3-hydroxyisobutyrate Dehydrogenase Research Articles

Accessory gland protein regulates pairing process and oviposition in the subterranean termite Reticulitermes chinensis after swarming.

Swarming and pairing behaviors are significant to population dispersal of termites. Tandem running is a key process in pairing behavior of dealates to find a mate. Succinylation can lead to significant changes in protein structure and function, which is widely involved in metabolism and behavior regulation in many organisms. However, whether succinylation modification regulates termites' tandem running is currently unknown. In this research, we performed quantitative modified proteomics of the subterranean termite Reticulitermes chinensis Snyder before and after alate swarming. The succinylation levels of accessory gland protein (ACP) were significantly altered after alate swarming. We found that ACP is enriched in male accessory gland and female oocytes of termites. The acetylation and succinylation sites of ACP affected tandem running of dealates. The transcriptome and metabolome analyses of alates injected with ACP and its mutant proteins showed that β-alanine metabolism pathway was the major downstream pathway of ACP. Silencing the significantly differentially expressed genes in the β-alanine metabolic pathway (acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxyisobutyrate dehydrogenase, methylmalonate-semialdehyde dehydrogenase) suppressed tandem running and altered oviposition of paired dealates. These findings demonstrate that protein translation modification is an important regulator of tandem running behavior of termites, which implies that the succinylation and acetylation modification sites of ACP could be potential targets for insecticide action. Our research offers a potential approach for developing novel dispersal inhibitors against social insect pests.

Abstract 16157: Impaired Branched Chain Amino Acid Metabolism in Porcine Right Atrial Dysfunction

Background: Right atrial (RA) dysfunction is prevalent and an early predictor of mortality in pulmonary arterial hypertension (PAH). At present, the underlying pathobiology of RA dysfunction is not well defined, and thus approaches are needed to delineate potential therapeutic pathways to hopefully improve outcomes in PAH. Methods: Surgical pulmonary artery banding induced right ventricular and right atrial dysfunction in male piglets. 6 weeks post-banding, cardiovascular magnetic resonance imaging quantified cardiac structure/function. Right atrial and ventricular tissue was subjected to quantitative proteomics and targeted metabolomics. Results: PAB piglets had decreased RA ejection fraction (RAEF) and increased RA end-systolic volume. Pathway analysis revealed proteins responsible for branched chain amino acid (BCAA) degradation, including MCEE (Methylmalonyl CoA Epimerase), DLD (Dihydrolipoamide Dehydrogenase), HIBADH (3-hydroxyisobutyrate Dehydrogenase), ACAT1 (Acetyl-CoA Acetyltransferase 1), and IVD (Isovaleryl-CoA Dehydrogenase), were reduced in PAB RAs, which was not observed in RV tissue. Targeted metabolomics analysis demonstrated BCAAs were elevated in the RA of PAB piglets and BCAA levels were strongly and inversely associated with RAEF. Conclusions: Deranged BCAA metabolism is uniquely observed in the dysfunctional RA, which offers a new therapeutic target for this important clinical problem.

Role of a cysteine residue in substrate entry and catalysis in MtHIBADH: Analysis by chemical modifications and site-directed mutagenesis.

Despite sharing conserved substrate-binding residues, members of 3-hydroxyisobutyrate dehydrogenase (HIBADH) superfamily show remarkable differences in substrate preference. Cysteine residues were identified within a radius of 6Å surrounding both the active site and the substrate entry site of HIBADH enzyme from Mycobacterium tuberculosis (MtHIBADH). Chemical modification with thiol-modifying reagents, pCMB and DTNB, abrogated the dehydrogenase activity of the enzyme. The loss in activity followed pseudo-first-order kinetics as a function of the concentration of pCMB. S-HIBA (substrate) binding provided partial protection, while NAD (cofactor) binding provided ~70% protection from thiol-modifying reagent. Site-directed mutagenesis of cysteine residues present in the MtHIBADH enzyme identified the indispensable role of Cys-210 residue, located at C-terminal domain, for its dehydrogenase activity. Cys-210 mutation to serine reduced the dehydrogenase activity by ~2-fold while mutation to alanine strikingly reduced the activity by ~140-fold. C210A mutation did not perturb the state of oligomerization of the enzyme but perturbed the secondary structure content. Structural analysis revealed the involvement of Cys-210 residue in inter-chain interaction with Gln-178, which acts as hydrogen bond donor and coordinates with Cys-210 and Gly-208 of the adjacent subunit. The data demonstrate a critical role of Cys-210 residue in maintaining the conformation and rigidity of loop composed of substrate-interacting residues involved in the entry of S-HIBA substrate in MtHIBADH.

Functional and Structural Analysis of a Novel Acyltransferase from Pathogenic Phytophthora melonis.

This investigation characterizes an acyltransferase enzyme responsible for the pathogenicity of Phytophthora melonis. The protein was characterized in vitro for its physicochemical properties. The biochemical characterization, including thermal and pH stability, revealed the 35 °C temperature and 7.0 pH as the optimum conditions for the enzyme. Applying the Tween-80 solution enhanced the activity up to 124.9%. Comprehensive structural annotation revealed two domains, A (ranging from residues 260 to 620) and B (ranging from 141 to 219). Domain A had transglutaminase (T-Gase) elicitor properties, while B possessed antifreeze features. Rigorous sequence characterization of the acyltransferase tagged it as a low-temperature-resistant protein. Further, the taxonomic distribution analysis of the protein highlighted three genera in Oomycetes, i.e., Pythium, Phytophthora, and Plasmopara, bearing this protein. However, some taxonomic groups other than Oomycetes (i.e., archaea and bacteria) also contained the protein. Functional studies of structurally analogous proteins spanned 10 different taxonomic groups. These revealed TGase elicitors (10%), phytopathogen effector proteins RxLR (4%), transporter family proteins (3%), and endonucleases (1%). Other analogues having one percent of their individual share were HIV tat-specific factor 1, protocadherin fat 4, transcription factor 1, and 3-hydroxyisobutyrate dehydrogenase. Because the plant infection by P. melonis is a complex process regulated by a profusion of extracellular signals secreted by both host plants and the pathogen, this study will be of help in interpreting the cross-talk in the host–pathogen system.

Characterization of a novel class of glyoxylate reductase belonging to the β-hydroxyacid dehydrogenase family in Acetobacter aceti.

Enzymes related to β-hydroxyacid dehydrogenases/3-hydroxyisobutyrate dehydrogenases are ubiquitous, but most of them have not been characterized. An uncharacterized protein with moderate sequence similarities to Gluconobacter oxydans succinic semialdehyde reductase and plant glyoxylate reductases/succinic semialdehyde reductases was found in the genome of Acetobacter aceti JCM20276. The corresponding gene was cloned and expressed in Escherichia coli. The gene product was purified and identified as a glyoxylate reductase that exclusively catalyzed the NAD(P)H-dependent reduction of glyoxylate to glycolate. The strict substrate specificity of this enzyme to glyoxylate, the diverged sequence motifs for its binding sites with cofactors and substrates, and its phylogenetic relationship to homologous enzymes suggested that this enzyme represents a novel class of enzymes in the β-hydroxyacid dehydrogenase family. This study may provide an important clue to clarify the metabolism of glyoxylate in bacteria. Abbreviations: GR: glyoxylate reductase; GRHPR: glyoxylate reductase/hydroxypyruvate reductase; HIBADH: 3-hydroxyisobutyrate dehydrogenase; SSA: succinic semialdehyde; SSAR: succinic semialdehyde reductase.

Analysis of methylated genomic cytosines of maize inbred line W22 in response to drought stress

Plants employ DNA methylation to maintain genomic plasticity that allows rapid adaptation to environmental stresses without alteration nucleotide sequence of DNA. The present investigation was carried out to detect DNA methylation changes induced by drought stress in inbred line W22 by using amplified methylation-polymorphism technique. The maize inbred line W22 encountered extensive DNA methylation variations, including hypo- and hypermethylation as a response to drought stress. Differential methylation of many genes involved in plant response to drought stress such as Antagonist of Like Heterochromatin Protein1, peptide methionine sulfoxide reductase (MSRA4), 3-isopropylmalate dehydratase large subunit 2, adenylyl cyclase-associated protein, 3-hydroxyisobutyrate dehydrogenase gene, and others was observed.

Fasting differentially alters the hypothalamic proteome of chickens from lines with the propensity to be anorexic or obese

BackgroundThe hypothalamus is the ultimate modulator of appetite and energy balance and therefore sensitive to changes in nutritional state. Chicks from lines selected for low (LWS) and high (HWS) body weight are hypophagic and compulsive eaters, respectively, and differ in their propensity to become obese and in their hypothalamic mRNA response to fasting.MethodsAs fasting-induced changes in hypothalamic proteins are unknown, we investigated the hypothalamic proteomes of 5-day old LWS and HWS chicks in the fed and fasted states using a label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach.ResultsA total of 744 proteins were identified in the chicken hypothalamus, and 268 differentially abundant proteins were identified among four pairwise comparisons. Ninety-five proteins were associated with the response to fasting in HWS chicks, and 23 proteins were associated with the response to fasting in LWS chicks. Fasting-responsive proteins in HWS chicks were significantly enriched in ATP metabolic processes, glyoxylate/dicarboxylate metabolism, and ribosome function. There was no enrichment for any pathways in LWS chicks in response to fasting. In the fasted and fed states, 159 and 119 proteins differed between HWS and LWS, respectively. Oxidative phosphorylation, citric acid cycle, and carbon metabolism were the main pathways associated with differences between the two lines of chicks. Enzymes associated with metabolic pathways differed between HWS and LWS in both nutritional states, including fumarase, aspartate aminotransferase, mitochondrial GOT2, 3-hydroxyisobutyrate dehydrogenase, chondrogenesis associated lipocalin, sialic acid synthase, arylamine N-acetyltransferase, pineal gland isozyme NAT-3, and succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial.ConclusionsThese results provide insights into the hypothalamic metabolic pathways that are affected by nutritional status and the regulation of appetite and eating behavior.

Restoration of polar-flagellum motility and biofilm-forming capacity in the mmsB1 mutant of the alphaproteobacterium Azospirillum brasilense Sp245 points to a new role for a homologue of 3-hydroxyisobutyrate dehydrogenase.

The bacterium Azospirillum brasilense can swim and swarm owing to the rotation of a constitutive polar flagellum (Fla) and inducible lateral flagella, respectively. They also form biofilms on various interfaces. Experimental data on flagellar assembly and social behaviours in these bacteria are scarce. Here, for the first time, the chromosomal coding sequence mmsB1 for a homologue of 3-hydroxyisobutyrate dehydrogenase (protein accession Nos. ADT80774 and E7CWE2) was shown to play a role in the assembly of motile Fla and in biofilm biomass accumulation. In the previously obtained mutant SK039 of A. brasilense Sp245, an Omegon-Km insertion in mmsB1 was concurrent with changes in cell-surface properties and with suppression of Fla assembly (partial) and Fla-dependent motility (complete). Here, the immotile leaky Fla- mutant SK039 was complemented with the expression vector pRK415-borne mmsB1 gene of Sp245. In the complemented mutant, the elevated relative cell hydrophobicity and changed relative membrane fluidity of SK039 returned to the wild-type levels; also, biofilm biomass accumulation increased and even reached Sp245's levels under nutritionally rich conditions. In strain SK039 (pRK415-mmsB1), the percentage of cells with Fla became significantly higher than that in mutant SK039, and the Fla-driven swimming velocity was equal to that in strain Sp245.

3-Hydroxyisobutyrate Dehydrogenase Is Involved in Both, Valine and Isoleucine Degradation in Arabidopsis thaliana.

In plants, amino acid catabolism is especially relevant in metabolic stress situations (e.g. limited carbohydrate availability during extended darkness). Under these conditions, amino acids are used as alternative substrates for respiration. Complete oxidation of the branched-chain amino acids (BCAAs) leucine, isoleucine (Ile), and valine (Val) in the mitochondria efficiently allows the formation of ATP by oxidative phosphorylation. However, the metabolic pathways for BCAA breakdown are largely unknown so far in plants. A systematic search for Arabidopsis (Arabidopsis thaliana) genes encoding proteins resembling enzymes involved in BCAA catabolism in animals, fungi, and bacteria as well as proteomic analyses of mitochondrial fractions from Arabidopsis allowed the identification of a putative 3-hydroxyisobutyrate dehydrogenase, AtHDH1 (At4g20930), involved in Val degradation. Systematic substrate screening analyses revealed that the protein uses 3-hydroxyisobutyrate but additionally 3-hydroxypropionate as substrates. This points to a role of the enzyme not only in Val but possibly also in Ile metabolism. At4g20930 knockdown plants were characterized to test this conclusion. Root toxicity assays revealed increased root growth inhibition of the mutants if cultivated in the presence of Val or Ile but not in the presence of leucine. We conclude that AtHDH1 has a dual role in BCAA metabolism in plants.

Structural and biochemical characterization of the Bacillus cereus 3-hydroxyisobutyrate dehydrogenase

The 3-hydroxyisobutyrate dehydrogenase (HIBADH) family catalyzes the NAD+- or NADP+-dependent oxidation of various β-hydroxyacid substrates into their cognate semialdehydes for diverse metabolic pathways. Because HIBADH group members exhibit different substrate specificities, the substrate-recognition mode of each enzyme should be individually characterized. In the current study, we report the biochemical and structural analysis of a HIBADH group enzyme from Bacillus cereus (bcHIBADH). bcHIBADH mediates a dehydrogenation reaction on S-3-hydroxyisobutyrate substrate with high catalytic efficiency in an NAD+-dependent manner; it also oxidizes l-serine and 3-hydroxypropionate with lower activity. bcHIBADH consists of two domains and is further assembled into a functional dimer rather than a tetramer that has been commonly observed in other prokaryotic HIBADH group members. In the bcHIBADH structure, the interdomain cleft forms a putative active site and simultaneously accommodates both an NAD+ cofactor and a substrate mimic. Our structure-based comparative analysis highlights structural motifs that are important in the cofactor and substrate recognition of the HIBADH group.

Changes in cell surface properties and biofilm formation efficiency in Azospirillum brasilense Sp245 mutants in the putative genes of lipid metabolism mmsB1 and fabG1

The previously obtained insertion mutants ofAzospirillum brasilense Sp245 in the genes mmsBl and fabG1 (strains SK039 and Sp245.1610, respectively) were characterized by impaired flagellation and motility. The putative products of expression of these genes are 3-hydroxyisobutyrate dehydrogenase and 3-oxoacyl-[acyl-carrier protein] reductase, respectively. In the present work, A. brasilense- Sp245 strains SK039 and Sp245.1610 were found to have differences in the content of 3-hydroxyhexadecanoic, hexadecanoic, 3-hydroxytetradecanoic, hexadecenoic, octadecenoic, and nonadecanoic acids in their lipopolysaccharide prepa- rations, as well as in cell hydrophobicity and hemagglutination activity and dynamics of cell aggregation, in biomass amount, and in the relative content of lipopolysaccharide antigens in mature biofilms formed on hydrophilic or hydrophobic surfaces.

Identification of Vibrio harveyi proteins involved in the specific immune response of Senegalese sole (Solea senegalensis, Kaup).

Senegalese sole cultures are frequently affected by Vibrio harveyi disease outbreaks. Vaccines in aquaculture are one of the most successful methods of preventing fish pathologies; however, these vaccines are usually composed of inactivated whole cells containing a wide pool of antigens, and some do not induce any protection against pathogens. Thus, the aim of this study was to identify immunogenic proteins of V. harveyi involved in the specific antibody production by Senegalese sole. S. senegalensis specimens were immunized, by intraperitoneal injection, with V. harveyi bacterin supplemented with inactivated extracellular polymeric substances (ECP) and Freund incomplete adjuvant to obtain polyclonal antiserum. One month later, specimens were re-inoculated with the same antigens. Sera from immunized fish were collected two months post first immunization. Strong specific immune response to V. harveyi antigens was detected by ELISA using bacterin (limit dilutions of sera were 1:64000), ECP (1:4000) and outer membrane proteins (OMP) (1:4000) as antigens. Presence of immunogenic proteins in V. harveyi ECP and OMP were determined by 2D-PAGE. For Western Blot analysis some gels were transferred onto nitrocellulose membranes and incubated with sera from S. senegalensis specimens immunized against V. harveyi. 2D-PAGE and Western Blot showed at least five reactive proteins in the ECP and two in the OMP fraction. The spots that clearly reacted with the sole antiserum were excised from stained gel, and analyzed by mass spectrometry (MALDI/TOFTOF). A database search was then performed, using MASCOT as the search method. According to the results, the five ECP spots were identified as Maltoporine, protein homologous to Metal dependent phosphohydrolase, two porins isoforms of V. harveyi and a protein homologous to the cell division protein FtsH. Reactive proteins in the OMP fraction were identified as the protein 3-hydroxyisobutyrate dehydrogenase and a protein homologous to acid phosphatase.

The g.-165 T>C Rather than Methylation Is Associated with Semen Motility in Chinese Holstein Bulls by Regulating the Transcriptional Activity of the HIBADH Gene.

The 3-hydroxyisobutyrate dehydrogenase (HIBADH) is regarded as a human sperm-motility marker. However, the molecular mechanisms involved in the regulation of expression of the HIBADH gene in bulls remain largely unknown. HIBADH was detected in the testis, epididymis, and sperm via reverse transcription polymerase chain reaction and Western blot analysis. It is also expressed in the seminiferous epithelium, spermatids, and the entire epididymis, as detected by immunohistochemistry. Furthermore, HIBADH was expressed in the neck-piece and mid-piece of bull spermatids, as shown in the immunofluorescence assay. Using serially truncated bovine HIBADH promoters and luciferase constructs, we discovered an 878 bp (-703 bp to +175 bp) fragment that constitutes the core promoter region. One SNP g.-165 T>C of HIBADH was identified and genotyped in 307 Chinese Holstein bulls. Correlation analysis revealed that bulls with the TT genotype had higher initial sperm motility than those with the CC genotype (P < 0.05). Furthermore, the T- or C-containing loci (designated as pGL3-T and pGL3-C) were transiently transfected into MLTC-1 to test the effect of SNP on HIBADH expression. The luciferase reporter assay showed that the pGL3-T genotype exhibited 58% higher transcriptional activity than the pGL3-C genotype (P < 0.05). The bisulfite sequencing analysis revealed that the methylation pattern of the core promoter presented hypomethylation in the ejaculated semen in high-motility and low-motility bulls. The results demonstrated for the first time that the g.-165 T>C rather than methylation in the 5'-flanking region could affect the bovine sperm motility through the regulation of HIBADH gene transcriptional activity.

HIBADH Plays an Important Role in the Course of Liver Cell Necrosis

Abstract Objective To observe the biological function of human 3-hydroxyisobutyrate dehydrogenase (HIBADH). Methods Human 3-hydroxyisobutyrate dehydrogenase (HIBADH, 3-hydroxy-2-methyl propanoate: NAD+ oxidoreductase) recombinant protein was expressed in E. coli BL21, and purified by Ni+ column. The special antisera was obtained from rabbits immunized by this purified antigen. On the distribution of HIBADH, it was found that HIBADH over-expressed in the injured liver cells when serious hepatitis occurred. The phenomenon was confirmed in the animal models of SD rats with acute liver cell injury induced by CCl4, but this phenomenon did not exist in the models induced by endotoxin combined with galactosamine. Further more, HIBADH’s overexpression in liver cells will induce cell necrosis through the pathway of oxidative stress. Results When the liver cells injured by drug or other chemical materials, HIBADH will be compensationally over-expressed for the deficiency of energy, so liver cells can make enough ATP through brand-chain amino acid catabolism. However, the overexpression of HIBADH will be harmful for liver cells through the product of much more active oxygens which will induce the cell necrosis. Conclusions HIBADH over-expression is a signal of the liver cell metabolism injury, and it can aggravate the liver cell injury through oxidative stress.

Mutants of bacterium Azospirillum brasilense Sp245 with Omegon insertion in mmsB or fabG genes of lipid metabolism are defective in motility and flagellation

Bacteria Azospirillum brasilense have mixed flagellation: in addition to the polar flagellum, numerous lateral flagella are formed in their cells on medium with increased density. Flagella determine the active swimming and swarming capacities of azospirilla. Using A. brasilense Sp245 as an example, we showed that the Omegon-Km artificial transposon insertion into the chromosomal gene for 3-hydroxyisobutyrate dehydrogenase (mmsB) was concurrent with the appearance of significant defects in the formation of polar flagella and with the paralysis of lateral flagella. The Sp245 mutant with the Omegon insertion into the plasmid AZOBR_p1-borne gene for 3-oxoacyl-[acyl-carrier protein]-reductase (fabG) showed the complete loss of flagella and the swarming capacity, as well as significant defects in polar flagellar assembly (though some cells are still motile in liquid medium). The viability of the A. brasilense Sp245 mutants with the Omegon insertion into the mmsB or fabG gene was not reduced. No considerable differences in the fatty acid composition of whole cell lipid extracts were found for the A. brasilense Sp245 strain and its mmsB and fabG mutants.

Identification of catalytically important amino acid residues for enzymatic reduction of glyoxylate in plants

NADPH-dependent glyoxylate reductases from Arabidopsis thaliana (AtGLYR) convert both glyoxylate and succinic semialdehyde into their corresponding hydroxyacid equivalents. The primary sequence of cytosolic AtGLYR1 reveals several sequence elements that are consistent with the β-HAD (β-hydroxyacid dehydrogenase) protein family, whose members include 3-hydroxyisobutyrate dehydrogenase, tartronate semialdehyde reductase and 6-phosphogluconate dehydrogenase. Here, site-directed mutagenesis was utilized to identify catalytically important amino acid residues for glyoxylate reduction in AtGLYR1. Kinetic studies and binding assays established that Lys170 is essential for catalysis, Phe231, Asp239, Ser121 and Thr95 are more important in substrate binding than in catalysis, and Asn174 is more important in catalysis. The low activity of the mutant enzymes precluded kinetic studies with succinic semialdehyde. The crystal structure of AtGLYR1 in the absence of substrate was solved to 2.1Å by molecular replacement using a previously unrecognized member of the β-HAD family, cytokine-like nuclear factor, thereby enabling the 3-D structure of the protein to be modeled with substrate and co-factor. Structural alignment of AtGLYR1 with β-HAD family members provided support for the essentiality of Lys170, Phe173, Asp239, Ser121, Asn174 and Thr95 in the active site and preliminary support for an acid/base catalytic mechanism involving Lys170 as the general acid and a conserved active-site water molecule. This information established that AtGLYR1 is a member of the β-HAD protein family. Sequence and activity comparisons indicated that AtGLYR1 and the plastidial AtGLYR2 possess structural features that are absent in Arabidopsis hydroxypyruvate reductases and probably account for their stronger preference for glyoxylate over hydroxypyruvate.

Cloning, Expression and Characterization of 3-Hydroxyisobutyrate Dehydrogenase from Pseudomonas denitrificans ATCC 13867

The gene encoding an NAD+-dependent, 3-hydroxyisobutyrate dehydrogenase (3HIBDH-IV) from Pseudomonas denitrificans ATCC 13867 was cloned and expressed in Escherichia coli BL 21 (DE3) and characterized to understand its physiological relevance in the degradation of 3-hydroxypropionic acid (3-HP). The deduced amino acid sequence showed high similarity to other 3-hydroxyisobutyrate dehydrogenase isozymes (3HIBDHs) of P. denitrificans ATCC 13867. A comparison of 3HIBDH-IV with its relevant enzymes along with molecular docking studies suggested that Lys171, Asn175 and Gly123 are important for its catalytic function on 3-hydroxyacids. The recombinant 3HIBDH-IV was purified to homogeneity utilizing a Ni-NTA-HP resin column in high yield. 3HIBDH-IV was very specific to (S)-3-hydroxyisobutyrate, but also catalyzed the oxidation of 3-HP to malonate semialdehyde. The specific activity and half-saturation constant (K m) for 3-HP at 30°C and pH 9.0 were determined to be 17 U/mg protein and 1.0 mM, respectively. Heavy metals, such as Ag+ and Hg2+, completely inhibited the 3HIBDH-IV activity, whereas dithiothreitol, 2-mercaptoethanol and ethylenediaminetetraacetic acid increased its activity 1.5–1.8-fold. This paper reports the characteristics of 3HIBDH-IV as well as its probable role in 3-HP degradation.

Characterization of 3-hydroxyisobutyrate dehydrogenase, HIBADH, as a sperm-motility marker

Asthenozoospermia is a major cause of male infertility. However, the molecular mechanisms underlying sperm-motility defects remain largely unknown in the majority of cases. In our previous study, we applied a proteomic approach to identify unknown proteins that were downregulated in spermatozoa with low motility compared to spermatozoa with good motility. Several sperm motility- related proteins have been identified. In this study, 3-hydroxyisobutyrate dehydrogenase (HIBADH), one of the proteins identified using the proteomic tools, is further characterized. Reverse-transcription polymerase chain reactions (RT-PCR), western blotting, and immunofluorescence assays (IFA) were preformed to investigate the expression pattern. The enzymatic activity of HIBADH was evaluated in sperm with good (>50 %), moderate (< 50 %) and lower motility (< 20 %). Using RT-PCR, we found that transcripts of HIBADH are enriched in the cerebellum, heart, skeletal muscle, uterus, placenta, and testes of male humans. In western blotting, it is expressed in the placenta, testes, and spermatozoa. During spermiogenesis, HIBADH is located at the mid-piece (a specialized development from the mitochondria) of elongating, elongated, and mature sperm. The enzymatic activity of HIBADH in sperm with moderate and lower motility were significantly reduced compared with good motility (P<0.0001 and P<0.05, respectively). Our study indicated that HIBADH is involved in the mitochondrial function of spermatozoa, and maintains sperm motility. It may serve as a sperm-motility marker.

Proteomic analysis of Pseudomonas putida reveals an organic solvent tolerance-related gene mmsB.

Organic solvents are toxic to most microorganisms. However, some organic-solvent-tolerant (OST) bacteria tolerate the destructive effects of organic solvent through various accommodative mechanisms. In this work, we developed an OST adapted strain Pseudomonas putida JUCT1 that could grow in the presence of 60% (v/v) cyclohexane. Two-dimensional gel electrophoresis was used to compare and analyze the total cellular protein of P. putida JUCT1 growing with or without 60% (v/v) cyclohexane. Under different solvent conditions, five high-abundance protein spots whose intensity values show over 60% discrepancies were identified by MALDI-TOF/TOF spectra. Specifically, they are arginine deiminase, carbon-nitrogen hydrolase family putative hydrolase, 3-hydroxyisobutyrate dehydrogenase, protein chain elongation factor EF-Ts, and isochorismatase superfamily hydrolase. The corresponding genes of the latter three proteins, mmsB, tsf, and PSEEN0851, were separately expressed in Escherichia coli to evaluate their effect on OST properties of the host strain. In the presence of 4% (v/v) cyclohexane, E. coli harboring mmsB could grow to 1.70 OD660, whereas cell growth of E. coli JM109 (the control) was completely inhibited by 2% (v/v) cyclohexane. Transformants carrying tsf or PSEEN0851 also showed an increased resistance to cyclohexane and other organic solvents compared with the control. Of these three genes, mmsB exhibited the most prominent effect on increasing OST of E. coli. Less oxidation product of cyclohexane was detected because mmsB transformants might help keep a lower intracellular cyclohexane level. This study demonstrates a feasible approach for elucidating OST mechanisms of microorganisms, and provides molecular basis to construct organic-solvent-tolerant strains for industrial applications.

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.