Oligomerization states of the Mycobacterium tuberculosis RNA polymerase core and holoenzymes.

During the past few decades, a wealth of knowledge has been made available for the transcription machinery in bacteria from the structural, functional and mechanistic point of view. However, comparatively little is known about the homooligomerization of the multisubunit M. tuberculosis RNA polymerase (RNAP) enzyme and its functional relevance. While E. coli RNAP has been extensively studied, many aspects of RNAP of the deadly pathogenic M. tuberculosis are still unclear. We used biophysical and biochemical methods to study the oligomerization states of the core and holoenzymes of M. tuberculosis RNAP. By size exclusion chromatography and negative staining Transmission Electron Microscopy (TEM) studies and quantitative analysis of the TEM images, we demonstrate that the in vivo reconstituted RNAP core enzyme (α2ßß'ω) can also exist as dimers in vitro. Using similar methods, we also show that the holoenzyme (core + σA) does not dimerize in vitro and exist mostly as monomers. It is tempting to suggest that the oligomeric changes that we see in presence of σA factor might have functional relevance in the cellular process. Although reported previously in E. coli, to our knowledge we report here for the first time the study of oligomeric nature of M. tuberculosis RNAP in presence and absence of σA factor.

The Error-Prone Polymerase DnaE2 Mediates the Evolution of Antibiotic Resistance in Persister Mycobacterial Cells.

Applying antibiotics to susceptible bacterial cultures generates a minor population of persisters that remain susceptible to antibiotics but can endure them for extended periods. Recent reports suggest that antibiotic persisters (APs) of mycobacteria experience oxidative stress and develop resistance upon treatment with lethal doses of ciprofloxacin or rifampicin. However, the mechanisms driving the de novo emergence of resistance remained unclear. Here, we show that mycobacterial APs activate the SOS response, resulting in the upregulation of the error-prone DNA polymerase DnaE2. The sustained expression of dnaE2 in APs led to mutagenesis across the genome and resulted in the rapid evolution of resistance to antibiotics. Inhibition of RecA by suramin, an anti-Trypanosoma drug, reduced the rate of conversion of persisters to resistors in a diverse group of bacteria. Our study highlights suramin's novel application as a broad-spectrum agent in combating the development of drug resistance.

Optimization strategies for expression and purification of soluble N-terminal domain of human centriolar protein SAS-6 in Escherichia coli.

Spindle assembly abnormal protein 6 (SAS-6), a highly conserved centriolar protein, constitutes the center of the cartwheel assembly that scaffolds centrioles early in their biogenesis. Abnormalities in cartwheel assembly lead to chromosomal dysfunctions. The molecular structure of human SAS-6 (HsSAS-6) and cartwheel hub and how they direct centriole symmetry is unknown. No crystal structure of wildtype HsSAS-6 has been reported to date, since soluble recombinant partial/full-length HsSAS-6 expression and purification posed grand challenges. In the present study we have explored optimization of ten different N terminal SAS-6 fusion proteins expression in a variety of E. coli hosts. During optimization we have included some of the most commonly used purification tags: Histidine tag, maltose-binding protein (MBP), small ubiquitin-related modifier (SUMO) tag and modified MBP tag with surface entropy reduction mutations. We demonstrate several levels of tag assisted solubility and stable expression strategies. We find that the MBP tag accompanied by Surface Entropy Reduction mutations (MBP/SER) in a fixed arm approach rescues the folded SAS-6N protein with significantly improved solubility. This expression of HsSAS-6N in E. coli Rosetta DE3 pLysS expression strain gave rise to high protein expression yielding around 6.0-11.5 mg of soluble protein per liter of growth culture.

Phosphomimetic Mutation Destabilizes the Central Core Domain of Human p53.

Transcriptional activity of p53 is modulated by various posttranslational modifications. Earlier studies have reported that Aurora B phosphorylation of p53 leads to loss of its transcriptional activity, subsequently leading to its ubiquitin-mediated proteasomal degradation. To decipher the fate of structural and functional stature of p53 upon phosphorylation by Aurora B, we have generated five phosphomimetic mutants of p53 core domain and characterized their biophysicochemical properties. Our biophysical studies show that the T211E, S215E, and S269E mutants are thermally unstable and show a higher propensity toward aggregation than WT with the loss of DNA binding except for S183E. These results indicate structural and functional destabilization of p53 upon phosphomimetic substitution, which provides a molecular basis toward understanding the process that drives the fate of p53 upon phosphorylation by Aurora B kinase. © 2018 IUBMB Life, 70(10):1023-1031, 2018.

HrpY protein of Ralstonia solanacearum exhibits spontaneous formation of pilus like assembly: analysis of its stability.

Type 3 secretory system (T3SS), a complex protein machinery has a unique virulence mechanism that involves injecting effector proteins directly into host cells. The T3SS effector proteins are transported through an extracellular long hollow needle made up of multiple copies of a small protein. In T3SS of the plant pathogen Ralstonia solanacearum, the 8.6 kDa HrpY protein assembles into a large needle like apparatus (pilus) for transporting effector proteins. To study structural details of HrpY, we recombinantly expressed and purified HrpY in E. coli. The dynamic light scattering (DLS) analysis showed that rHrpY has spontaneously formed oligomers of large order (>100 nm). Transmission electron microscopy of rHrpY samples revealed that the large structures are tube like assembly having dimensions 86.3-166.6 nm and 5.8-6.8 nm in length and width respectively. Different molecular sizes of the purified rHrpY hindered the crystallization of the protein. The stability of oligomer assembly was studied with denaturants and surfactants. Denaturants like urea and guanidine HCl could not break them apart; however, detergents like SDS, sarkosyl, Octyl-ß-Glucoside, CHAPS, Tween 20, Tween 80 and Triton X-100 showed disassembly of the oligomer. rHrpY assembly was found to withstand up to 50 °C and the circular dichroism analysis revealed that there is no significant change in the secondary structural composition with increase in temperature. However, change in the secondary structure was observed with the addition of SDS.Communicated by Ramaswamy H. Sarma.

Role of unique loops in oligomerization and ATPase function of Plasmodium falciparum gyrase B.

DNA gyrase is an ATP dependent Type IIA topoisomerase that is unique to prokaryotes. Interestingly DNA gyrase has also been found in the apicoplasts of apicomplexan parasites like Plasmodium falciparum (Pf) the causative agent of Malaria. Gyrase B (GyrB), a subunit of gyrase A2 B2 complex has an N-terminal domain (GyrBN) which is endowed with ATPase activity. We reported earlier that PfGyrB exhibits ATP-independent dimerization unlike its bacterial counterparts. Here we report the role of two unique regions (L1 and L2) identified in PfGyrBN. Deletions of L1 alone (PfGyrBNΔL1), or L1 and L2 together (PfGyrBNΔL1ΔL2) have indicated that these regions may play an important role in ATPase activity and the oligomeric state of PfGyrBN. Our experiments show that the deletion of L1 region disrupts the dimer interface of PfGyrBN and reduces its ATPase activity. Further through ITC experiments we show that the binding affinity of ATP to PfGyrBN is reduced upon the deletion of L1 region. We have observed a reduction in ATPase activity for of all three proteins PfGyrBN, PfGyrBNΔL1, and PfGyrBNΔL1ΔL2 in presence of coumermycin. Our results suggests that L1 region of PfGyrBN is likely to be functionally important and may provide a unique dimer interface that affects its enzymatic activity. Since deletion of L1 region decreases the affinity of ATP to the protein, this region can be targeted toward designing novel inhibitors of ATP hydrolysis.

Hypomorphic mutations in human DNA ligase IV lead to compromised DNA binding efficiency, hydrophobicity and thermal stability.

Studies have shown that Lig4 syndrome mutations in DNA ligase IV (LigIV) are compromised in its function with residual level of double strand break ligation activity in vivo. It was speculated that Lig4 syndrome mutations adversely affect protein folding and stability. Though there are crystal structures of LigIV, there are no reports of crystal structures of Lig4 syndrome mutants and their biophysical characterization to date. Here, we have examined the conformational states, thermal stability, hydrophobicity and DNA binding efficiency of human DNA LigIV wild type and its hypomorphic mutants by far-UV circular dichroism, tyrosine and tryptophan fluorescence, and 1-anilino-8-naphthalene-sulfonate binding, dynamic light scattering, size exclusion chromatography, multi-angle light scattering and electrophoretic mobility shift assay. We show here that LigIV hypomorphic mutants have reduced DNA-binding efficiency, a shift in secondary structure content from the helical to random coil, marginal reduction in their thermal stability and increased hydrophobicity as compared to the wild-type LigIV.

Stability of p53 oligomers: Tetramerization of p53 impinges on its stability.

The p53 protein has been known to exist structurally in three different forms inside the cells. Earlier studies have reported the predominance of the lower oligomeric forms of p53 over its tetrameric form inside the cells, although only the tetrameric p53 contributes to its transcriptional activity. However, it remains unclear the functional relevance of the existence of other p53 oligomers inside the cells. In this study, we characterize the stability and conformational state of tetrameric, dimeric and monomeric p53 that spans both DNA Binding Domain (DBD) and Tetramerization Domain (TD) of human p53 (94-360 amino acid residues). Intriguingly, our studies reveal an unexpected drastic reduction in tetrameric p53 thermal stability in comparison to its dimeric and monomeric form with a higher propensity to aggregate at physiological temperature. Our EMSA study suggests that tetrameric p53, not their lower oligomeric counterpart, exhibit rapid loss of binding to their consensus DNA elements at the physiological temperature. This detrimental effect of destabilization is imparted due to the tetramerization of p53 that drives the DBDs to misfold at a faster pace when compared to its lower oligomeric form. This crosstalk between DBDs is achieved when it exists as a tetramer but not as dimer or monomer. Our findings throw light on the plausible reason for the predominant existence of p53 in dimer and monomer forms inside the cells with a lesser population of tetramer form. Therefore, the transient disruption of tetramerization between TDs could be a potential cue for the stabilization of p53 inside the cells.

Mycobacterial transcript cleavage factor Gre, exhibits chaperone-like activity.

Gre factors reactivate stalled elongation complexes by enhancing the intrinsic transcript cleavage activity of RNA polymerase. Previous work by us has shown that unlike in Escherichia coli (E.coli), Mycobacterium tuberculosis Gre factor is essential for its survival. Apart from their role in transcription regulation Gre factors have been implicated in stress response. A recent study has shown the role of E.coli GreA as a cellular chaperone, which inhibits aggregation of substrate proteins under heat stress condition. Moreover it was shown that GreA enables E.coli to survive heat shock and oxidative stress. In the current work, we have characterized the moonlighting chaperone activity and its plausible mechanism in Mycobacterium smegmatis Gre (MsGre) factor. We show here that MsGre prevents heat-induced aggregation of the substrate protein and also protects enzymatic activity. Interestingly Gre factor exists as a dimer in solution and does not undergo heat induced oligomerization. From the 8-anilino-1-naphthalene sulfonate (ANS) binding studies MsGre was shown to expose hydrophobic surface upon heat stress that would allow binding to unfolded or partially folded substrate protein. From Circular Dichroism (CD) studies, we also show that MsGre has a stable secondary structure under thermal stress. We propose that the presence of C-terminal FKBP-like fold in MsGre factor that might contribute to its chaperone-like function.

MSMEG_6292, a Mycobacterium smegmatis RNA polymerase secondary channel-binding protein: purification, crystallization and X-ray diffraction analysis.

The transcriptional activity of RNA polymerase (RNAP) is controlled by a diverse set of regulatory factors. A subset of these regulators modulate the activity of RNAP through its secondary channel. Gre factors reactivate stalled elongation complexes by enhancing the intrinsic cleavage activity of RNAP. In the present study, the protein MSMEG_6292, a Gre-factor homologue from Mycobacterium smegmatis, was expressed heterologously in Escherichia coli and purified using standard chromatographic techniques. The hanging-drop vapour-diffusion crystallization method yielded diffraction-quality crystals. The crystals belonged to the trigonal space group P3121 (or its enantiomorph P3221), with unit-cell parameters a = b = 83.15, c = 107.07 Å, α = ß = 90, γ = 120°. The crystals diffracted to better than 3.0 Šresolution. Molecular-replacement attempts did not yield any phasing models; hence, platinum derivatization was carried out with K2PtCl4 and derivative data were collected to 3.4 Šresolution.

A two-domain folding intermediate of RuBisCO in complex with the GroEL chaperonin.

The chaperonins (GroEL and GroES in Escherichia coli) are ubiquitous molecular chaperones that assist a subset of essential substrate proteins to undergo productive folding to the native state. Using single particle cryo EM and image processing we have examined complexes of E. coli GroEL with the stringently GroE-dependent substrate enzyme RuBisCO from Rhodospirillum rubrum. Here we present snapshots of non-native RuBisCO - GroEL complexes. We observe two distinct substrate densities in the binary complex reminiscent of the two-domain structure of the RuBisCO subunit, so that this may represent a captured form of an early folding intermediate. The occupancy of the complex is consistent with the negative cooperativity of GroEL with respect to substrate binding, in accordance with earlier mass spectroscopy studies.

Crystal structure of human carbonic anhydrase II at 1.95 A resolution in complex with 667-coumate, a novel anti-cancer agent.

CA (carbonic anhydrase) catalyses the reversible hydration of carbon dioxide into bicarbonate, and at least 14 isoforms have been identified in vertebrates. The role of CA type II in maintaining the fluid and pH balance has made it an attractive drug target for the treatment of glaucoma and cancer. 667-coumate is a potent inhibitor of the novel oncology target steroid sulphatase and is currently in Phase 1 clinical trials for hormone-dependent breast cancer. It also inhibits CA II in vitro. In the present study, CA II was crystallized with 667-coumate and the structure was determined by X-ray crystallography at 1.95 A (1 A=0.1 nm) resolution. The structure reported here is the first for an inhibitor based on a coumarin ring and shows ligation of the sulphamate group to the active-site zinc at 2.15 A through a nitrogen anion. The first two rings of the coumarin moiety are bound within the hydrophobic binding site of CA II. Important residues contributing to binding include Val-121, Phe-131, Val-135, Leu-141, Leu-198 and Pro-202. The third seven-membered ring is more mobile and is located in the channel leading to the surface of the enzyme. Pharmacokinetic studies show enhanced stability of 667-coumate in vivo and this has been ascribed to binding of CA II in erythrocytes. This result provides a structural basis for the stabilization and long half-life of 667-coumate in blood compared with its rapid disappearance in plasma, and suggests that reversible binding of inhibitors to CA may be a general method of delivering this type of labile drug.

Unique hydrophobic extension of the RGS2 amphipathic helix domain imparts increased plasma membrane binding and function relative to other RGS R4/B subfamily members.

RGS2 and RGS5 are inhibitors of G-protein signaling belonging to the R4/B subfamily of RGS proteins. We here show that RGS2 is a much more potent attenuator of M1 muscarinic receptor signaling than RGS5. We hypothesize that this difference is mediated by variation in their ability to constitutively associate with the plasma membrane (PM). Compared with full-length RGS2, the RGS-box domains of RGS2 and RGS5 both show reduced PM association and activity. Prenylation of both RGS-box domains increases activity to RGS2 levels, demonstrating that lipid bilayer targeting increases RGS domain function. Amino-terminal domain swaps confirm that key determinants of localization and function are found within this important regulatory domain. An RGS2 amphipathic helix domain mutant deficient for phospholipid binding (L45D) shows reduced PM association and activity despite normal binding to the M1 muscarinic receptor third intracellular loop and activated Galpha(q). Replacement of a unique dileucine motif adjacent to the RGS2 helix with corresponding RGS5 residues disrupts both PM localization and function. These data suggest that RGS2 contains a hydrophobic extension of its helical domain that imparts high efficiency binding to the inner leaflet of the lipid bilayer. In support of this model, disruption of membrane phospholipid composition with N-ethylmaleimide reduces PM association of RGS2, without affecting localization of the M1 receptor or Galpha(q). Together, these data indicate that novel features within the RGS2 amphipathic alpha helix facilitate constitutive PM targeting and more efficient inhibition of M1 muscarinic receptor signaling than RGS5 and other members of the R4/B subfamily.

Structure of testis ACE glycosylation mutants and evidence for conserved domain movement.

Human angiotensin-converting enzyme is an important drug target for which little structural information has been available until recent years. The slow progress in obtaining a crystal structure was due to the problem of surface glycosylation, a difficulty that has thus far been overcome by the use of a glucosidase-1 inhibitor in the tissue culture medium. However, the prohibitive cost of these inhibitors and incomplete glucosidase inhibition makes alternative routes to minimizing the N-glycan heterogeneity desirable. Here, glycosylation in the testis isoform (tACE) has been reduced by Asn-Gln point mutations at N-glycosylation sites, and the crystal structures of mutants having two and four intact sites have been solved to 2.0 A and 2.8 A, respectively. Both mutants show close structural identity with the wild-type. A hinge mechanism is proposed for substrate entry into the active cleft, based on homology to human ACE2 at the levels of sequence and flexibility. This is supported by normal-mode analysis that reveals intrinsic flexibility about the active site of tACE. Subdomain II, containing bound chloride and zinc ions, is found to have greater stability than subdomain I in the structures of three ACE homologues. Crystallizable glycosylation mutants open up new possibilities for cocrystallization studies to aid the design of novel ACE inhibitors.

Localization of an N-domain region of angiotensin-converting enzyme involved in the regulation of ectodomain shedding using monoclonal antibodies.

ACE chimeric proteins and N domain monoclonal antibodies (mAbs) were used to determine the influence of the N domain, and particular regions thereof, on the rate of ACE ectodomain shedding. Somatic ACE (having both N and C domains) was shed at a rate of 20%/24 h. Deletion of the C domain of somatic ACE generated an N domain construct (ACEDeltaC) which demonstrated the lowest rate of shedding (12%). However, deletion of the N domain of somatic ACE (ACEDeltaN) dramatically increased shedding (212%). Testicular ACE (tACE) having 36 amino acid residues (heavily O-glycosylated) at the N-terminus of the C domain shows a 4-fold decrease in the rate of shedding (49%) compared to that of ACEDeltaN. When the N-terminal region of the C domain was replaced with the corresponding homologous 141 amino acids of the N domain (N-delACE) the rate of shedding of the ACEDeltaN was only slightly decreased (174%), but shedding was still 3.5-fold more efficient than wild-type testicular ACE. Monoclonal antibodies specific for distinct, but overlapping, N-domain epitopes altered the rate of ACE shedding. The mAb 3G8 decreased the rate of shedding by 30%, whereas mAbs 9B9 and 3A5 stimulated ACE shedding 2- to 4-fold. Epitope mapping of these mAbs in conjunction with a homology model of ACE N domain structure, localized a region in the N-domain that may play a role in determining the relatively low rate of shedding of somatic ACE from the cell surface.

Crystal structures of Erythrina cristagalli lectin with bound N-linked oligosaccharide and lactose.

Erythrina cristagalli lectin (ECL) is a galactose-specific legume lectin. Although its biological function in the legume is unknown, ECL exhibits hemagglutinating activity in vitro and is mitogenic for T lymphocytes. In addition, it has been recently shown that ECL forms a novel conjugate when coupled to a catalytically active derivative of the type A neurotoxin from Clostridium botulinum, thus providing a therapeutic potential. ECL is biologically active as a dimer in which each protomer contains a functional carbohydrate-combining site. The crystal structure of native ECL was recently reported in complex with lactose and 2'-fucosyllactose. ECL protomers adopt the legume lectin fold but form non-canonical dimers via the handshake motif as was previously observed for Erythrina corallodendron lectin. Here we report the crystal structures of native and recombinant forms of the lectin in three new crystal forms, both unliganded and in complex with lactose. For the first time, the detailed structure of the glycosylated hexasaccharide for native ECL has been elucidated. The structure also shows that in the crystal lattice the glycosylation site and the carbohydrate binding site are involved in intermolecular contacts through water-mediated interactions.

Roles of active site tryptophans in substrate binding and catalysis by alpha-1,3 galactosyltransferase.

Aromatic amino acids are frequent components of the carbohydrate binding sites of lectins and enzymes. Previous structural studies have shown that in alpha-1,3 galactosyltransferase, the binding site for disaccharide acceptor substrates is encircled by four tryptophans, residues 249, 250, 314, and 356. To investigate their roles in enzyme specificity and catalysis, we expressed and characterized variants of the catalytic domain of alpha-1,3 galactosyltransferase with substitutions for each tryptophan. Substitution of glycine for tryptophan 249, whose indole ring interacts with the nonpolar B face of glucose or GlcNAc, greatly increases the K(m) for the acceptor substrate. In contrast, the substitution of tyrosine for tryptophan 314, which interacts with the beta-galactosyl moiety of the acceptor and UDP-galactose, decreases k(cat) for the galactosyltransferase reaction but does not affect the low UDP-galactose hydrolase activity. Thus, this highly conserved residue stabilizes the transition state for the galactose transfer to disaccharide but not to water. High-resolution crystallographic structures of the Trp(249)Gly mutant and the Trp(314)Tyr mutant indicate that the mutations do not affect the overall structure of the enzyme or its interactions with ligands. Substitutions for tryptophan 250 have only small effects on catalytic activity, but mutation of tryptophan 356 to threonine reduces catalytic activity for both transferase and hydrolase activities and reduces affinity for the acceptor substrate. This residue is adjacent to the flexible C-terminus that becomes ordered on binding UDP to assemble the acceptor binding site and influence catalysis. The results highlight the diverse roles of these tryptophans in enzyme action and the importance of k(cat) changes in modulating glycosyltransferase specificity.

Crystal structure of the human angiotensin-converting enzyme-lisinopril complex.

Angiotensin-converting enzyme (ACE) has a critical role in cardiovascular function by cleaving the carboxy terminal His-Leu dipeptide from angiotensin I to produce a potent vasopressor octapeptide, angiotensin II. Inhibitors of ACE are a first line of therapy for hypertension, heart failure, myocardial infarction and diabetic nephropathy. Notably, these inhibitors were developed without knowledge of the structure of human ACE, but were instead designed on the basis of an assumed mechanistic homology with carboxypeptidase A. Here we present the X-ray structure of human testicular ACE and its complex with one of the most widely used inhibitors, lisinopril (N2-[(S)-1-carboxy-3-phenylpropyl]-L-lysyl-L-proline; also known as Prinivil or Zestril), at 2.0 A resolution. Analysis of the three-dimensional structure of ACE shows that it bears little similarity to that of carboxypeptidase A, but instead resembles neurolysin and Pyrococcus furiosus carboxypeptidase--zinc metallopeptidases with no detectable sequence similarity to ACE. The structure provides an opportunity to design domain-selective ACE inhibitors that may exhibit new pharmacological profiles.

Structural details on the binding of antihypertensive drugs captopril and enalaprilat to human testicular angiotensin I-converting enzyme.

Angiotensin converting enzyme (ACE) plays a critical role in the circulating or endocrine renin-angiotensin system (RAS) as well as the local regulation that exists in tissues such as the myocardium and skeletal muscle. Here we report the high-resolution crystal structures of testis ACE (tACE) in complex with the first successfully designed ACE inhibitor captopril and enalaprilat, the Phe-Ala-Pro analogue. We have compared these structures with the recently reported structure of a tACE-lisinopril complex [Natesh et al. (2003) Nature 421, 551-554]. The analyses reveal that all three inhibitors make direct interactions with the catalytic Zn(2+) ion at the active site of the enzyme: the thiol group of captopril and the carboxylate group of enalaprilat and lisinopril. Subtle differences are also observed at other regions of the binding pocket. These are compared with N-domain models and discussed with reference to published biochemical data. The chloride coordination geometries of the three structures are discussed and compared with other ACE analogues. It is anticipated that the molecular details provided by these structures will be used to improve the binding and/or the design of new, more potent domain-specific inhibitors of ACE that could serve as new generation antihypertensive drugs.

Roles of individual enzyme-substrate interactions by alpha-1,3-galactosyltransferase in catalysis and specificity.

The retaining glycosyltransferase, alpha-1,3-galactosyltransferase (alpha3GT), is mutationally inactivated in humans, leading to the presence of circulating antibodies against its product, the alpha-Gal epitope. alpha3GT catalyzes galactose transfer from UDP-Gal to beta-linked galactosides, such as lactose, and in the absence of an acceptor substrate, to water at a lower rate. We have used site-directed mutagenesis to investigate the roles in catalysis and specificity of residues in alpha3GT that form H-bonds as well as other interactions with substrates. Mutation of the conserved Glu(317) to Gln weakens lactose binding and reduces the k(cat) for galactosyltransfer to lactose and water by 2400 and 120, respectively. The structure is not perturbed by this substitution, but the orientation of the bound lactose molecule is changed. The magnitude of these changes does not support a previous proposal that Glu(317) is the catalytic nucleophile in a double displacement mechanism and suggests it acts in acceptor substrate binding and in stabilizing a cationic transition state for cleavage of the bond between UDP and C1 of the galactose. Cleavage of this bond also linked to a conformational change in the C-terminal region of alpha3GT that is coupled with UDP binding. Mutagenesis indicates that His(280), which is projected to interact with the 2-OH of the galactose moiety of UDP-Gal, is a key residue in the stringent donor substrate specificity through its role in stabilizing the bound UDP-Gal in a suitable conformation for catalysis. Mutation of Gln(247), which forms multiple interactions with acceptor substrates, to Glu reduces the catalytic rate of galactose transfer to lactose but not to water. This mutation is predicted to perturb the orientation or environment of the bound acceptor substrate. The results highlight the importance of H-bonds between enzyme and substrates in this glycosyltransferase, in arranging substrates in appropriate conformations and orientation for efficient catalysis. These factors are manifested in increases in catalytic rate rather than substrate affinity.