic acid derivatives, Neu5Ac and Neu5Gc. Amino-acid sequence and structural comparison of the active site of VcCMAS with those of eukaryotic and other bacterial counterparts reveal a diverse hydrophobic pocket that interacts with the C5 substituents of sialic acid. Analyses of the thermodynamic signatures obtained from the binding of the nucleotide (CTP) and the product (CMP-sialic acid) to VcCMAS provide fundamental information on the energetics of the binding process.

The primary role of bacterial periplasmic binding proteins is sequestration of essential metabolites present at a low concentration in the periplasm and making them available for active transporters that transfer these ligands into the bacterial cell. The periplasmic binding proteins (SiaPs) from the tripartite ATP-independent periplasmic (TRAP) transport system that transports mammalian host–derived sialic acids have been well studied from different pathogenic bacteria, including Haemophilus influenzae, Fusobacterium nucleatum, Pasteurella multocida, and Vibrio cholerae. SiaPs bind the sialic acid N-acetylneuraminic acid (Neu5Ac) with nanomolar affinity by forming electrostatic and hydrogen-bonding interactions. Here, we report the crystal structure of a periplasmic binding protein (SatA) of the ATP-binding cassette (ABC) transport system from the pathogenic bacterium Haemophilus ducreyi. The structure of Hd-SatA in the native form and sialic acid–bound forms (with Neu5Ac and N-glycolylneuraminic acid (Neu5Gc)), determined to 2.2, 1.5, and 2.5 Å resolutions, respectively, revealed a ligand-binding site that is very different from those of the SiaPs of the TRAP transport system. A structural comparison along with thermodynamic studies suggested that similar affinities are achieved in the two classes of proteins through distinct mechanisms, one enthalpically driven and the other entropically driven. In summary, our structural and thermodynamic characterization of Hd-SatA reveals that it binds sialic acids with nanomolar affinity and that this binding is an entropically driven process. This information is important for future structure-based drug design against this pathogen and related bacteria.

Sandercyanin, a blue homo-tetrameric lipocalin protein purified from Canadian walleye (Stizostedion vitreus), is the first far-red fluorescent protein reported in vertebrates(1–3). Sandercyanin binds non-covalently to biliverdin IXα (BLA) and fluoresces at 675nm on excitation at 375nm and 635nm(1). Sandercyanin fluorescence can be harnessed for many in vivo applications when engineered into a stable monomeric form. Here, we report the spectral properties and crystal structures of engineered monomeric Sandercyanin-BLA complexes. Compared to wild-type protein, monomeric Sandercyanin (∼18kDa) binds BLA with similar affinities and show a broad red-shifted absorbance spectra but possess reduced quantum efficiency. Crystal structures reveal D-ring pyrrole of BLA rotated around the C14-C15 bond, which is stabilized by neighboring aromatic residues and increased water-mediated polar contacts in the BLA-binding pocket. A tetrameric Sandercyanin variant (Tyr-142-Ala) co-displaying red- and far-red absorbing states, and reduced fluorescence shows similar conformational changes in BLA binding pocket. Our results suggest that D-ring flexibility of BLA and its rearrangement reduces the fluorescence quantum-yield of monomeric Sandercyanin. Structures of monomeric Sandercyanin could be utilized as prototypes to generate bright BLA-inducible fluorescent proteins. Further, our study postulates a mechanism for modulating photo-states in BLA-bound lipocalins, known only in phytochromes till date.Significance StatementSandercyanin is a tetrameric red fluorescent protein from a blue variant of walleye (Stizostedion vitreum) that binds to biliverdin IXα (BLA). Its biophysical properties and structures have been published earlier(1). A bright and stable monomeric Sandercyanin could be utilized as a fusion protein for fluorescence-based applications. Here we report the first structures and spectral properties of fluorescent monomeric Sandercyanin-BLA complexes and describe the molecular basis of modulated spectral properties due to rotated D-ring pyrrole around C14-C15 bond and re-shuffling of BLA-binding pocket. BLA-bound monomeric Sandercyanin could be engineered into brighter variants for in-vivo applications. Our study also reveals an unfamiliar mechanism in BLA-binding lipocalins that regulates red- and far-red absorbance states.

Structures of yeast alcohol dehydrogenase determined by X-ray crystallography show that the subunits have two different conformational states in each of the two dimers that form the tetramer. Apoenzyme and holoenzyme complexes relevant to the catalytic mechanism were described, but the asymmetry led to questions about the cooperativity of the subunits in catalysis. This study used cryo-electron microscopy (cryo-EM) to provide structures for the apoenzyme, two different binary complexes with NADH, and a ternary complex with NAD+ and 2,2,2-trifluoroethanol. All four subunits in each of these complexes are identical, as the tetramers have D2 symmetry, suggesting that there is no preexisting asymmetry and that the subunits can be independently active. The apoenzyme and one enzyme-NADH complex have "open" conformations and the inverted coordination of the catalytic zinc with Cys-43, His-66, Glu-67, and Cys-153, whereas another enzyme-NADH complex and the ternary complex have closed conformations with the classical coordination of the zinc with Cys-43, His-66, Cys-153, and a water or the oxygen of trifluoroethanol. The conformational change involves interactions of Arg-340 with the pyrophosphate group of the coenzyme and Glu-67. The cryo-EM and X-ray crystallography studies provide structures relevant for the catalytic mechanism.

Enzymes typically have high specificity for their substrates, but the structures of substrates and products differ, and multiple modes of binding are observed. In this study, high resolution X-ray crystallography of complexes with NADH and alcohols show alternative modes of binding in the active site. Enzyme crystallized with the good substrates NAD(+) and 4-methylbenzyl alcohol was found to be an abortive complex of NADH with 4-methylbenzyl alcohol rotated to a “non-productive” mode as compared to the structures that resemble reactive Michaelis complexes with NAD(+) and 2,2,2-trifluoroethanol or 2,3,4,5,6-pentafluorobenzyl alcohol. The NADH is formed by reduction of the NAD(+) with the alcohol during the crystallization. The same structure was also formed by directly crystallizing the enzyme with NADH and 4-methylbenzyl alcohol. Crystals prepared with NAD(+) and 4-bromobenzyl alcohol also form the abortive complex with NADH. Surprisingly, crystals prepared with NAD(+) and the strong inhibitor 1H,1H-heptafluorobutanol also had NADH, and the alcohol was bound in two different conformations that illustrate binding flexibility. Oxidation of 2-methyl-2,4-pentanediol during the crystallization apparently led to reduction of the NAD(+). Kinetic studies show that high concentrations of alcohols can bind to the enzyme-NADH complex and activate or inhibit the enzyme. Together with previous studies on complexes with NADH and formamide analogues of the carbonyl substrates, models for the Michaelis complexes with NAD(+)-alcohol and NADH-aldehyde are proposed.