Analysis of N-glycosylation in fungal l-amino acid oxidases expressed in the methylotrophic yeast Pichia pastoris.

l-amino acid oxidases (LAAOs) catalyze the oxidative deamination of l-amino acids to corresponding α-keto acids. Here, we describe the heterologous expression of four fungal LAAOs in Pichia pastoris. cgLAAO1 from Colletotrichum gloeosporioides and ncLAAO1 from Neurospora crassa were able to convert substrates not recognized by recombinant 9His-hcLAAO4 from the fungus Hebeloma cylindrosporum described earlier thereby broadening the substrate spectrum for potential applications. 9His-frLAAO1 from Fibroporia radiculosa and 9His-laLAAO2 from Laccaria amethystine were obtained only in low amounts. All four enzymes were N-glycosylated. We generated mutants of 9His-hcLAAO4 lacking N-glycosylation sites to further understand the effects of N-glycosylation. All four predicted N-glycosylation sites were glycosylated in 9His-hcLAAO4 expressed in P. pastoris. Enzymatic activity was similar for fully glycosylated 9His-hcLAAO4 and variants without one or all N-glycosylation sites after acid activation of all samples. However, activity without acid treatment was low in a variant without N-glycans. This was caused by the absence of a hypermannosylated N-glycan on asparagine residue N54. The lack of one or all of the other N-glycans was without effect. Our results demonstrate that adoption of a more active conformation requires a specific N-glycosylation during biosynthesis.

APOBEC3A Loop 1 Is a Determinant for Single-Stranded DNA Binding and Deamination.

The apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3 or A3) family of proteins functions in the innate immune system. The A3 proteins are interferon inducible and hypermutate deoxycytidine to deoxyuridine in foreign single-stranded DNA (ssDNA). However, this deaminase activity cannot discriminate between foreign and host ssDNA at the biochemical level, which presents a significant danger when A3 proteins gain access to the nucleus. Interestingly, this A3 capability can be harnessed when coupled with novel CRISPR-Cas9 proteins to create a targeted base editor. Specifically, A3A has been used in vitro to revert mutations associated with disease states. Recent structural studies have shown the importance of loop regions of A3A and A3G in ssDNA recognition and positioning for deamination. In this work, we further examined loop 1 of A3A to determine how it affects substrate selection, as well as the efficiency of deamination, in the hopes of advancing the potential of A3A in base editing technology. We found that mutating residue H29 enhanced deamination activity without changing substrate specificity. Also interestingly, we found that increasing the length of loop 1 decreases substrate specificity. Overall, these results lead to a better understanding of substrate recognition and deamination by A3A and the A3 family of proteins.

Mutating for Good: DNA Damage Responses During Somatic Hypermutation.

Somatic hypermutation (SHM) of immunoglobulin (Ig) genes plays a key role in antibody mediated immunity. SHM in B cells provides the molecular basis for affinity maturation of antibodies. In this way SHM is key in optimizing antibody dependent immune responses. SHM is initiated by targeting the Activation-Induced Cytidine Deaminase (AID) to rearranged V(D)J and switch regions of Ig genes. The mutation rate of this programmed mutagenesis is ~10-3 base pairs per generation, a million-fold higher than the non-AID targeted genome of B cells. AID is a processive enzyme that binds single-stranded DNA and deaminates cytosines in DNA. Cytosine deamination generates highly mutagenic deoxy-uracil (U) in the DNA of both strands of the Ig loci. Mutagenic processing of the U by the DNA damage response generates the entire spectrum of base substitutions characterizing SHM at and around the initial U lesion. Starting from the U as a primary lesion, currently five mutagenic DNA damage response pathways have been identified in generating a well-defined SHM spectrum of C/G transitions, C/G transversions, and A/T mutations around this initial lesion. These pathways include (1) replication opposite template U generates transitions at C/G, (2) UNG2-dependent translesion synthesis (TLS) generates transversions at C/G, (3) a hybrid pathway comprising non-canonical mismatch repair (ncMMR) and UNG2-dependent TLS generates transversions at C/G, (4) ncMMR generates mutations at A/T, and (5) UNG2- and PCNA Ubiquitination (PCNA-Ub)-dependent mutations at A/T. Furthermore, specific strand-biases of SHM spectra arise as a consequence of a biased AID targeting, ncMMR, and anti-mutagenic repriming. Here, we review mammalian SHM with special focus on the mutagenic DNA damage response pathways involved in processing AID induced Us, the origin of characteristic strand biases, and relevance of the cell cycle.

Oxidative Deamination of Emixustat by Human Vascular Adhesion Protein-1/Semicarbazide-Sensitive Amine Oxidase.

Emixustat potently inhibits the visual cycle isomerase retinal pigment epithelium protein 65 (RPE65) to reduce the accumulation of toxic bisretinoid by-products that lead to various retinopathies. Orally administered emixustat is cleared rapidly from the plasma, with little excreted unchanged. The hydroxypropylamine moiety that is critical in emixustat's inhibition of RPE65 is oxidatively deaminated to three major carboxylic acid metabolites that appear rapidly in plasma. These metabolites greatly exceed the plasma concentrations of emixustat and demonstrate formation-rate-limited metabolite kinetics. This study investigated in vitro deamination of emixustat in human vascular membrane fractions, plasma, and recombinant human vascular adhesion protein-1 (VAP-1), demonstrating single-enzyme kinetics for the formation of a stable aldehyde intermediate (ACU-5201) in all in vitro systems. The in vitro systems used herein established sequential formation of the major metabolites with addition of assay components for aldehyde dehydrogenase and cytochrome P450. Reaction phenotyping experiments using selective chemical inhibitors and recombinant enzymes of monoamine oxidase, VAP-1, and lysyl oxidase showed that only VAP-1 deaminated emixustat. In individually derived human vascular membranes from umbilical cord and aorta, rates of emixustat deamination were highly correlated to VAP-1 marker substrate activity (benzylamine) and VAP-1 levels measured by enzyme-linked immunosorbent assay. In donor-matched plasma samples, soluble VAP-1 activity and levels were lower than in aorta membranes. A variety of potential comedications did not strongly inhibit emixustat deamination in vitro.

RNA editing in bacteria: occurrence, regulation and significance.

DNA harbors the blueprint for life. However, the instructions stored in the DNA could be altered at the RNA level before they are executed. One of these processes is RNA editing, which was shown to modify RNA sequences in many organisms. The most abundant modification is the deamination of adenosine (A) into inosine (I). In turn, inosine can be identified as a guanosine (G) by the ribosome and other cellular machineries such as reverse transcriptase. In multicellular organisms, enzymes from the ADAR (adenosine deaminase acting on RNA) family mediate RNA editing in mRNA, whereas enzymes from the ADAT family mediate A-to-I editing on tRNAs. In bacteria however, until recently, only one editing site was described, in tRNAArg, but never in mRNA. The tRNA site was shown to be modified by tadA (tRNA specific adenosine deaminase) which is believed to be the ancestral enzyme for the RNA editing family of enzymes. In our recent work, we have shown for the first time, editing on multiple sites in bacterial mRNAs and identified tadA as the enzyme responsible for this editing activity. Focusing on one of the identified targets - the self-killing toxin hokB, we found that editing is physiologically regulated and that it increases protein activity. Here we discuss possible modes of regulation on hokB editing, potential roles of RNA editing in bacteria, possible implications, and future research directions.

Molecular Interactions of a DNA Modifying Enzyme APOBEC3F Catalytic Domain with a Single-Stranded DNA.

The single-stranded DNA (ssDNA) cytidine deaminase APOBEC3F (A3F) deaminates cytosine (C) to uracil (U) and is a known restriction factor of HIV-1. Its C-terminal catalytic domain (CD2) alone is capable of binding single-stranded nucleic acids and is important for deamination. However, little is known about how the CD2 interacts with ssDNA. Here we report a crystal structure of A3F-CD2 in complex with a 10-nucleotide ssDNA composed of poly-thymine, which reveals a novel positively charged nucleic acid binding site distal to the active center that plays a key role in substrate DNA binding and catalytic activity. Lysine and tyrosine residues within this binding site interact with the ssDNA, and mutating these residues dramatically impairs both ssDNA binding and catalytic activity. This binding site is not conserved in APOBEC3G (A3G), which may explain differences in ssDNA-binding characteristics between A3F-CD2 and A3G-CD2. In addition, we observed an alternative Zn-coordination conformation around the active center. These findings reveal the structural relationships between nucleic acid interactions and catalytic activity of A3F.

New insights into the mechanisms of acetic acid resistance in Acetobacter pasteurianus using iTRAQ-dependent quantitative proteomic analysis.

Acetobacter pasteurianus is the main starter in rice vinegar manufacturing due to its remarkable abilities to resist and produce acetic acid. Although several mechanisms of acetic acid resistance have been proposed and only a few effector proteins have been identified, a comprehensive depiction of the biological processes involved in acetic acid resistance is needed. In this study, iTRAQ-based quantitative proteomic analysis was adopted to investigate the whole proteome of different acidic titers (3.6, 7.1 and 9.3%, w/v) of Acetobacter pasteurianus Ab3 during the vinegar fermentation process. Consequently, 1386 proteins, including 318 differentially expressed proteins (p<0.05), were identified. Compared to that in the low titer circumstance, cells conducted distinct biological processes under high acetic acid stress, where >150 proteins were differentially expressed. Specifically, proteins involved in amino acid metabolic processes and fatty acid biosynthesis were differentially expressed, which may contribute to the acetic acid resistance of Acetobacter. Transcription factors, two component systems and toxin-antitoxin systems were implicated in the modulatory network at multiple levels. In addition, the identification of proteins involved in redox homeostasis, protein metabolism, and the cell envelope suggested that the whole cellular system is mobilized in response to acid stress. These findings provide a differential proteomic profile of acetic acid resistance in Acetobacter pasteurianus and have potential application to highly acidic rice vinegar manufacturing.

Degradation of spent craft brewer's yeast by caprine rumen hyper ammonia-producing bacteria.

UNLABELLED: Spent yeast from craft beers often includes more hops (Humulus lupulus L.) secondary metabolites than traditional recipes. These compounds include α- and ß- acids, which are antimicrobial to the rumen hyper ammonia-producing bacteria (HAB) that are major contributors to amino acid degradation. The objective was to determine if the hops acids in spent craft brewer's yeast (CY; ~ 3·5 mg g(-1) hops acids) would protect it from degradation by caprine rumen bacteria and HAB when compared to a baker's yeast (BY; no hops acids). Cell suspensions were prepared by harvesting rumen fluid from fistulated goats, straining and differential centrifugation. The cells were re-suspended in media with BY or CY. After 24 h (39°C), HAB were enumerated and ammonia was measured. Fewer HAB and less ammonia was produced from CY than from BY. Pure culture experiments were conducted with Peptostreptococcus anaerobiusBG1 (caprine HAB). Ammonia production by BG1 from BY was greater than from CY. Ammonia production was greater when exogenous amino acids were included, but similar inhibition was observed in CY treatments. These results indicate that rumen micro-organisms deaminated the amino acids in CY to a lesser degree than BY. SIGNIFICANCE AND IMPACT OF THE STUDY: Spent brewer's yeast has long been included in ruminant diets as a protein supplement. However, modern craft beers often include more hops (Humulus lupulus L.) than traditional recipes. These compounds include α- and ß- acids, which are antimicrobial to the rumen hyper ammonia-producing bacteria (HAB) that are major contributors to amino acid degradation. This study demonstrated that hops acids in spent craft brewer's yeast protected protein from destruction by HABin vitro. These results suggest that the spent yeast from craft breweries, a source of beneficial hops secondary metabolites, could have value as rumen-protected protein.

Combined QM(DFT)/MM molecular dynamics simulations of the deamination of cytosine by yeast cytosine deaminase (yCD).

Extensive combined quantum mechanical (B3LYP/6-31G*) and molecular mechanical (QM/MM) molecular dynamics simulations have been performed to elucidate the hydrolytic deamination mechanism of cytosine to uracil catalyzed by the yeast cytosine deaminase (yCD). Though cytosine has no direct binding to the zinc center, it reacts with the water molecule coordinated to zinc, and the adjacent conserved Glu64 serves as a general acid/base to shuttle protons from water to cytosine. The overall reaction consists of several proton-transfer processes and nucleophilic attacks. A tetrahedral intermediate adduct of cytosine and water binding to zinc is identified and similar to the crystal structure of yCD with the inhibitor 2-pyrimidinone. The rate-determining step with the barrier of 18.0 kcal/mol in the whole catalytic cycle occurs in the process of uracil departure where the proton transfer from water to Glu64 and nucleophilic attack of the resulting hydroxide anion to C2 of the uracil ring occurs synchronously. © 2016 Wiley Periodicals, Inc.

A vesicular sequestration to oxidative deamination shift in myocardial sympathetic nerves in Parkinson's disease.

In Parkinson's disease (PD), profound putamen dopamine (DA) depletion reflects denervation and a shift from vesicular sequestration to oxidative deamination of cytoplasmic DA in residual terminals. PD also involves cardiac sympathetic denervation. Whether PD entails myocardial norepinephrine (NE) depletion and a sequestration-deamination shift have been unknown. We measured apical myocardial tissue concentrations of NE, DA, and their neuronal metabolites 3,4-dihydroxyphenylglycol (DHPG), and 3,4-dihydroxyphenylacetic acid (DOPAC) from 23 PD patients and 23 controls and ascertained the extent of myocardial NE depletion in PD. We devised, validated in VMAT2-Lo mice, and applied 5 neurochemical indices of the sequestration-deamination shift-concentration ratios of DOPAC:DA, DA:NE, DHPG:NE, DOPAC:NE, and DHPG:DOPAC-and used a kinetic model to estimate the extent of the vesicular storage defect. The PD group had decreased myocardial NE content (p < 0.0001). The majority of patients (70%) had severe NE depletion (mean 2% of control), and in this subgroup all five indices of a sequestration-deamination shift were increased compared to controls (p < 0.001 for each). Vesicular storage in residual nerves was estimated to be decreased by 84-91% in this subgroup. We conclude that most PD patients have severe myocardial NE depletion, because of both sympathetic denervation and decreased vesicular storage in residual nerves. We found that the majority (70%) of Parkinson's disease (PD) patients have profound (98%) myocardial norepinephrine depletion, because of both cardiac sympathetic denervation and a shift from vesicular sequestration to oxidative deamination of cytoplasmic catecholamines in the residual nerves. This shift may be part of a final common pathogenetic pathway in the loss of catecholaminergic neurons that characterizes PD.

Epigenetic reprogramming: is deamination key to active DNA demethylation?

DNA demethylation processes are important for reproduction, being central in epigenetic reprogramming during embryonic and germ cell development. While the enzymes methylating DNA have been known for many years, identification of factors capable of mediating active DNA demethylation has been challenging. Recent findings suggest that cytidine deaminases may be key players in active DNA demethylation. One of the most investigated candidates is activation-induced cytidine deaminase (AID), best known for its role in generating secondary antibody diversity in B cells. We evaluate evidence for cytidine deaminases in DNA demethylation pathways in vertebrates and discuss possible models for their targeting and activity regulation. These findings are also considered along with alternative demethylation pathways involving hydroxymethylation.

Altering the spectrum of immunoglobulin V gene somatic hypermutation by modifying the active site of AID.

High-affinity antibodies are generated by somatic hypermutation with nucleotide substitutions introduced into the IgV in a semirandom fashion, but with intrinsic mutational hotspots strategically located to optimize antibody affinity maturation. The process is dependent on activation-induced deaminase (AID), an enzyme that can deaminate deoxycytidine in DNA in vitro, where its activity is sensitive to the identity of the 5'-flanking nucleotide. As a critical test of whether such DNA deamination activity underpins antibody diversification and to gain insight into the extent to which the antibody mutation spectrum is dependent on the intrinsic substrate specificity of AID, we investigated whether it is possible to change the IgV mutation spectrum by altering AID's active site such that it prefers a pyrimidine (rather than a purine) flanking the targeted deoxycytidine. Consistent with the DNA deamination mechanism, B cells expressing the modified AID proteins yield altered IgV mutation spectra (exhibiting a purine-->pyrimidine shift in flanking nucleotide preference) and altered hotspots. However, AID-catalyzed deamination of IgV targets in vitro does not yield the same degree of hotspot dominance to that observed in vivo, indicating the importance of features beyond AID's active site and DNA local sequence environment in determining in vivo hotspot dominance.