Evolutionary significance and functional characterization of streptomycin adenylyltransferase from Serratia marcescens

Abstract

Complete functional annotations of proteins are essential to understand the role and mechanisms in pathogenesis. Aminoglycoside nucleotidyltransferases are the subclasses of aminoglycosides modifying enzymes conferring resistance to organisms. Insight into the structural and functional understanding of nucleotidyltransferase family protein provides vital information to combat pathogenesis. Phylogenetic analysis is employed to identify the evolutionary significance and common motif’s present among the homologs of nucleotidyltransferase family protein. Structure, sequence based approaches and molecular docking were implemented to predict the exact function of the protein. Wide distribution of the nucleotidyltransferase family protein in gram-positive and gram-negative organisms are evidenced from phylogenetic analysis. Five common motifs were present in all the homolog’s of nucleotidyltransferase family protein. Sequence-structure based functional annotations predicts that the targeted protein function as ATP-Mg dependent streptomycin adenylyltransferase. Structural comparisons and docking studies correlate well with the identified function. The complete function of nucleotidyltransferase family protein was identified as Streptomycin adenylyltransferase and it could be targeted as a potential therapeutic target to overcome antibiotic resistance.

Communicated by Ramaswamy H. Sarma

Abbreviations AAC aminoglycoside acetyltransferases

AME aminoglycoside modifying enzyme

ANT aminoglycoside nucleotidyltransferases

APH aminoglycoside phosphotransferases

ATP adenosine triphosphate

CASTp computer atlas and surface topography of proteins

DUF domains of unknown function

Glide grid-based ligand docking with energetic

HMM hidden Markov model

MAST motif alignment and search tool

MEGA molecular evolutionary genetics analysis

MEME multiple Em for motif elicitation

MSA multiple sequence alignment

NMP nucleoside monophosphate

NTP nucleoside triphosphate

NT nucleotidyltransferase

OPLS optimized potential for liquid simulation

XP extra precision

     

Biophysical properties of Opuntia ficus-indica mucilage

The purified mucilage from Opuntia ficus-indica is a high MW polysaccharide which behaves as a polyelectrolyte. Viscosity of its solution is dependent on the Ca²⁺ ion concentration and on pH, being greatest at alkaline pH. The sedimentation coefficient was dependent on concentration. The molecule had an estimated axial ratio of 256 in water, and this was reduced at low pH and in the presence of high concentrations of Ca²⁺. The molecule was studied with light scattering and CD techniques and its UV spectrum was recorded. All these parameters were influenced by pH and by ion concentration. The gelation properties also changed with pH and with Ca²⁺ giving dense gels in its presence and loose ones in its absence. The results are interpreted in terms of changes in conformation of the molecule, changes in Ca²⁺ binding and degree of ionization of the molecule. An attempt is made to relate the molecular properties to the physiological function of the mucilage in the calcium and water economy of the plant.     

Crystal structure of nicotinic acid mononucleotide adenylyltransferase from Pseudomonas aeruginosa in its Apo and substrate-complexed forms reveals a fully open conformation

The enzyme nicotinic acid mononucleotide adenylyltransferase (NaMN AT; EC 2.7.7.18) is essential for the synthesis of nicotinamide adenine dinucleotide and is a potential target for antibiotics. It catalyzes the transfer of an AMP moiety from ATP to nicotinic acid mononucleotide to form nicotinic acid adenine dinucleotide. In order to provide missing structural information on the substrate complexes of NaMN AT and to assist structure-based design of specific inhibitors for antibacterial discovery, we have determined the crystal structure of NaMN AT from Pseudomonas aeruginosa in three distinct states, i.e. the NaMN-bound form at 1.7A resolution and ATP-bound form at 2.0A as well as its apo-form at 2.0A. They represent crucial structural information necessary for better understanding of the substrate recognition and the catalytic mechanism. The substrate-unbound and substrate-complexed structures are all in the fully open conformation and there is little conformational change upon binding each of the substrates. Our structures indicate that a conformational change is necessary to bring the two substrates closer together for initiating the catalysis. We suggest that such a conformational change likely occurs only after both substrates are simultaneously bound in the active site.     

多药耐药铜绿假单胞菌氨基糖苷类修饰酶和16S rRNA甲基化酶基因分析

目的了解临床分离的铜绿假单胞菌对氨基糖苷类、β-内酰胺类、喹诺酮类抗菌药物耐药情况、氨基糖苷类耐药相关基因和16S rRNA甲基化酶基因存在情况以及菌株之间的亲缘性。方法采用琼脂稀释法测定临床分离的30株铜绿假单胞菌对7种临床常用于治疗铜绿假单胞菌感染的抗菌药物的敏感性,采用聚合酶链反应分析氨基糖苷类修饰酶、16S rRNA甲基化酶基因型及其他基因型,运用SPSS统计分析软件对菌株样本亲缘性做聚类分析。结果30株铜绿假单胞菌对临床常用抗生素的耐药率分别是奈替米星70%、妥布霉素63.3%、庆大霉素63.3%、环丙沙星53.3%、亚氨培南40%和阿米卡星13.3%,而多黏菌素B的耐药率为0。21株氨基糖苷类耐药菌株中（其中20株为多药耐药菌株）,氨基糖苷类耐药基因型aac（6＇）-Ⅰ阳性13株（61.9%）、aac（6＇）-Ⅱ阳性13株（61.9%）、ant（2＇＇）-Ⅰ阳性10株（47.6%）、ant（3＇＇）-Ⅰ阳性9株（42.9%）、aac（3）-Ⅱ阳性1株（4.8%）,另有1株菌oprD2基因缺失,未检出基因型aac（6＇）-Ⅰae、aph（3＇）-Ⅲ、aac（6＇）-aph（2＇＇）和ant（4＇）-Ⅰ;16S rRNA甲基化酶基因rmtA基因型阳性19株（90.4%）、armA基因型阳性有8株（38.1%）,未检出基因型rmtC、rmtD。聚类分析结果显示分离的菌株中存在克隆传播。结论大部分测试的铜绿假单胞菌对临床常用的铜绿假单胞菌抗感染药物已产生广泛耐药,尤其对氨基糖苷类抗生素。这些菌株的氨基糖苷类修饰酶常见耐药基因型检出率高,16SrRNA甲基化酶基因型rmtA和armA的检出率亦较高。30株测试菌株中存在克隆传播。     

A spontaneous point mutation i the aac(6')-lb' gene results in altered substrate specificity of aminoglycoside 6'-N-acetyltransferase of a Pseudomonas fluorescens strain

Abstract The aac(6')-lb' gene from Pseudomonas fluorescens BM2687, encoding an aminoglycoside 6'- N -acetyltransferase type II which confers resistance to gentamicin but not to amikacin, was characterized. Nucleotide sequence determination indicated total identity between aac6')-lb and the aac(6')-lb gene from Pseudomonas aeruginosa BM2656 [1] with the exception of a C-to-T transition that results in a serine to lecine substitution at position 83 of the deduced polypeptide. The aac(6')-lb gene specifies a type I enzyme which confers resistance to amikacin but not to gentamicin [2]. It thus appears that the point mutation detected is responsible for enzymic altered substrate specificity.     

Spectroscopic characterization and structural modeling of prolamin from maize and pearl millet

Biophysical methods and structural modeling techniques have been used to characterize the prolamins from maize (Zea mays) and pearl millet (Pennisetum americanum). The alcohol-soluble prolamin from maize, called zein, was extracted using a simple protocol and purified by gel filtration in a 70% ethanol solution. Two protein fractions were purified from seed extracts of pearl millet with molecular weights of 25.5 and 7 kDa, as estimated by SDS-PAGE. The high molecular weight protein corresponds to pennisetin, which has a high -helical content both in solution and the solid state, as demonstrated by circular dichroism and Fourier transform infrared spectra. Fluorescence spectroscopy of both fractions indicated changes in the tryptophan microenvironments with increasing water content of the buffer. Low-resolution envelopes of both fractions were retrieved by ab initio procedures from small-angle X-ray scattering data, which yielded maximum molecular dimensions of about 14 nm and 1 nm for pennisetin and the low molecular weight protein, respectively, and similar values were observed by dynamic light scattering experiments. Furthermore, ¹H nuclear magnetic resonance spectra of zein and pennisetin do not show any signal below 0.9 ppm, which is compatible with more extended solution structures. The molecular models for zein and pennisetin in solution suggest that both proteins have an elongated molecular structure which is approximately a prolate ellipsoid composed of ribbons of folded -helical segments with a length of about 14 nm, resulting in a structure that permits efficient packing within the seed endosperm.     

Spectroscopic investigations of the binding mechanisms between antimicrobial peptides and membrane models of Pseudomonas aeruginosa and Klebsiella pneumoniae

CD spectroscopy was used to investigate the interactions of a series of synthetic AMPs with LPS isolated from Pseudomonas aeruginosa and Klebsiella pneumoniae, as well as with various phospholipids to better approximate the chemical composition of the membranes of these two strains of Gram-negative bacteria. This investigation was conducted in order to probe how the contributions of key physicochemical properties of an AMP vary in different regions of the membranes of these two bacteria. The conclusions from this study are as follows. (1) The binding interactions between the AMP and the membranes are defined by the complementarity of delocalization of positive charge density of the basic amino side chains (i.e., electrostatics), molecular flexibility of the peptide backbone, and overall hydrophobicity. (2) The binding interactions of these AMPs to LPS seem to be predominantly with the lipid A region of the LPS. (3) Incorporation of phospholipids into the LPS containing SUVs resulted in dramatic changes in the conformational equilibrium of the bound AMPs. (4) For the LPS-phospholipid models of Pseudomonas aeruginosa, delocalization of the side chain positive charge plays a major role in determining the number of conformers that contribute to the binding conformational equilibrium. This relationship was not observed for the models of the outer and inner membranes of Klebsiella pneumoniae.     

Induction of bacteriolytic enzyme from pyocinogenic Pseudomonas aeruginosa and its enzymatic properties.

Mitomycin C induced a pyocinogenic Pseudomonas aeruginosa P15 to produce a bacteriolytic enzyme, PR1-lysozyme, together with pyocin R1. No significant accumulation of the enzyme was observed inside the induced cells. The enzyme was partially purified by acrinol treatment and Amberlie CG-50 column chromatography. The mode of action of the enzyme on the host bacterial cells as well as on Micrococcus lysodeikticus cells or peptidoglycan isolated from Salmonella typhimurium, was compared with that of hen egg-white lysozyme or phage lambda-lysozyme. It is suggested that PR1-lysozyme should be classified as a glycosidase, rather than an amidase or an endopeptidase.     

In vitro and in vivo antimicrobial activity of a synthetic peptide derived from the C-terminal region of human chemokine CCL13 against Pseudomonas aeruginosa

Chemokines are important mediators of immunological responses during inflammation and under steady-state conditions. In addition to regulating cell migration, some chemotactic cytokines have direct effects on bacteria. Here, we characterized the antibacterial ability of the synthetic oligopeptide CCL13_57-75, which corresponds to the carboxyl-terminal region of the human chemokine CCL13. In vitro measurements indicated that CCL13_57-75 disrupts the cell membrane of Pseudomonas aeruginosa through a mechanism coupled to an unordered-helicoidal conformational transition. In a murine pneumonic model, CCL13_57-75 improved mouse survival and bacterial clearance and decreased neutrophil recruitment, proinflammatory cytokines and lung pathology compared with that observed in untreated infected animals. Overall, our study supports the ability of chemokines and/or chemokine-derived oligopeptides to act as direct defense agents against pathogenic bacteria and suggests their potential use as alternative antibiotics.     

Cloning,expression, and characterization of a thermostable and pH-stable laccase from Klebsiella pneumoniae and its application to dye decolorization

A thermostable and pH-stable laccase from Klebsiella pneumoniae was cloned and expressed in Escherichia coli. The recombinant laccase (rLac) achieved a specific activity of 7.12 U/mg after purification by Ni-affinity chromatography. Optimal enzyme activity was observed at pH 4.0 and 35 °C for 2,2′-azino-bis (3-ethylbenzthiazoline sulfonic acid) (ABTS) oxidization and pH 8.0 and 70 °C for 2,6-dimethoxyphenol (2,6-DMP) oxidization. Thermostability and pH stability studies showed that the rLac was stable over the range of 30–70 °C and pH 5.0–9.0 using 2,6-DMP as substrate. Circular dichroism analysis suggested that the secondary structure of the rLac mainly consisted of α-helix that played a vital role in maintaining laccase activity and revealed the potential mechanisms for the changes in laccase activity under varying pHs (3.0–11.0) and temperatures (20–90 °C). Finally, the rLac could decolorize the tested dyes with high decolorization efficiency.     

Biochemical and structural studies of uncharacterized protein PA0743 from Pseudomonas aeruginosa revealed NAD+-dependent L-serine dehydrogenase

The β-hydroxyacid dehydrogenases form a large family of ubiquitous enzymes that catalyze oxidation of various β-hydroxy acid substrates to corresponding semialdehydes. Several known enzymes include β-hydroxyisobutyrate dehydrogenase, 6-phosphogluconate dehydrogenase, 2-(hydroxymethyl)glutarate dehydrogenase, and phenylserine dehydrogenase, but the vast majority of β-hydroxyacid dehydrogenases remain uncharacterized. Here, we demonstrate that the predicted β-hydroxyisobutyrate dehydrogenase PA0743 from Pseudomonas aeruginosa catalyzes an NAD⁺-dependent oxidation of l-serine and methyl-l-serine but exhibits low activity against β-hydroxyisobutyrate. Two crystal structures of PA0743 were solved at 2.2–2.3-Å resolution and revealed an N-terminal Rossmann fold domain connected by a long α-helix to the C-terminal all-α domain. The PA0743 apostructure showed the presence of additional density modeled as HEPES bound in the interdomain cleft close to the predicted catalytic Lys-171, revealing the molecular details of the PA0743 substrate-binding site. The structure of the PA0743-NAD⁺ complex demonstrated that the opposite side of the enzyme active site accommodates the cofactor, which is also bound near Lys-171. Site-directed mutagenesis of PA0743 emphasized the critical role of four amino acid residues in catalysis including the primary catalytic residue Lys-171. Our results provide further insight into the molecular mechanisms of substrate selectivity and activity of β-hydroxyacid dehydrogenases.     

Anti-aggregation properties of trehalose on heat-induced secondary structure and conformation changes of bovine serum albumin

During our experimental work, aggregation of bovine serum albumin was obtained incubating the protein solution at 60 °C to investigate temperature-induced secondary structure, conformation changes and anti-aggregative activity of trehalose. IR-measurements suggested that in the presence of 1.0 M of trehalose there is a little increase in short segment connecting ?α-helical and a clearly decrease in the loss of ?α-helix structure and in the formation of intermolecular and antiparellel β-sheet up to 78 and 55%, respectively. Useful information also arose following the temperature evolution of Amide I′ band profile in the range of temperature between 25 and 90 °C in absence or in presence of 1.0 M trehalose. Complementary information is obtained by electrophoresis, circular dichroism, fluorescence spectroscopy, titration of SH groups and light scattering measurements. Results encouraged biotechnology and pharmaceutical application of the disaccharide and provided evidence for its utilization in degenerative diseases evolving via aggregation process.     

Solution structure and properties of AlgH from Pseudomonas aeruginosa

In Pseudomonas aeruginosa, the algH gene regulates the cellular concentrations of a number of enzymes and the production of several virulence factors, and is suggested to serve a global regulatory function. The precise mechanism by which the algH gene product, the AlgH protein, functions is unknown. The same is true for AlgH family members from other bacteria. In order to lay the groundwork for understanding the physical underpinnings of AlgH function, we examined the structure and physical properties of AlgH in solution. Under reducing conditions, results of NMR, electrophoretic mobility, and sedimentation equilibrium experiments indicate AlgH is predominantly monomeric and monodisperse in solution. Under nonreducing conditions intra and intermolecular disulfide bonds form, the latter promoting AlgH oligomerization. The high‐resolution solution structure of AlgH reveals alpha/beta‐sandwich architecture fashioned from ten beta strands and seven alpha helices. Comparison with available structures of orthologues indicates conservation of overall structural topology. The region of the protein most strongly conserved structurally also shows the highest amino acid sequence conservation and, as revealed by hydrogen‐deuterium exchange studies, is also the most stable. In this region, evolutionary trace analysis identifies two clusters of amino acid residues with the highest evolutionary importance relative to all other AlgH residues. These frame a partially solvent exposed shallow hydrophobic cleft, perhaps identifying a site for intermolecular interactions. The results establish a physical foundation for understanding the structure and function of AlgH and AlgH family proteins and should be of general importance for further investigations of these and related proteins. Proteins 2015; 83:1137–1150. © 2015 Wiley Periodicals, Inc.     

Structural characterization of aminoglycoside 4′‐O‐adenylyltransferase ANT(4′)‐IIb from Pseudomonas aeruginosa

Aminoglycosides were one of the first classes of broad‐spectrum antibacterial drugs clinically used to effectively combat infections. The rise of resistance to these drugs, mediated by enzymatic modification, has since compromised their utility as a treatment option, prompting intensive research into the molecular function of resistance enzymes. Here, we report the crystal structure of aminoglycoside nucleotidyltransferase ANT(4′)‐IIb in apo and tobramycin‐bound forms at a resolution of 1.6 and 2.15 Å, respectively. ANT(4′)‐IIb was discovered in the opportunistic pathogen Pseudomonas aeruginosa and conferred resistance to amikacin and tobramycin. Analysis of the ANT(4′)‐IIb structures revealed a two‐domain organization featuring a mixed β‐sheet and an α‐helical bundle. ANT(4′)‐IIb monomers form a dimer required for its enzymatic activity, as coordination of the aminoglycoside substrate relies on residues contributed by both monomers. Despite harbouring appreciable primary sequence diversity compared to previously characterized homologues, the ANT(4′)‐IIb structure demonstrates a surprising level of structural conservation highlighting the high plasticity of this general protein fold. Site‐directed mutagenesis of active site residues and kinetic analysis provides support for a catalytic mechanism similar to those of other nucleotidyltransferases. Using the molecular insights provided into this ANT(4′)‐IIb‐represented enzymatic group, we provide a hypothesis for the potential evolutionary origin of these aminoglycoside resistance determinants.     

Structural and transfection properties of amine-substituted gemini surfactant-based nanoparticles

BACKGROUND: Increases in DNA transfection efficiencies for non-viral vectors can be achieved through rational design of novel cationic building blocks. Based on previous results examining DNA condensation by polyamines, novel gemini surfactants have been designed that incorporate aza or imino substituents within the spacer group in order to increase interactions with DNA and potentially improve their DNA transfection ability. METHODS: Transfection efficiencies and cell toxicity of gemini nanoparticles constructed from plasmid DNA, gemini surfactant, and a neutral lipid were measured in COS7 cells using a luciferase assay. Structural properties of nanoparticles were examined by using circular dichroism, particle size, zeta potential, and small-angle X-ray scattering (SAXS) measurements. RESULTS: The incorporation of aza and imino substituents within the spacer group was observed to enhance the transfection ability of gemini surfactants. Incorporation of an imino group in the structure of the 1,9-bis(dodecyl)-1,1,9,9-tetramethyl-5-imino-1,9-nonanediammonium dibromide surfactant (12-7NH-12) resulted in a statistically significant (p < 0.01) 9-fold increase in transfection compared to an unsubstituted gemini surfactant and a 3-fold increase compared to the corresponding aza-substituted compound. A pH-dependent transition in size and zeta potential was observed to occur at pH 5.5 for complexes formed from the 12-7NH-12 compound. SAXS results show weakly ordered structures and the presence of multiple phases. CONCLUSIONS: The incorporation of a pH-active imino group within the spacer of the gemini surfactant results in a significant increase in transfection efficiency that can be related to both pH-induced changes in nanoparticle structure and the formation of multiple phases that more readily allow for membrane fusion that may facilitate DNA release.     

Formation of 5- and 6-Aminocytosine Nucleosides and Nucleotides from the Corresponding 5-Bromocytosine Derivatives: Synthesis and Reaction Mechanism

Abstract

An improved method for the synthesis of 5-aminocytidine (3a), 5-amino-2′-deoxycytidine (3b), and their 5′-monophosphates (3c,d) from the corresponding 5-bromo pyrimidines, using liquid ammonia, is described. The respective 6-aminocytosine derivatives (4a,b,c,d), minor products of the amination reaction, were isolated and characterized. A plausible mechanism is proposed to account for the formation of both 5-and 6-substituted products.     

Characterization and site-directed mutagenesis of the putative novel acyl carrier protein Rv0033 and Rv1344 from Mycobacterium tuberculosis

Mycolic acids are generated in Mycobacterium tuberculosis as a result of the interaction of two fatty acid biosynthetic systems: type I fatty acid synthase (FAS) and type II fatty acid synthase. Acyl carrier protein (ACP) is a small, acidic protein in type II FAS systems. It plays a central role in mycolic acid biosynthesis by transferring the acyl groups from one enzyme to another for the completion of the fatty acid synthesis cycle. The nature of the proper recognition between ACPs and its many interactive proteins is not understood. Here, we report the over-expression, purification, and characterization of two putative ACPs: Rv0033 and Rv1344 in M. tuberculosis. In order to study the role of the conserved residues and the conformation of whole protein, some site-directed mutations of recombinant Acp1344 were made and the 3D structure of Acp1344 was modeled.     

Domain dissection and characterization of the aminoglycoside resistance enzyme ANT(3″)-Ii/AAC(6′)-IId from Serratia marcescens

Aminoglycosides (AGs) are broad-spectrum antibiotics whose constant use and presence in growth environments has led bacteria to develop resistance mechanisms to aid in their survival. A common mechanism of resistance to AGs is their chemical modification (nucleotidylation, phosphorylation, or acetylation) by AG-modifying enzymes (AMEs). Through evolution, fusion of two AME-encoding genes has resulted in bifunctional enzymes with broader spectrum of activity. Serratia marcescens, a human enteropathogen, contains such a bifunctional enzyme, ANT(3″)-Ii/AAC(6′)-IId. To gain insight into the role, effect, and importance of the union of ANT(3″)-Ii and AAC(6′)-IId in this bifunctional enzyme, we separated the two domains and compared their activity to that of the full-length enzyme. We performed a thorough comparison of the substrate and cosubstrate profiles as well as kinetic characterization of the bifunctional ANT(3″)-Ii/AAC(6′)-IId and its individually expressed components.     

The disulfiram metabolites S-methyl-N,N-diethyldithiocarbamoyl sulfoxide and S-methyl-N,N-diethylthiocarbamoyl sulfone irreversibly inactivate betaine aldehyde dehydrogenase from Pseudomonas aeruginosa, both in vitro and in situ, and arrest bacterial growth

Betaine aldehyde dehydrogenase from the human opportunistic pathogen Pseudomonas aeruginosa (PaBADH) catalyzes the irreversible, NAD(P)⁺-dependent oxidation of betaine aldehyde, producing glycine betaine, an osmoprotectant. PaBADH participates in the catabolism of choline and likely in the defense against the osmotic and oxidative stresses to which the bacterium is exposed when infecting human tissues. Given that choline or choline precursors are abundant in infected tissues, PaBADH is a potential drug target because its inhibition will lead to the build up of the toxic betaine aldehyde inside bacterial cells. We tested the thiol reagents, disulfiram (DSF) and five DSF metabolites—diethyldithiocarbamic acid (DDC), S-methyl-N,N-diethyldithiocarbamoyl sulfoxide (MeDDTC-SO) and sulfone (MeDDTC-SO₂), and S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO) and sulfone (MeDTC-SO₂)—as inhibitors of PaBADH and P. aeruginosa growth. As in vitro PaBADH inhibitors, their order of potency was: MeDDTC-SO₂ > DSF > MeDTC-SO₂ > MeDDTC-SO > MeDTC-SO. DDC did not inactivate the enzyme. PaBADH inactivation by DSF metabolites (i) was not affected by NAD(P)⁺, (ii) could not be reverted by dithiothreitol, and (iii) did not affect the quaternary structure of the enzyme. Of the DSF metabolites tested, MeDTC-SO₂ and MeDDTC-SO produced significant in situ PaBADH inactivation and arrest of P. aeruginosa growth in choline containing media, in which the expression of PaBADH is induced. They had no effect in media lacking choline, indicating that PaBADH is their main intracellular target, and that arrest of growth is due to accumulation of betaine aldehyde. The in vitro and in situ kinetics of enzyme inactivation by these two compounds were very similar, indicating no restriction on their uptake by the cells. MeDDTC-SO₂ and DSF have no inhibitory effects in situ, probably because their high reactivity towards intracellular nonessential thiols causes their depletion. Our results support that PaBADH is a promising target to treat P. aeruginosa infections, and that some DSF metabolites might be of help in this aim.     

Isolation of cDNA and enzymatic properties of betaine aldehyde dehydrogenase from Zoysia tenuifolia

We isolated cDNAs encoding betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8) from the salt-tolerant Poaceae, Zoysia tenuifolia by polymerase chain reactions. Zoysia betaine aldehyde dehydrogenase 1 (ZBD1) is 1892bp long and codes for 507 amino acids. The deduced amino acid sequence of ZBD1 is 88% similar to the sequence of rice BADH. Ten cDNA clones were isolated from a cDNA Library of salt-treated Z. tenuifolia by using the ZBD1 fragment as a probe. The proteins coded in some clones were more homologous to BBD2, the cytosolic BADH of barley, than to ZBD1. To investigate their enzymatic properties, ZBD1 and spinach BADH were expressed in Escherichia coli and purified. The optimal pH of ZBD1 was 9.5, which was more alkaline than that of spinach BADH. ZBD1 was less tolerant to NaCl than spinach BADH. ZBD1 showed not only BADH activity but also aminoaldehyde dehydrogenase activity. The Km values of ZBD1 for betaine aldehyde, 4-aminobutyraldehyde (AB-ald), and 3-aminopropionaldehyde (AP-ald) were 291, 49, and 4.0 microM, respectively. ZBD1 showed higher specific activities for AB-ald and AP-ald than did spinach BADH.