Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria. III) The primary structure of thermophilic lactate dehydrogenase from Bacillus stearothermophilus. Hydroxylamine-, o-iodosobenzoic acid- and tryptic-fragments. The complete amino-acid sequence

Based on the partial sequence of the cyanogen bromide fragments [Tratschin, J.D., Wirz, B., Frank, G. and Zuber, H. (1983) Hoppe-Seyler's Z. Physiol. Chem. 364, 879-892], the amino-acid sequence of thermophilic lactate dehydrogenase from B. stearothermophilus was completed by the preparation and sequencing (sequenator, carboxypeptidase A and Y) of further overlapping fragments. Suitable peptide fragments were obtained by lactate dehydrogenase cleavage with hydroxylamine, o-iodosobenzoic acid and trypsin. The polypeptide chain of thermophilic lactate dehydrogenase from B. stearothermophilus consists of 317 amino-acid residues. While sequence homology with mesophilic lactate dehydrogenase of higher organisms reaches 35%, it is substantially higher with this mesophilic enzyme of bacillae (greater than 60%, B. megaterium, B. subtilis). The secondary structure elements and amino-acid residues of the active site of thermophilic lactate dehydrogenase deducted from primary structure data were compared with those from the mesophilic enzyme, the same was done for the internal sequence homology at the nucleotide-binding units. A comparative structure analysis (matrix system) based on the primary structure data of thermophilic enzyme should provide insight into the characteristic structure differences between thermophilic and mesophilic lactate dehydrogenase.     

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, IV. The primary structure of the mesophilic lactate dehydrogenase from Bacillus subtilis

The complete amino-acid sequence of lactate dehydrogenase from the mesophilic Bacillus subtilis (B. X1) was determined. Approximately 70% of the sequence was obtained by sequence analysis of intact protein (N-terminal sequence) and of four CNBr fragments (CNBr3, CNBr4, CNBr5 and CNBr6). Sequences overlapping the CNBr fragments were determined from polypeptide fragments obtained by cleavage using o-iodosobenzoic acid (cleavage at Trp) or clostripain (cleavage at Arg). The C-terminal amino-acid residue (Asn) was detected by carboxypeptidase Y-degradation. Lactate dehydrogenase from B. subtilis shows a 69% sequence homology to that from the thermophilic strain B. stearothermophilus, and a 34% sequence homology to those from higher organism. The homology of these enzymes is particularly high at the active site regions (the coenzyme and substrate binding sites). The relatively high sequence conservation of the lactate dehydrogenases from B. subtilis and B. stearothermophilus (and from other bacilli) allows a structural comparison of this temperature variants.     

Structure and function of L-lactate dehydrogenases from thermophilic, mesophilic and psychrophilic bacteria, IX. Identification, isolation and nucleotide sequence of two L-lactate dehydrogenase genes of the psychrophilic bacterium Bacillus psychrosaccharolyticus

Two genes encoding for L-lactate dehydrogenase (LDH) from the psychrophilic bacterium Bacillus psychrosaccharolyticus (DSM 6) were cloned and their nucleotide sequence determined using a pEMBL vector and gene hybridization probes. The deduced amino-acid sequence of the gene from clone pLDH(X), which is located on a 5.87-kb HindIII-fragment, shows an identity of 86% as compared with the sequence of the wildtype LDH(P) from B. psychrosaccharolyticus and consists of 319 amino acids. Clone pLDH(P) contained a gene on a 4-kb HindIII-EcoRI fragment, of which the amino-acid sequence is identical with the enzyme isolated from B. psychrosaccharolyticus. The nucleotide sequences of LDH(P) and LDH(X) show 77% identity. Both genes are expressed in E. coli and the proteins could be isolated as shown by enzyme activity tests and determination of the N-terminal amino-acid sequence. However no expression of LDH(X) could be detected in B. psychrosaccharolyticus itself under the conditions chosen for oxygen induction of LDH. The function of the additional, non-expressed enzyme is not known.     

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria. I) Isolation and characterization of lactate dehydrogenases from thermophilic and mesophilic bacilli.

Lactate dehydrogenases from thermophilic bacilli (Bacillus stearothermophilus, Bacillus caldotenax) and from mesophilic bacilli (Bacillus X1, Bacillus subtilis) have been isolated by a two-step purification procedure. Only one type (LDH-P4) composed of four identical subunits (Mr 34 000 or 36 000) was found in each bacillus. The tetrameric enzymes were characterized with respect to thermostability, pH and temperature dependence of the pyruvate reduction and the L-lactate oxidation, substrate specificity, saturation kinetics (Km values of pyruvate, lactate, NAD, NADH), pyruvate and oxamate inhibition, and activation by fructose bisphosphate. The thermophilic and mesophilic enzymes differ characteristically in these parameters. Preliminary structural data (amino acid composition, comparative N-terminal sequence analysis) show the expected close phylogenetic relationship (high degree of sequence homology), but also typical differences between thermophilic and mesophilic dehydrogenases, a suitable basis for further comparative studies.     

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, VI. Nucleotide sequences of lactate dehydrogenase genes from the thermophilic bacteria Bacillus stearothermophilus, B. caldolyticus and B. caldotenax

Based on the previously determined amino-acid sequence of lactate dehydrogenase from B. stearothermophilus, an oligonucleotide probe was synthesized and used to clone the structural genes for lactate dehydrogenase from B. stearothermophilus, B. caldolyticus and B. caldotenax. The nucleotide sequences of the entire LDH genes from these three thermophilic bacilli were determined by the method of Maxam and Gilbert. The nucleotide sequence of the LDH gene from B. stearothermophilus is exactly identical to the one published recently; it agrees with the experimentally determined amino-acid sequence except at three positions. The amino-acid homologies among these thermophilic enzymes are 90% or more. The LDH genes are efficiently expressed in E. coli.     

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, XI. Engineering thermostability and activity of lactate dehydrogenases from bacilli

An extensive comparative structural analysis of lactate dehydrogenase (LDH) sequences from thermophilic, mesophilic and psychrophilic bacilli revealed characteristic primary structural differences. These specific amino-acid substitutions were found in the entire LDH molecule. However, in certain regions of the LDH an accumulation of these exchanges could be detected. These regions seem to be particularly important for the temperature adaptation of the enzyme. The influence of one of such regions at the N-terminus on stability and activity of LDHs was analysed by the construction of hybrid mutants between LDH sequences from thermophilic, mesophilic and psychrophilic bacilli and also by site-directed mutagenesis experiments at five different positions. The substitutions of Thr-29 or Ser-39 to Ala residues in the LDH from the mesophilic B. megaterium increased the thermostability of the enzyme drastically (15 degrees C). An increase of 20 degrees C could be observed when both amino-acid substitutions were introduced. These amino-acid substitutions resulted in an increase of Km for pyruvate and led to a three-fold reduction of the activity (kcat/Km) at 40 degrees C compared with the wild type enzyme. The influence of these amino-acid substitutions was also investigated in the LDHs from thermophilic and psychrophilic bacilli. The high heat resistance of the LDH from the thermophilic B. stearothermophilus was not altered by the Ala to Thr and Ser substitutions at positions 29 and 39, respectively. This indicates a cooperatively stabilized conformation of this LDH. However, in this mutant of the B. stearothermophilus LDH the activity (kcat/Km) was increased two-fold.     

嗜热菌的耐热L—乳酸脱氢酶的研究

About 200 strains of extreme thermophilic bacteria were isolated from hot springs in Guandong province. A strain, HG25, was found to produce thermostable intracellular L-lactate dehydrogenase (EC. 1.1.1.27). It has the characteristic of Thermus sp. The cells were gram-negative, non-sporulating, nonmotile, aerobic rods containing yellow pigment. The optimum temperature for growth was between 65 degrees C to 75 degrees C, the maximum 85 degrees C, and minimum 40 degrees C. The generation time at the optimum was about 80 min. Starch was not hydrolyzed. Acid was not produced from glucose. The G+C content in DNA was 62-65 mol% (Tm). As the properties of strain HG25 is similar to those of Thermus aquaticus and T. thermophilus HB 8 belonging to the genus Thermus. The thermostable L-lactate dehydrogenase was partially purified by ammonium sulfate fractionation and DEAE-cellulose column chromatography. For pyruvate reduction, the optimum temperature of the enzyme was 60 degrees C and pH 8.0. After incubation in 0.1 mol/L phosphate buffer pH 7.4 at 70 degrees C for 10 min, the enzyme retained about 85% of its original activity. The half-live time (t1/2) at 85 degrees C was 10 min.     

Isocitrate dehydrogenase from thermophilic and mesophilic bacteria. Isolation and some characteristics

1. Simple methods incorporating the principle of selective enzyme elution from a triazinyl dye adsorbent with a mixture of NADP+ and isocitrate are described for isolating NADP+-linked isocitrate dehydrogenase in pure state from several mesophilic and thermophilic bacteria. 2. Several characteristics of the isocitrate dehydrogenases have been examined, viz. molecular size, amino acid composition including the content of sulphydryl groups, thermostability and structural homology by the criterion of immunological cross-section.     

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, X. Analysis of structural elements responsible for the differences in thermostability and activation by fructose 1,6-bisphosphate in the lactate dehydrogenases from B. stearothermophilus and B. caldolyticus by protein engineering

The amino-acid sequences of the lactate dehydrogenases (LDH) from B. stearothermophilus and B. caldolyticus differ at only 10 positions. The properties of these enzymes however show substantial differences. The LDH from B. stearothermophilus is activated by Fru-P2 and has a higher thermostability (10 degrees C) than the enzyme from B. caldolyticus which cannot be activated by Fru-P2. To correlate these functional differences to the structural properties, we have constructed a set of hybrid- and point-mutants of the two LDHs. The amino acids at positions 207, 209B, and 209C could be identified to confer the property of activation by Fru-P2 to the enzymes. This part of the enzyme is to a large extent also responsible for the different thermostabilities of these two proteins.     

Simple efficient methods for the isolation of malate dehydrogenase from thermophilic and mesophilic bacteria.

Malate dehydrogenase from a number of bacteria drawn from several genera and representing the mesophilic, moderately thermophilic and extremely thermophilic classes was isolated by procedures which involve only a small number of steps (in most cases only two), of which the key one is affinity chromatography on 5'-AMP--Sepharose and/or on NAD+--hexane--agarose. Electrophoretic analysis of the native enzymes in polyacrylamide gel and of the denaturated enzymes in sodium dodecyl sulphate/polyacrylamide gel revealed no significant protein impurity in the purified preparations. The yields ranged from about 40% to over 80%. The malate dehydrogenases from the extreme thermophiles and from some of the moderate thermophiles are appreciably less efficient catalytically than their mesophilic homologues.     

Phosphofructokinase: complete amino-acid sequence of the enzyme from Bacillus stearothermophilus

The entire amino acid sequence of the protein subunit of phosphofructokinase from Bacillus stearothermophilus has been established mainly by sequence analysis of cyanogen bromide fragments and of peptides derived from these fragments by further digestion with proteolytic enzymes. Overlaps of the cyanogen bromide fragments as well as peptide sequences necessary to complement and to confirm tentative assignments within the larger peptide fragments were obtained from the sequences of selected peptides isolated from tryptic and chymotryptic digests of the intact S-[14C]-carboxymethylated protein. Sequence information was also provided by automated sequence analysis of the intact protein subunit and of some of the larger peptide fragments. The sequence is as follows: (See Text).     

Purification and properties of histidinol dehydrogenases from psychrophilic, mesophilic and thermophilic bacilli.

As a first step in elucidating one molecular mechanism of adaptation to life at extreme temperatures, we purified and characterized the enzyme histidinol dehydrogenase (EC 1.1.1.23) from a number of bacilli whose growth temperatures range from 5 degrees t to 90 degrees C. The enzymes were purified by (NH4)2SO4 precipitation, ion-exchange chromatography on Sephadex, affinity chromatography on histamine- or histidine-Sepharose and preparative gradient gel electrophoresis. All had similar mol.wts. (29200), sedimentation coefficients (S20,w 2.56S), affinities for histidinol and NAD+ (Km = 48 micron and 0.2 mM respectively) and all had pH optima at 9.6. Marked differences were observed in stability with respect to temperature and the temperature at which the initial velocity for histidinol dehydrogenation was optimal. These optima range from 25 degrees C for the enzyme from the psychrophilic species through to 41 degrees C for the mesophiles to 85-92 degrees C for the extreme thermophiles. It is concluded that the ability of the enzymes to operate at their various optimum temperatures is an intrinsic property of their amino acid sequences.     

Conservation of antigenic determinants in leucine dehydrogenase from thermophilic and mesophilic Bacillus strains

Abstract Antibodies against the purified octameric l -leucine dehydrogenase (LeuDH) from the mesophilic Bacillus cereus have been used to screen 16 thermophilic Bacillus strains for LeuDH. 4 of these strains, Bacillus sphaericus 461 and Bacillus sp. 405, 406, and 411, showed a particularly strong cross reaction of the partial identity type when examined by Ouchterlony double diffusion assay, thus indicating that they were immunologically related to the B. cereus enzyme. The LeuDH from the thermophilic strains were very stable and highly active at elevated temperatures, and gave a downward bend at about 55°C in the Arrhenius plot. The pH optimum for l -leucine deamination was around pH 11 for all strains examined.     

The thermostability of DNA-binding protein HU from mesophilic, thermophilic, and extreme thermophilic bacteria

Based on primary structure comparison between four highly homologous DNA-binding proteins (HUs) displaying differential thermostability, we have employed in vitro site-directed mutagenesis to decipher their thermostability mechanism at the molecular level. The contribution of the 11 amino acids that differ between the thermophilic HUBst from Bacillus stearothermophilus (Tm = 61.6 degrees C) and the mesophilic HUBsu from Bacillus subtilis (Tm = 39.7 degrees C) was evaluated by replacing these amino acids in HUBst with their mesophilic counterparts. Among 11 amino acids, three residues, Gly-15, Glu-34, and Val-42, which are highly conserved in the thermophilic HUs, have been found to be responsible for the thermostability of HUBst. These amino acids in combination (HUBst-G15E/E34D/V42I) reduce the thermostability of the protein (Tm = 45.1 degrees C) at the level of its mesophilic homologue HUBsu. By replacing these amino acids in HUBsu with their thermophilic counterparts, the HUBsu-E15G/D34E/142V mutant was generated with thermostability (Tm = 57.8 degrees C) at the level of thermophilic HUBst. Employing the same strategy, we generated several mutants in the extremely thermophilic HUTmar from Thermotoga maritima (Tm = 80.5 degrees C), and obtained data consistent with the previous results. The triplet mutant HUTmar-G15E/E34D/V421 (Tm = 35.9 degrees C) converted the extremely thermophilic protein HUTmar to mesophilic. The various forms of HU proteins were overproduced in Escherichia coli, highly purified, and the thermostability of the mutants confirmed by circular dichroism spectroscopy. The results presented here were elucidated on the basis of the X-ray structure of HUBst and HUTmar (our unpublished results), and their mechanism was proposed at the molecular level. The results clearly show that three individual local interactions located at the helix-turn-helix part of the protein are responsible for the stability of HU proteins by acting cooperatively in a common mechanism for thermostability.     

Structure and function of SirC from Bacillus megaterium: a metal-binding precorrin-2 dehydrogenase

In Bacillus megaterium, the synthesis of vitamin B(12) (cobalamin) and sirohaem diverges at sirohydrochlorin along the branched modified tetrapyrrole biosynthetic pathway. This key intermediate is made by the action of SirC, a precorrin-2 dehydrogenase that requires NAD(+) as a cofactor. The structure of SirC has now been solved by X-ray crystallography to 2.8 A (1 A = 0.1 nm) resolution. The protein is shown to consist of three domains and has a similar topology to the multifunctional sirohaem synthases Met8p and the N-terminal region of CysG, both of which catalyse not only the dehydrogenation of precorrin-2 but also the ferrochelation of sirohydrochlorin to give sirohaem. Guided by the structure, in the present study a number of active-site residues within SirC were investigated by site-directed mutagenesis. No active-site general base was identified, although surprisingly some of the resulting protein variants were found to have significantly enhanced catalytic activity. Unexpectedly, SirC was found to bind metal ions such as cobalt and copper, and to bind them in an identical fashion with that observed in Met8p. It is suggested that SirC may have evolved from a Met8p-like protein by loss of its chelatase activity. It is proposed that the ability of SirC to act as a single monofunctional enzyme, in conjunction with an independent chelatase, may provide greater control over the intermediate at this branchpoint in the synthesis of sirohaem and cobalamin.