Hyphomicrobium Methylovorum Research Articles

A novel in-silico model explores LanM homologs among Hyphomicrobium spp

Investigating microorganisms in metal-enriched environments holds the potential to revolutionize the sustainable recovery of critical metals such as lanthanides (Ln3+). We observe Hyphomicrobium spp. as part of a Fe2+/Mn2+-oxidizing consortia native to the ferruginous bottom waters of a Ln3+-enriched lake in Czechia. Notably, one species shows similarities to recently discovered bacteria expressing proteins with picomolar Ln3+ affinity. This finding was substantiated by developing an in-silico ionic competition model and recombinant expression of a homolog protein (Hm-LanM) from Hyphomicrobium methylovorum. Biochemical assays validate Hm-LanM preference for lighter Ln3+ ions (from lanthanum to gadolinium). This is comparable to established prototypes. Bioinformatics analyses further uncover additional H. methylovorum metabolic biomolecules in genomic proximity to Hm-LanM analogously dependent on Ln3+, including an outer membrane receptor that binds Ln3+-chelating siderophores. These combined observations underscore the remarkable strategy of Hyphomicrobium spp. for thriving in relatively Ln3+ enriched zones of metal-polluted environments.

Synergy effects of Methylomonas koyamae and Hyphomicrobium methylovorum under methanethiol stress.

Methanotrophs are able to metabolize volatile organic sulfur compounds (VOSCs), excrete organic carbon during CH4 oxidation, and influence microbial community structure and function of the ecosystem. In return, microbial community structure and environmental factors can affect the growth metabolism of methanotrophs. In this study, Methylomonas koyamae and Hyphomicrobium methylovorum were used for model organisms, and methanethiol (MT) was chosen for a typical VOSC to investigate the synergy effects under VOSC stress. The results showed that when Hyphomicrobium methylovorum was co-cultured with Methylomonas koyamae in the medium with CH4 used as the carbon source, the co-culture had better MT tolerance relative to Methylomonas koyamae and oxidized all CH4 within 120h, even at the initial MT concentration of 2000mgm-3. The optimal co-culture ratios of Methylomonas koyamae to Hyphomicrobium methylovorum were 4:1-12:1. Although MT could be converted spontaneously to dimethyl disulfide (DMDS), H2S, and CS2 in air, faster losses of MT, DMDS, H2S, and CS2 were observed in each strain mono-culture and the co-culture. Compared with Hyphomicrobium methylovorum, MT was degraded more quickly in the Methylomonas koyamae culture. During the co-culture, the CH4 oxidation process of Methylomonas koyamae could provide carbon and energy sources for the growth of Hyphomicrobium methylovorum, while Hyphomicrobium methylovorum oxidized MT to help Methylomonas koyamae detoxify. These findings are helpful to understand the synergy effects of Methylomonas koyamae and Hyphomicrobium methylovorum under MT stress and enrich the role of methanotrophs in the sulfur biogeochemical cycle. KEY POINTS: • The co-culture of Methylomonas and Hyphomicrobium has better tolerance to CH3SH. • Methylomonas can provide carbon sources for the growth of Hyphomicrobium. • The co-culture of Methylomonas and Hyphomicrobium enhances the removal of CH4 and CH3SH.

A novel serine hydroxymethyltransferase from Arthrobacter nicotianae: characterization and improving catalytic efficiency by rational design.

BackgroundSerine hydroxymethyltransferase (SHMT) is the key enzyme in L-serine enzymatic production, suggesting the importance of obtaining a SHMT with high activity.ResultsHere, a novel SHMT gene, glyA, was obtained through degenerate oligonucleotide-primed PCR and encoded a novel SHMT with 54.3% similarity to the known SHMT from Escherichia coli. The obtained protein AnSHMT showed the optimal activity at 40°C and pH 7.5, and was more stable in weakly alkali conditions (pH 6.5-8.5) than Hyphomicrobium methylovorum’s SHMT (pH 6.0-7.5), In order to improve the catalytic efficiency of the wild type, the site-directed mutagenesis based on sequences alignment and bioinformatics prediction, was used and the catalytic efficiency of the mutant I249L was found to be 2.78-fold higher than that of the wild-type, with the replacement of isoleucine by leucine at the 249 position.ConclusionsThis research provides useful information about the interesting site, and the application of DOP-PCR in cloning a novel glyA gene.

A serine hydroxymethyltransferase from marine bacterium Shewanella algae: Isolation, purification, characterization and l-serine production

Currently, l-serine is mainly produced by enzymatic conversion, in which serine hydroxymethyltransferase (SHMT) is the key enzyme, suggesting the importance of searching for a SHMT with high activity. Shewanella algae, a methanol-utilizing marine bacterium showing high SHMT activity, was selected based on screening bacterial strains and comparison of the activities of SHMTs. A glyA was isolated from the S. algae through thermal asymmetric interlaced PCR (TAIL-PCR) and it encoded a 417 amino acid polypeptide. The SaSHMT, encoded by the glyA, showed the optimal activity at 50°C and pH 7.0, and retained over 45% of its maximal activity after incubation at 40°C for 3h. The enzyme showed better stability under alkaline environment (pH 6.5–9.0) than Hyphomicrobium methylovorum GM2's SHMT (pH 6.0–7.5). The SaSHMT can produce 77.76mM of l-serine by enzymatic conversion, with the molecular conversion rate in catalyzing glycine to l-serine being 1.41-fold higher than that of Escherichia coli. Therefore, the SaSHMT has the potential for industrial applications due to its tolerance of alkaline environment and a relatively high enzymatic conversion rate.

Detection of a gem-diamine and a stable quinonoid intermediate in the reaction catalyzed by serine–glyoxylate aminotransferase from Hyphomicrobium methylovorum

BackgroundThe enzyme l-serine–glyoxylate aminotransferase (SGAT) from Hyphomicrobium methylovorum is a PLP-containing enzyme that catalyzes the conversion of l-serine and glyoxylate to hydroxypyruvate and glycine. The cloned enzyme expressed in Escherichia coli is isolated as a mixture of the E:PLP and E:PMP forms. The PLP form of the enzyme has a maximum absorbance at 413 nm. MethodsUv–visible spectra of SGAT were obtained using an HP-8453 diode array spectrophotometer in the absence and presence of substrates and substrate analogs. Pre-steady state kinetic studies were carried out using an OLIS rapid scanning spectrophotometer in the rapid scanning mode. ResultsIncubation of the enzyme with a saturating concentration D-serine leads to a shift in the 413 nm peak to 421 nm that is ascribed to the external aldimine. The reverse stereochemistry of D-serine does not allow for abstraction of the Cα proton by the ε-amine of the active site lysine residue leading to an abortive external aldimine intermediate. Pre-steady state studies pushing SGAT against d-serine leads to a rapid decrease in the 413 nm peak and an increase at ∼330 nm with an associated rate constant of 47 s−1 at pH 7.6. This is followed by a slower decrease (0.26 s−1) at 330 nm and an increase and shift of the 413 nm peak to 421 nm. The intermediate species that absorbs at ∼330 nm is attributed to the gem-diamine intermediate. The rate of the fast phase increases with pH and increase in rate is likely due to the deprotonation of an enzymatic group that accepts a proton from the α-amine of d-serine. In the presence of hydroxypyruvate and ammonia the enzyme spectra display an increase in absorbance at 521 nm that occurs on the order of minutes. The shape and position of the 521 nm species is consistent with a quinonoid intermediate. General significanceThe data suggest a non-enzymatic reaction between hydroxypyruvate and ammonia to form an imine which will be in equilibrium with the enamine. A mechanism is proposed by which the enamine reacts with the PLP form of SGAT to generate the stable highly conjugated quinonoid intermediate.

Cloning and analysis of methanol oxidation genes in the methylotroph Hyphomicrobium methylovorum GM2.

The gene encoding the alpha-subunit of methanol dehydrogenase (mxaF) and its flanking region was isolated from a methylotrophic bacterium, Hyphomicrobium methylovorum GM2. The deduced amino acid sequence of MxaF showed 80, 80, 74 and 66% identity with those of Methylobacterium extorquens AM1, M. organophilum XX, Paracoccus denitrificans and Methylophilus methylotrophus, respectively. The putative mxaF promoter sequence (-35 -AAAGACA-, -10 -TAGAA-) observed in other methylotrophs was not found in the region 5' of H. methylovorum GM2 mxaF. Downstream of mxaF, five open reading frames and one partial open reading frame were detected which had high identity with the genes mxaJ, mxaG, mxaI, mxaR, mxaS and mxaA. This indicated the existence of a mxaFJGIRSA gene cluster in H. methylovorum GM2, as previously observed in M. extorquens AM1.

Reaction of Serine-Glyoxylate Aminotransferase with the Alternative Substrate Ketomalonate Indicates Rate-Limiting Protonation of a Quinonoid Intermediate

Serine-glyoxylate aminotransferase (SGAT) from Hyphomicrobium methylovorum is a pyridoxal 5'-phosphate (PLP) enzyme that catalyzes the interconversion of L-serine and glyoxylate to hydroxypyruvate and glycine. The primary deuterium isotope effect using L-serine 2-D is one on (V/K)serine and V in the steady state. Pre-steady-state experiments also indicate that there is no primary deuterium isotope effect with L-serine 2-D. The results suggest there is no rate limitation by abstraction of the alpha proton of L-serine in the SGAT reaction. In the steady-state a solvent deuterium isotope effect of about 2 was measured on (V/K)L-serine and (V/K)ketomalonate and about 5.5 on V. Similar solvent isotope effects were observed in the pre-steady-state for the natural substrates and the alternative substrate ketomalonate. In the pre-steady-state, no reaction intermediates typical of PLP enzymes were observed with the substrates L-serine, glyoxylate, and hydroxypyruvate. The data suggest that breakdown and formation of the ketimine intermediate is the primary rate-limiting step with the natural substrates. In contrast, using the alternative substrate ketomalonate, pre-steady-state experiments display the transient formation of a 490 nm absorbing species typical of a quinonoid intermediate. The solvent isotope effect results also suggest that with ketomalonate as substrate protonation at C(alpha) is the slowest step in the SGAT reaction. This is the first report of a rate-limiting protonation of a quinonoid at C(alpha) of the external Schiff base in an aminotransferase reaction.

Molecular detection and isolation of facultatively methylotrophic bacteria, including Methylobacterium podarium sp. nov., from the human foot microflora.

This is the first study to demonstrate that diverse methylotrophic bacteria occur in the human foot microflora. Polymerase chain reaction (PCR) amplification of DNA from the soles and toe clefts of feet of five subjects indicated Methylobacterium strains to be present in all cases. Polymerase chain reaction amplification also showed the gene for the alpha-subunit of methanol dehydrogenase (mxaF) to be present in all samples. Two types of mxaF were recovered, one closest to that of Methylobacterium extorquens and the other most similar to that of Hyphomicrobium methylovorum. Numerous methylotrophic strains able to grow on methylamine were isolated with ease from the feet of nine volunteers. These were found by 16S rRNA analysis to be most closely related to Methylobacterium species, Brevibacterium casei, Pseudomonas strain NZ099 and P. migulae. Three strains from two subjects were of a novel species, Methylobacterium podarium sp. nov. This facultatively methylotrophic, obligately aerobic, pink-pigmented, non-motile rod grew with a wide range of multicarbon and one-carbon compounds including citrate, xylose, mono-, di-, and trimethylamine, dimethylsulphide, methanethiol, dimethylsulphoxide, dimethylsulphone and methanol.

Initial Velocity, Spectral, and pH Studies of the Serine-Glyoxylate Aminotransferase from Hyphomicrobiuim methylovorum

Serine-glyoxylate aminotransferase (SGAT) from Hyphomicrobium methylovorum is a pyridoxal 5′-phosphate (PLP) enzyme that catalyzes the interconversion of l-serine and glyoxylate to hydroxypyruvate and glycine. The initial velocity and dead-end inhibition patterns are consistent with a ping-pong kinetic mechanism. The Km values for l-serine and the alternative substrate ketomalonate are 0.28 ± 0.02 and 1.13 ± 0.08 mM, respectively. The spectrum of SGAT at pH 7.5 shows an absorbance maximum at 413 nm and a shoulder centered at 330 nm corresponding to the ketoenamine and enolimine forms of the protonated Schiff's base with the enolimine tautomer predominating. As determined by the changes in the enzyme absorbance spectrum the enzyme can be converted from the E–PLP to the E–pyridoxamine 5′-phosphate (E–PMP) form on addition of l-serine. The enzyme can subsequently be converted back to E–PLP by addition of glyoxylate or hydroxypyruvate. The enzyme displays a pH-dependent spectral change with a pK of about 8.2 which is ascribed to the ionization of an enzymatic residue that effects the tautomeric equilibrium between the ketoenamine and enolimine tautomers of the protonated aldimine. The V/KL-serine pH profile displays two pK values at pH 7.5 and 8.5 with limiting slopes of 1 and −1. The V/Kketomalonate pH profile displays one pK at 8.2 on the basic side with a limiting slope of 1 and the log KI oxalate pH profile shows one pK on the basic side at pH 7.2. The data suggest the active enzyme is the protonated aldimine and an enzymatic base with a pK of 7.5 accepts a proton from the α-amine of substrate to initiate catalysis.

Phylogenetic analysis and intrageneric structure of the genus Hyphomicrobium and the related genus Filomicrobium.

Almost complete 16S rDNA sequences from the type strains of seven species of the genus Hyphomicrobium and of Filomicrobium fusiforme have been determined. The Hyphomicrobium species from two phylogenetic clusters that are only moderately related to each other. While cluster I contains the type species Hyphomicrobium vulgare, Hyphomicrobium aestuarii, Hyphomicrobium hollandicum and Hyphomicrobium zavarzinii, cluster II comprises Hyphomicrobium facilis, Hyphomicrobium denitrificans and Hyphomicrobium methylovorum. Within the two species cluster, the species are highly related. Phylogenetically, Filomicrobium fusiforme clusters moderately with Hyphomicrobium species. The lack of distinguishing phenotypical properties presently excludes the possibility of describing cluster II as a new genus.

Characterization, gene cloning and expression of isocitrate lyase involved in the assimilation of one-carbon compounds in Hyphomicrobium methylovorum GM2.

Isocitrate lyase of an obligate methylotrophic bacterium, Hyphomicrobium methylovorum GM2, was purified to homogeneity and characterized. The enzyme is a homotetramer of identical 62-kDa subunits. After the enzyme had been incubated at temperatures up to 25 degrees C for 30 min, no loss of activity was observed. The enzyme was stable in the pH range of 7.5-9.0. Maximum activity was observed at pH 7.5 and around 45 degrees C. The Km value for Ds-isocitrate was 0.51 mM. The activity required Mg2+ and was inhibited by oxalate, succinate and glycolate. The gene encoding the isocitrate lyase and its flanking regions were isolated from H. methylovorum GM2. Nucleotide sequencing of recombinant plasmids revealed that the isocitrate lyase gene codes for a 540-amino-acid protein. The amino acid sequence of the enzyme is similar to those of the enzymes from Escherichia coli (40% identity) and cucumber (37% identity). The recombinant plasmid, which was constructed by ligation of the cloned gene and an expression vector, pKK223-3, was introduced into E. coli HB101. The transformed E. coli cells expressed isocitrate lyase, which was indistinguishable from the purified H. methylovorum GM2 isocitrate lyase on analysis by SDS/PAGE.

Cloning and analysis of methanol oxidation genes in the methylotroph Hyphomicrobium methylovorum GM2

The gene encoding the α-subunit of methanol dehydrogenase ( mxaF) and its flanking region was isolated from a methylotrophic bacterium, Hyphomicrobium methylovorum GM2. The deduced amino acid sequence of MxaF showed 80, 80, 74 and 66% identity with those of Methylobacterium extorquens AM1, M. organophilum XX, Paracoccus denitrificans and Methylophilus methylotrophus, respectively. The putative mxaF promoter sequence (−35 -AAAGACA-, −10 -TAGAA-) observed in other methylotrophs was not found in the region 5′ of H. methylovorum GM2 mxaF. Downstream of mxaF, five open reading frames and one partial open reading frame were detected which had high identity with the genes mxaJ, mxaG, mxaI, mxaR, mxaS and mxaA. This indicated the existence of a mxaFJGIRSA gene cluster in H. methylovorum GM2, as previously observed in M. extorquens AM1.

Cloning and expression of the gene for serine-glyoxylate aminotransferase from an obligate methylotroph Hyphomicrobium methylovorum GM2.

The gene encoding serine-glyoxylate aminotransferase, one of key enzymes for the assimilation of one-carbon compounds in methylotrophs, and its flanking regions were isolated from an obligate methylotrophic bacterium, Hyphomicrobium methylovorum GM2. Nucleotide sequencing of the recombinant plasmids revealed that the serine-glyoxylate aminotransferase gene encodes a 405-amino-acid protein with a calculated molecular mass of 43880 Da. The amino acid sequence of the enzyme showed identity to the sequences of serine-glyoxylate aminotransferase of Methylobacterium extorquens AM1 (57%), aspartate aminotransferase of Methanobacterium thermoformicicum (31%), human peroxisomal alanine-glyoxylate aminotransferase (27%), and serine-pyruvate aminotransferase of rat liver mitochondria (33%). The recombinant plasmid, which was constructed by ligation of the cloned gene and an expression vector pKK223-3, was introduced into Escherichia coli HB101. The recombinant enzyme was purified from transformed E. coli cells and analyzed by immunological and enzymological methods. The overexpressed enzyme was indistinguishable from the wild-type enzyme isolated from H. methylovorum GM2.

Immunological characterization of serine-glyoxylate aminotransferase and hydroxypyruvate reductase from a methylotrophic bacterium, Hyphomicrobium methylovorum GM2

Immunological characterization of serine-glyoxylate aminotransferase and hydroxypyruvate reductase, key enzymes for the assimilation of one-carbon compounds in methylotrophs, was performed using antibodies raised against these enzymes purified from Hyphomicrobium methylovorum GM2. Immunodiffusion studies indicated that serine-glyoxylate aminotransferase and hydroxypyruvate reductase of all seven Hyphomicrobium strains tested were immunochemically similar. In immunotitration experiments and Western blot analyses of both enzymes in the genera Hyphomicrobium and Methylobacterium, the serineglyoxylate aminotransferase of the genus Methylobacterium exhibited low similarity to that of the genus Hyphomicrobium. For hydroxypyruvate reductase, no immunological relationship was observed between the genera Hyphomicrobium and Methylobacterium, which was in agreement with the differences in primary structure and enzymological properties.

CoASAc-independent Phosphoenolpyruvate Carboxylase from an Obligate MethylotrophHyphomicrobium methylovorumGM2-Purification and Characterization-

CoASAc-independent phosphoenolpyruvate (PEP) carboxylase was purified from a serine-producing methylotroph, Hyphomicrobium methylovorum GM2, and compared with those from other methylotrophs. The activity of thie enzyme was not affected by CoASAc, a well-known activator for enzymes from various heterotrophs, NADH and ADP, effectors for PEP carboxylase from methylamine-grown Pseudomonas MA. This suggested that the H. methylovorum enzyme should be classified into the second group according to Utter and Kolenbrander (ref. 1) and different from that of Pseudomonas MA.

Crystal structure of a NAD-dependent d-glycerate dehydrogenase at 2·4 Å resolution

d-Glycerate dehydrogenase (GDH) catalyzes the NADH-linked reduction of hydroxypyruvate to d-glycerate. GDH is a member of a family of NAD-dependent dehydrogenases that is characterized by a specificity for the d-isomer of the hydroxyacid substrate. The crystal structure of the apoenzyme form of GDH from Hyphomicrobium methylovorum has been determined by the method of isomorphous replacement and refined at 2·4 Å resolution using a restrained least-squares method. The crystallographic R-factor is 19·4% for all 24,553 measured reflections between 10·0 and 2·4 Å resolution. The GDH molecule is a symmetrical dimer composed of subunits of molecular mass 38,000, and shares significant structural homology with another NAD-dependent enzyme, formate dehydrogenases The GDH subunit consists of two structurally similar domains that are approximately related to each other by 2-fold symmetry. The domains are separated by a deep cleft that forms the putative NAD and substrate binding sites. One of the domains has been identified as the NAD-binding domain based on its close structural similarity to the NAD-binding domains of other NAD-dependent dehydrogenases. The topology of the second domain is different from that found in the various catalytic domains of other dehydrogenases. A model of a ternary complex of GDH has been built in which putative catalytic residues are identified based on sequence homology between the d-isomer specific dehydrogenases. A structural comparison between GDH and l-lactate dehydrogenase indicates a convergence of active site residues and geometries for these two enzymes. The reactionscatalyzed are chemically equivalent but of opposing stereospecificity. A hypothesis is presented to explain how the two enzymes may exploit the same coenzyme stereochemistry and a similar spatial arrangement of catalytic residues to carry out reactions that proceed to opposite enantiomers.

L-Serine production by a methylotroph and its related enzymes

The production process of L-serine from methanol and glycine has been developed using a methylotroph with the serine pathway. Consecutive reactions of two enzymes, methanol dehydrogenase (MDH) and serine hydroxymethyltransferase (SHMT) are involved in the production. We screened a high producer, Hyphomicrobium methylovorum, which is an obligate methylotroph. With resting cells of the bacterium, 24 mg/ml of L-serine was produced from 100 mg/ml of glycine and 48 mg/ml of methanol in 3 days under optimal conditions. Next, a glycine-resistant mutant GM2 showed improved serine production (32-34 mg/ml). The mutant GM2 was found to have elevated activities of MDH and SHMT. Since there has so far been little report on the systematic characterization of enzymes of the serine pathway in methylotrophs, not only the above two enzymes but also the other three enzymes in H. methylovorum were purified and characterized: MDH, SHMT and hydroxypyruvate reductase (HPR) were crystallized; serine-glyoxylate aminotransferase (SGAT) and glycerate kinase (GK) were purified to homogeneity. As a result, all these enzymes were found to be stable against preservation and to exist abundantly in the bacterium. The gene of SHMT was cloned and its deduced amino acid sequence had homology to those of Escherichia coli (55%) and rabbit liver (44%), whereas the enzyme of the bacterium was immunochemically distinguishable from those of microorganisms other than Hyphomicrobium strains and mammalian livers.

Enzymatic Assay for L-Serine and Glyoxylate Involving the Enzymes in the Serine Pathway of a Methylotroph

An easy, rapid, and accurate enzymatic assay method for L-serine was established involving two enzymes, serine-glyoxylate aminotransferase (SGAT, EC 2.6.1.45) and hydroxypyruvate reductase (HPR, EC 1.1.1.81), in the serine pathway of the methylotrophic bacterium, Hyphomicrobium methylovorum (IFO 14180), from which they were purified. This method consists of two reaction steps: the first is the nearly irreversible transamination of L-serine and glyoxylate by SGAT, and the second is the HPR reaction involving NADH, which comprises the absolutely irreversible reduction of hydroxypyruvate derived from L-serine by SGAT. The amounts of L-serine were determined spectrophotometrically as the decrease in the amount of NADH. When the values determined with the present enzymatic method were compared with those obtained with an amino acid analyzer, the correlation coefficient was found to be 0.9963. This method can also be applied to the assaying of glyoxylate.

Purification and characterization of glycerate kinase from a serine-producing methylotroph, Hyphomicrobium methylovorum GM2.

The glycerate kinase of a serine-producing methylotroph, Hyphomicrobium methylovorum GM2, was purified to complete homogeneity and characterized, the first time for an enzyme from a methylotroph. The enzyme was a monomer with a molecular mass about 41-52 kDa. The enzyme was stable against heating at 35 degrees C for 30 min at pH values over 6-10. Maximum activity was observed at pH 8.0 and around 50 degrees C. The Km values for D-glycerate and ATP were 0.13 mM and 0.13 mM, respectively. The enzyme showed high specificity for D-glycerate, and was activated by potassium and ammonium ions. The reaction product of the enzyme was identified as 2-phosphoglycerate.

Crystallization and preliminary diffraction studies of hydroxypyruvate reductase ( d-glycerate dehydrogenase) from Hyphomicrobium methylovorum

Two crystal forms of hydroxypyruvate reductase ( d-glycerate dehydrogenase) from the methylotrophic bacterium Hyphomicrobium methylovorum have been grown from ammonium sulphate solutions. One crystal form is triclinic, with unit cell parameters a = 60.4 A ̊ , b = 60.5 A ̊ , c = 66.3 A ̊ , α = 102.3 °, β = 113.7 ° and γ = 102.7 ° , suggesting that a dimer (monomer M r 38,000) occupies the unit cell. This crystal form diffracts to beyond 2.4 Å resolution and is suitable for crystallographic structure analysis.