Kinetic mechanism of the serine acetyltransferase from Haemophilus influenzae

The kinetic mechanism of serine acetyltransferase from Haemophilus influenzae was studied in both reaction directions. The enzyme catalyzes the conversion of acetyl CoA and L-serine to O-acetyl-L-serine (OAS) and coenzyme A (CoASH). In the direction of L-serine acetylation, an equilibrium ordered mechanism is assigned at pH 6.5. The initial velocity pattern in the absence of added inhibitors is best described by a series of lines converging on the ordinate when L-serine is varied at different fixed levels of acetyl CoA. The initial velocity pattern at pH 7.5 is also intersecting, but the lines are nearly parallel. Product inhibition by OAS is noncompetitive against acetyl CoA, while it is uncompetitive against L-serine. Product inhibition by L-serine in the reverse reaction direction is noncompetitive with respect to both OAS and CoASH. Glycine and S-methyl-L-cysteine (SMC) were used as dead-end analogs of L-serine and OAS, respectively. Glycine is competitive versus L-serine and uncompetitive versus acetyl CoA, while SMC is competitive against OAS and uncompetitive against CoASH. Desulfo-CoA was used as a dead-end analog of both acetyl CoA and CoASH, and is competitive versus both substrates in the direction of L-serine acetylation; while it is competitive against CoASH and noncompetitive against OAS in the direction of CoASH acetylation. All of the above kinetic parameters are consistent with those predicted for an ordered mechanism at pH 6.5 with the exception of the uncompetitive inhibition by OAS vs. serine. The latter inhibition pattern suggests combination of OAS with the central E:acetyl CoA:serine complex. Cysteine is known to regulate its own biosynthesis at the level of SAT. As a dead-end inhibitor, L-cysteine is competitive against both substrates in both reaction directions. These results are discussed in terms of the mechanism of regulation.     

Chemical mechanism of Haemophilus influenzae diaminopimelate epimerase

Seven unique enzymatic steps lead to the biosynthesis of L-lysine from L-aspartate semialdehyde and pyruvate in bacteria. The immediate precursor to L-lysine is D,L-diaminopimelate, a diamino acid which is incorporated into the pentapeptide of the Gram-negative peptidoglycan moiety. D,L-Diaminopimelate is generated from the corresponding L,L-isomer by the dapF-encoded epimerase. Diaminopimelate epimerase is a representative of the pyridoxal phosphate-independent amino acid racemases, for which substantial evidence exists supporting the role of two cysteine residues as the catalytic acid and base. The pH dependencies of the maximum velocities in the L,L --> D,L and D,L --> L,L direction depend on a single catalytic group exhibiting pK values of 7.0 and 6.1, respectively, which must be unprotonated for activity. The pH dependencies of the V/K values in both directions depend on the ionization of two groups, one exhibiting a pK value of 6.7 which must be unprotonated and one exhibiting a pK value of 8.5 which must be protonated. Primary kinetic isotope effects on V and V/K are unequal, with D(V/K) being larger than DV in both the forward and reverse directions. Solvent kinetic isotope effects in both directions are inverse on V/K, but normal on V. Both of these isotopic observations support a model in which proton isomerization after catalysis and substrate dissociation is kinetically significant. A single solvent "overshoot" is observed when L, L-diaminopimelate is incubated with enzyme in D2O; however, an unprecedented double overshoot is observed when D,L-diaminopimelate is incubated with enzyme in D2O. A model has been developed which allows these two overshoots to be simulated. A chemical mechanism is proposed invoking the function of two cysteine residues, Cys73 and Cys217, observed in the recently determined three-dimensional structure of this enzyme [Cirilli, M., et al. (1998) Biochemistry 37, 16452-16458], as the acid and base in the mechanism.     

Roles of histidines 154 and 189 and aspartate 139 in the active site of serine acetyltransferase from Haemophilus influenzae

A crystal structure of serine acetyltransferase (SAT) with cysteine bound in the serine subsite of the active site shows that both H154 and H189 are within hydrogen-bonding distance to the cysteine thiol [Olsen, L. R., Huang, B., Vetting, M. W., and Roderick, S. L. (2004) Biochemistry 43, 6013 -6019]. In addition, H154 is in an apparent dyad linkage with D139. The structure suggests that H154 is the most likely catalytic general base and that H189 and D139 may also play important roles during the catalytic reaction. Site-directed mutagenesis was performed to mutate each of these three residues to Asn, one at a time. The V1/Et value of all of the single mutant enzymes decreased, with the largest decrease (approximately 1240-fold) exhibited by the H154N mutant enzyme. Mutation of both histidines, H154N/H189N, gave a V1/Et approximately 23700-fold lower than that of the wild-type enzyme. An increase in K Ser was observed for the H189N, D139N, and H154N/H189N mutant enzymes, while the H154N mutant enzyme gave an 8-fold decrease in K Ser. For all three single mutant enzymes, V1/Et and V1/K Ser Et decrease at low pH and give a pKa of about 7, while the V1/Et of the double mutant enzyme was pH independent. The solvent deuterium kinetic isotope effects on V 1 and V1/K Ser decreased compared to wild type for the H154N mutant enzyme and increased for the H189N mutant enzyme but was about the same as that of wild type for D139N and H154N/H189N. Data suggest that H154, H189, and D139 play different catalytic roles for SAT. H154 likely serves as a general base, accepting a proton from the beta-hydroxyl of serine as the tetrahedral intermediate is formed upon nucleophilic attack on the thioester carbonyl of acetyl-CoA. However, activity is not completely lost upon elimination of H154, and thus, H189 may be able to serve as a backup general base at a lower efficiency compared to H154; it also aids in binding and orienting the serine substrate. Aspartate 139, in dyad linkage with H154, likely facilitates catalysis by increasing the basicity of H154.     

The structure and mechanism of serine acetyltransferase from Escherichia coli

Serine acetyltransferase (SAT) catalyzes the first step of cysteine synthesis in microorganisms and higher plants. Here we present the 2.2 A crystal structure of SAT from Escherichia coli, which is a dimer of trimers, in complex with cysteine. The SAT monomer consists of an amino-terminal alpha-helical domain and a carboxyl-terminal left-handed beta-helix. We identify His(158) and Asp(143) as essential residues that form a catalytic triad with the substrate for acetyl transfer. This structure shows the mechanism by which cysteine inhibits SAT activity and thus controls its own synthesis. Cysteine is found to bind at the serine substrate site and not the acetyl-CoA site that had been reported previously. On the basis of the geometry around the cysteine binding site, we are able to suggest a mechanism for the O-acetylation of serine by SAT. We also compare the structure of SAT with other left-handed beta-helical structures.     

Chemical synthesis of Haemophilus influenzae glycopeptide conjugates

A simple procedure for conjugating synthetic fragments of the capsular polysaccharide of Haemophilus influenzae type b, poly-3--D-ribose-(1, 1)-D-ribitol-5-phosphate (sPRP) to linear peptides is described. The procedure consists of (i) reacting the amino group of amino-heptyl sPRP with m-maleimidobenzoyl-N-hydroxysuccinimide (MBS) in phosphate buffer, pH 7.5; (ii) selectively coupling the MBS-modified sPRP to the sulfhydryl group of the cysteine residue of peptides containing functional T-helper cell epitope(s). The glycopeptide conjugates were purified by gel filtration chromatography, biochemically characterized, and elicited protective level of anti-PRP antibody responses in rabbits. Abbreviations: PRP, poly-3--D-ribose-(1, 1)-D-ribitol-5-phosphate; sPRP, synthetic oligo-3--D-ribose-(1, 1)-D-ribitol-5-phosphate; Hib, Haemophilus influenzae type b; MBS, m-maleimidobenzoyl-N-hydroxysuccinimide; PEG, polyethylene glycol monomethyl ether; CRM 197, a non-toxic diphtheria toxin mutant; TT, tetanus toxoid; DT, diphtheria toxoid; OMP, outer membrane protein; RP-HPLC reverse phase high pressure liquid chromatograph     

Random-order ternary complex reaction mechanism of serine acetyltransferase from Escherichia coli

Although serine acetyltransferase (SAT) from Escherichia coli is homologous with a number of bacterial enzymes that catalyze O-acetyl transfer by a sequential (ternary complex) mechanism, it has been suggested, from experiments with the nearly identical enzyme from Salmonella typhimurium, that the reaction could proceed via an acetyl-enzyme intermediate. To resolve the matter, the E. coli gene for SAT was overexpressed and the enzyme purified 13-fold to homogeneity. The results of a steady-state kinetic analysis of the forward reaction are diagnostic for a ternary complex mechanism, and the response of SAT to dead-end inhibitors indicates a random order for the addition of substrates. The linearity of primary double-reciprocal plots, in the presence and absence of dead-end inhibitors, argues that interconversion of ternary complexes is not significantly faster than kcat, whereas substrate inhibition by serine suggests that breakdown of the SAT.CoA binary complex is rate-determining. The results of equilibrium isotope exchange experiments, for both half-reactions, rule out a "ping-pong" mechanism involving an acetyl-enzyme intermediate, and a pre-steady-state kinetic analysis of the turnover of AcCoA supports such a conclusion. Kinetic data for the reverse reaction (acetylation of CoA by O-acetylserine) are also consistent with a steady-state random-order mechanism, wherein both the breakdown of the SAT*serine complex and the interconversion of ternary complexes are partially rate-determining.     

Methylase activities from Haemophilus influenzae that protect Haemophilus parainfluenzae transforming deoxyribonucleic acid from inactivation by Haemophilus influenzae endonuclease R.

Specific methylases that have the properties of deoxyribonucleic acid (DNA) modification enzymes have been isolated from Haemophilus influenzae strain Rd. Two activities ((Methylase IIa and methylase III) were found to protect transforming DNA of H. parainfluenzae from the action of H. influenzae restriction enzymes. To determine the specificty of the protection, a procedure based on biological activity was developed for the separation and purification of the restriction endonucleases from H. influenzae strain Rd. Two endonuclease R activities presumably corresponding to Hind II and Hind III (P. H. Roy and H. O. Smith, 1973; H. O. Smith and K. W. Wilcox, 1970) were characterized by differences in their chromatographic properties, ability to attack T7 DNA, and inactivation of the transforming activity of different markers of H. parainfluenzae DNA. One endonuclease R enzyme (Hind II) attacked T7 DNA and was found to inactivate the dalacin resistance marker (smaller than 0.01% activity remaining) with only a slight effect on the streptomycin resistance marker (83% activity remaining). Methylase IIa treatment protected 40% of the dalacin resistance marker of H. parainfluenzae DNA from inactivation by Hind II. The other restriction activity (Hind III) was inert towards T7 DNA and inactivated the streptomycin resistance marker of H. parainfluenzae DNA (smaller than 0.01% activity remaining) without any effect on the dalacin resistance marker. The methylation of H. parainfluenzae DNA accomplished by methylase III protected 60% of the transforming activity of the streptomycin resistance marker of H. parainfluenzae DNA from the action of Hind III.     

In vitro validation of acetyltransferase activity of GlmU as an antibacterial target in Haemophilus influenzae

GlmU is a bifunctional enzyme that is essential for bacterial growth, converting D-glucosamine 1-phosphate into UDP-GlcNAc via acetylation and subsequent uridyl transfer. A biochemical screen of AstraZeneca's compound library using GlmU of Escherichia coli identified novel sulfonamide inhibitors of the acetyltransferase reaction. Steady-state kinetics, ligand-observe NMR, isothermal titration calorimetry, and x-ray crystallography showed that the inhibitors were competitive with acetyl-CoA substrate. Iterative chemistry efforts improved biochemical potency against gram-negative isozymes 300-fold and afforded antimicrobial activity against a strain of Haemophilus influenzae lacking its major efflux pump. Inhibition of precursor incorporation into bacterial macromolecules was consistent with the antimicrobial activity being caused by disruption of peptidoglycan and fatty acid biosyntheses. Isolation and characterization of two different resistant mutant strains identified the GlmU acetyltransferase domain as the molecular target. These data, along with x-ray co-crystal structures, confirmed the binding mode of the inhibitors and explained their relative lack of potency against gram-positive GlmU isozymes. This is the first example of antimicrobial compounds mediating their growth inhibitory effects specifically via GlmU.     

Haemophilus influenzae biochemotyping

Biochemotyping was performed in 129 strains of Haemophilus influenzae. 95% of strains could be assigned to one of the five biochemotypes proposed by Kilian. Most of the serotype B strains isolated in meningitis belonged to biochemotype I. The biochemical differentiation of Haemophilus influenzae is regarded as a reliable technique deserving further application.     

流感嗜血杆菌的分型研究

目的分析同济医院分离的流感嗜血杆菌的生物学分型及荚膜基因分型,了解本地区分离的流感嗜血杆菌的主要流行株。方法2012年1月1日至2012年12月31日从华中科技大学同济医学院附属同济医院分离流感嗜血杆菌100株。根据脲酶、吲哚和鸟氨酸脱羧酶试验对流感嗜血杆菌进行传统的生物学分型,分为Ⅰ～Ⅷ八个生物型。回顾患者病史资料,分析生物学分型和流感嗜血杆菌所引起的疾病之间的关系。用流感嗜血杆菌荚膜编码基因（bexA）和a—f型特异性荚膜基因设计引物,采用PCR技术对流感嗜血杆菌进行荚膜基因检测。通过生物学分型和荚膜基因分型结果的比对,探讨两者之间的关联。结果分离的100株流感嗜血杆菌生物学分型结果如下：Ⅲ型6株,Ⅳ型28株,Ⅴ型1株,Ⅵ型54株,Ⅶ型11株。未分离到Ⅰ型、Ⅱ型和Ⅷ型。分析患者的临床诊断,发现主要流行株Ⅵ型流感嗜血杆菌主要引起患者肺炎（包括支气管肺炎和新生儿肺炎）和支气管炎（包括毛细支气管炎和喘息性支气管炎）。荚膜基因分型结果显示,未分离到b型和b-型流感嗜血杆菌。共分离到1株f型,其余99株均为无荚膜抗原的不可分型流感嗜血杆菌。生物学分型和荚膜分型之间无明显的相关性。结论该院分离的流感嗜血杆菌主要为生物型Ⅵ型。回顾患者病史,发现Ⅵ型主要引起肺炎和支气管炎。荚膜基因分型显示,本地区分离的流感嗜血杆菌主要为不可分型流感嗜血杆菌。生物学分型和荚膜基因分型之间无明显相关性。     

A new structural type for Haemophilus influenzae lipopolysaccharide. Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain 486.

Structural elucidation of the sialylated lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae (NTHi) strain 486 has been achieved by the application of high-field NMR techniques and ESI-MS along with composition and linkage analyses on O-deacylated LPS and oligosaccharide samples. It was found that the LPS contains the common element of H. influenzae, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-L-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A, but instead of glycosyl substitution of the terminal heptose residue (HepIII) at the O2 position observed in other H. influenzae strains, HepIII is chain elongated at the O3 position by either lactose or sialyllactose (i.e. alpha-Neu5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp). The LPS is substituted by an O-acetyl group linked to the O2 position of HepIII and phosphocholine (PCho) which was located at the O6 position of a terminal alpha-D-Glcp residue attached to the central heptose, a molecular environment different from what has been reported earlier for PCho. In addition, minor substitution by O-linked glycine to the LPS was observed. By investigation of LPS from a lpsA mutant of NTHi strain 486, it was demonstrated that the lpsA gene product also is responsible for chain extension from HepIII in this strain. The involvement of lic1 in expression of PCho was established by investigation of a lic1 mutant of NTHi strain 486.