Influence of growth environment on tolerance to UV-B radiation, germination speed, and morphology of Metarhizium anisopliae var. acridum conidia

We previously reported wide variability in UV-B tolerance among different Metarhizium anisopliae isolates [Braga, G.U.L., Flint, S.D., Miller, C.D., Anderson, A.J., Roberts, D.W., 2001a. Variability in response to UV-B among species and strains of Metarhizium isolated from sites at latitudes from 61 degrees N to 54 degrees S. J. Invertebr. Pathol. 78, 98-108] as well as wide phenotypic variability in some of these isolates in response to alterations in their growth environments [Rangel, D.E.N., Braga, G.U.L., Flint, S.D., Anderson, A.J., Roberts, D.W., 2004. Variations in UV-B tolerance and germination speed of M. anisopliae conidia produced on artificial and natural substrates. J. Invertebr. Pathol. 87, 77-83]. Studies on other biological systems have demonstrated that strong selective pressure for tolerance to a stress factor may reduce the phenotypic variability of this trait. In the present study, conidia of the isolate most tolerant to radiation and heat, ARSEF 324, presented very little phenotypic variability in UV-B tolerance in response to production on either artificial culture medium or infected insects. The phenotypic plasticities in two other traits (conidial morphology and germination speed), however, were considerably higher.     

Growth of Metarhizium anisopliae on non-preferred carbon sources yields conidia with increased UV-B tolerance

Conidia of the insect-pathogenic fungus Metarhizium anisopliae var. anisopliae produced on different growth substrates (culture media or insect cadavers) demonstrate reproducibly altered tolerance to UV-B radiation [Rangel, D.E.N., Braga, G.U.L., Flint, S.D., Anderson, A.J., Roberts, D.W., 2004. Variations in UV-B tolerance and germination speed of M. anisopliae conidia produced on artificial and natural substrates. J. Invertebr. Pathol. 87, 77-83]. In the current study, the fungus was grown on potato dextrose agar with yeast extract (PDAY), on minimal medium [(MM)=Czapek medium without saccharose], or on MM with one of 16 different carbon sources. The conidia produced on these media were exposed to UV-B radiation. Great amplitude in phenotypic plasticity for UV-B tolerance was demonstrated, viz., conidia produced under nutritive stress [MM or MM supplemented with non-preferred carbon sources (e.g., fructose, galactose, lactose etc.)] had at least two times higher tolerance than conidia produced on the rich medium (PDAY). Endogenous trehalose and mannitol accumulated at least two times more in conidia produced on MM (or MM with lactose, a non-preferred carbon source), as compared to conidia from MM plus glucose. High accumulations of these two carbohydrates in fungal spores are known to protect them against a wide range of stresses. Sporulation, however, was most profuse on PDAY, second best on MM plus d-mannose and least on MM or MM containing non-preferred carbon sources. Taken together, the results illustrate that nutritive stress generated by MM or MM plus a non-preferred carbon source greatly improved UV-B tolerance, but reduced conidial yield; while, on the other hand, preferred carbon sources improved conidial yield, but reduced UV-B tolerance.     

Investigations on the destruxin production of the entomopathogenic fungus Metarhizium anisopliae

The dynamics of cyclic peptide destruxins (dtxs) produced by Metarhizium anisopliae strains V245 and V275 were monitored both on solid and in liquid media. The results showed that both strains did not produce dtxs in large-scale fermenter cultures or solid Czapek Dox (CD) agar. Production of the major dtxs A and B could be determined in both strains when grown on rice for up to 10-30 days. The main dtxs A, B, E, and E diol were detected in CD liquid culture filtrate from both strains after three days post-inoculation on. Parallel decrease of dtx E and increase of E diol in the culture medium were found, indicating that the latter is the hydrolytic product from the former. Production of dtxs A and B was significantly positively correlated. A negative correlation was observed between the production of the metabolites and pH value of the medium. The influence of different nutrient sources on dtx production was evaluated by using media with different carbon and nitrogen ratios as well as with different insect homogenates. The findings showed that the amount of dtxs A, B, and E increased with the increasing content of peptone in the medium. When insect homogenate was used as single nutrient source or added to CD medium, no toxins were detected in the culture filtrate. The potential risk posed by the toxic metabolites during mass production is discussed.     

Concurrence of losing a chromosome and the ability to produce destruxins in a mutant of Metarhizium anisopliae

In a previous study, a spontaneous subtilisin pr1A and pr1B gene-deficient mutant of the entomopathogenic fungus Metarhizium anisopliae strain V275 has been identified [Wang, C.-S. et al. (2002) FEMS Microbiol. Lett. 213, 251-255]. The insecticidal metabolites of this mutant were studied further. High-performance liquid chromatography (HPLC) analysis indicated that the mutant isolate lost the ability to produce cyclic peptide toxins, destruxins, both in vitro and in vivo. Pulsed-field gel electrophoresis revealed that the mutant concurrently lost a 1.05 Mb (approximately) chromosome, demonstrating for the first time that a conditionally dispensable (CD) chromosome exists in the insect pathogenic fungus, M. anisopliae. Concurrence of losing the ability to produce destruxins and a CD chromosome in the mutant suggests that the toxin synthetase genes of M. anisopliae are located on this CD chromosome, as similarly described for plant pathogenic fungi. Semi-quantitative api ZYM analysis showed more biochemical disparities between the mutant and the wild-type strain.     

绿僵菌素的分离制备及其对蛴螬的毒力

以金龟子绿僵菌金龟子变种Metarhizium anisopliae var. anisopliae菌株MaQ10发酵液为材料,采用萃取、浓缩、制备色谱及重结晶技术,分离纯化出5种绿僵菌素的晶体,经与标准样品以及参考文献相对照,此5种晶体与绿僵菌素A、A2、B、C和E相吻合。进而,采用浸渍法测定了绿僵菌素A和B对大等鳃金龟Exolontha serrulata (Gyllenhall) 和卵圆齿爪鳃金龟Holotrichia ovata Chang 1龄幼虫的触杀毒力。结果表明： 绿僵菌素A和B对大等鳃金龟的触杀活性,在处理后96～120 h内最高,LC₅₀分别为78.1571 mg/L和88.7562 mg/L; 处理浓度为300 mg/L时,LT₅₀分别为13.4159 h和10.5331 h。绿僵菌素A和B对卵圆齿爪鳃金龟的触杀活性,在处理后96 h时最高,LC₅₀分别为66.5308 mg/L和79.4309 mg/L;处理浓度为300 mg/L时,LT₅₀分别是13.6399 h和9.9451 h。     

Effect of the cyclopeptolide 90-215 on the production of destruxins and helvolic acid by Metarhizium anisopliae

The effect of the cyclopeptolide 90-215 on the production of destruxins and helvolic acid in various Metarhizium anisopliae strains was investigated. Addition of 10.0 mg L⁻¹ of the cyclopeptolide to the production media increased the production of destruxins by 1.3-fold to 12.5-fold, whereas the production of helvolic acid was decreased by 1.6- to 11.0-fold. Fifty liters were fermented in Erlenmeyer flasks with the strain 86-23766 grown in medium supplemented with cyclopeptolide 90-215. The procedure for isolation and purification of destruxins was simplified due to the higher yield of destruxins. Good quantities of destruxins were obtained from this fermentation. The results of our studies show that the addition of small quantities of a suitable compound can drastically alter the production and relative ratios of secondary metabolites. This can have a wide range of potential applications in the area of metabolite production. Received 09 December 1996/ Accepted in revised form 09 April 1997     

Effects of the entomopathogenic fungus Metarhizium anisopliae and its secondary metabolites on morphology and cytoskeleton of plasmatocytes isolated from the greater wax moth,Galleria mellonella

The effects of Metarhizium anisopliae infection and three different secondary metabolites released by the fungus, destruxin A and E and cytochalasin D, on the morphology and cytoskeleton of plasmatocytes of the greater wax moth Galleria mellonella were studied. Plasmatocytes isolated from M. anisopliae infected larvae exhibited impairment of attachment, spreading and cytoskeleton formation accompanied with the occurrence of blebbing and pycnotic nuclei. Plasmatocytes treated with destruxin in vitro exhibited similar morphological and cytoskeleton alterations. The corresponding effects were characterized by inhibition of attachment, spreading and filopodia formation as well as by impaired formation of actin filaments and microtubules. Cytochalasin was shown to affect plasmatocytes in vitro in a different manner than destruxin A and E. The results of our comparative study strongly suggested that the morphology and cytoskeleton alterations of plasmatocytes observed in M. anisopliae infected larvae were predominantly caused by destruxins released by the fungus during mycosis. Its mode of action is discussed with regard to present knowledge about its effects on target cells.     

A new destruxin as inhibitor of vacuolar-type H+-ATPase of Saccharomyces cerevisiae

In the course of our screening efforts to discover small molecules as selective inhibitors of vacuolar-type H+-ATPase of Saccharomyces cerevisiae, we have identified eight active destruxins, 1-8, from the fungus Metarhizium anisopliae. The structures were elucidated by extensive 1D- and 2D-NMR spectroscopy, and MS spectrometry. One of these compounds, 8, a regioisomer of chlorohydrin destruxin E (7), is a new destruxin.     

Depsipeptides from Metarhizium anisopliae

From the culture medium of a strain of Metarhizium anisopliae, 14 depsipeptides have been isolated. Five of them were identified as known destruxins A, B, C, D and desmethyldestruxin B. The structures of the new compounds, named destruxins E, A₁, A₂, B₁, B₂, C₂, D₁, D₂ and E₁,were established mainly from the mass spectral analysis of their corresponding open-chain derivatives.     

Nitrogenase-catalyzed ethane production and CO-sensitive hydrogen evolution from MoFe proteins having amino acid substitutions in an alpha-subunit FeMo cofactor-binding domain.

Unlike wild type, certain Mo-dependent nitrogenases, which are expressed in non-N2-fixing mutant strains of Azotobacter vinelandii and have single amino acid substitutions within a region of the MoFe protein alpha-subunit proposed to encompass an FeMo cofactor-binding domain, are able to catalyze the reduction of acetylene by both two and four electrons to yield ethylene and ethane, respectively (Scott, D. J., May, H. D., Newton, W. E., Brigle, K. E., and Dean, D. R. (1990) Nature 343, 188-190). Although the V-dependent nitrogenase is also able to catalyze the reduction of acetylene to the same two- and four-electron products (Dilworth, M. J., Eady, R. R., Robson, R. L., and Miller, R. W. (1987) Nature 327, 167-168), we find that ethane formation from acetylene catalyzed by the altered Mo-dependent nitrogenases occurs by a different mechanism, which is distinguished by: (i) an increased sensitivity to CO; (ii) the absence of a lag; and (iii) no temperature dependence of product distribution among ethylene and ethane during acetylene reduction. An altered MoFe protein, which was purified from one such mutant strain having the alpha-subunit glutaminyl 191 residue substituted by lysyl, exhibited both a changed S = 3/2 EPR spectrum and changes in the distribution of electrons to various products when compared to wild type. Also, unlike wild type, this altered MoFe protein catalyzed proton reduction that is inhibited by carbon monoxide (CO). Because proton reduction catalyzed by a nitrogenase that has a FeMo cofactor with citrate rather than homocitrate as its organic constituent (Liang, J., Madden, M., Shah, V. K., and Burris, R. H. (1990) Biochemistry 29, 8577-8581) is also inhibited by CO, the possibility arose that changes in the polypeptide environment of FeMo cofactor might have caused a rearrangement in its molecular structure or composition. However, this possibility was ruled out by biochemical reconstitution studies (using FeMo cofactor isolated from both the wild-type and altered MoFe proteins), which were monitored by EPR spectroscopy and resulting catalytic activity.     

Site-specific mutagenesis of the alpha-helices of calmodulin. Effects of altering a charge cluster in the helix that links the two halves of calmodulin

Alteration of residues 82-84 in the alpha-helix that links the two halves of calmodulin results in a differential effect on activator activity. Previous studies (Lukas, T. J., Burgess, W. H., Prendergast, F. G., Lau, W., and Watterson, D. M. (1986) Biochemistry 25, 1458-1464) indicated the importance of positive charge clusters in the calmodulin-binding protein, myosin light chain kinase. This suggested the possible importance of complementary negative charge clusters in calmodulin. By using an efficient cassette mutagenesis approach and a synthetic calmodulin gene (Roberts, D. M., Crea, R., Malecha, M., Alvarado-Urbina, G., Chiarello, R. H., and Watterson, D. M. (1985) Biochemistry 24, 5090-5098), this possibility was directly addressed by engineering a new calmodulin, VU-8 calmodulin, in which the glutamate cluster at residues 82-84 in the synthetic gene product (VU-1 calmodulin) was replaced by three lysines. VU-8 calmodulin activated phosphodiesterase to the same maximal extent as VU-1 calmodulin, although there was an alteration in the concentration of calmodulin required for half-maximal stimulation. In contrast, myosin light chain kinase was activated to only 30% of maximal activity and NAD kinase was not activated. These results provide insight into the functional role of the unusual central helix structure found in the calmodulin family of proteins and indicate that different, although possibly overlapping, chemical complementarities are employed in the interaction between calmodulin and its various physiological targets.     

Toxicity testing of destruxins and crude extracts from the insect-pathogenic fungus Metarhizium anisopliae

Increasing sensitivity towards secondary metabolites from fungal biological control agents (BCAs) has prompted the toxicological risk assessment of metabolites produced by the insect pathogenic fungus Metarhizium anisopliae. Viability studies on one human and one insect cell line were used to compare the two approaches of testing individual metabolites (destruxins A, B and E) or the complete crude extract from liquid cultures. Furthermore, crude extract was separated into fractions, which did not contain the main destruxins A, B and E. Evaluation of the cytotoxic activity of these different compounds suggested that a wide range of metabolites with synergistic or adverse effects are present in the crude extract. The results indicate that identification and toxicological assessment of each individual metabolite produced by a BCA is not only time and cost-intensive, but also does not convey the whole picture. Testing of the crude extract offers an alternative approach and is recommended when assessing the risks of metabolites for registration purposes.     

Mono-sulfated globotetraosylceramide from human kidney

A novel sulfated glycosphingolipid that belongs to "globo-series" was isolated from human kidney. This lipid was purified from a pooled kidney preparation by chloroform-methanol extraction, mild alkaline treatment, DEAE-Sephadex and silicic acid column chromatographies, and preparative TLC. The structure and the properties were studied by IR spectroscopy, proton NMR spectroscopy, negative secondary ion-mass spectrometry, solvolysis, periodate oxidation, compositional and methylation analyses, monoclonal antibodies, and a sulfatide-binding protein. From the results of the above analyses, the structure of this glycolipid was proposed to be HSO3-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1ceramide. This sulfated lipid reacted with a monoclonal anti-SSEA-3 (stage-specific embryonic antigen-3) (MC-631) (Kannagi, R., Cochran, N.A., Ishigami, F., Hakomori, S., Andrews, P.W., Knowles, B.B., & Solter, D. (1983) EMBO J. 2, 2355-2361), whose epitope is R-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-R', on TLC and solid-phase radioimmunoassay. This lipid also bound to the 125I-labeled sulfatide-binding protein, thrombospondin. The yield of this sulfated glycolipid was 34 pmol/g of tissue, which was about 0.028, 0.16, and 18 mol% of galactosyl- and lactosylceramide sulfates, and globopentosylceramide sulfate (Nagai, K.-i., Roberts, D.D., Toida, T., Matsumoto, H., Kushi, Y., Handa, S., & Ishizuka, I. (1989) J. Biol. Chem. 264, in press), respectively, in human kidney.     

Development and applications of destruxins: A review

The insecticidal and phytotoxic activities of destruxins (dtxs) have been well studied. The cyclodepsipeptides, which are dtxs mainly isolated from the fungus Metarhizium anisopliae and other fungi, have been well characterized in vitro and in vivo. A succession of important function, such as antitumoral, antiviral, insecticidal, cytotoxic, immunosuppressant, phytotoxic, and antiproliferative effects have been observed. To date, 39 dtxs derivatives have been identified. Dtxs possess a variety of biological activities, including acting as virulence factors for specific insects, a V-ATPase inhibitor that provides a basis for the development of new drug to against osteoporosis, cancer, or biological control agents, etc. Here, we focus on some of the research progress made on understanding dtxs during the last decade, introduce some of the newly identified dtx members, especially from M. anisopliae, and give an overview of the applications of dtxs. Using the dtxs to learn about and moderate biological events has advanced significantly during the past year. We believe that several ongoing dtx application fields may benefit from the reviewed information herein.     

Chemical synthesis and expression of a calmodulin gene designed for site-specific mutagenesis

A gene coding for a calmodulin was synthesized and expressed in Escherichia coli. The gene was produced by the enzymatic ligation of 61 chemically synthesized DNA fragments. The gene possesses 27 unique, regularly spaced, restriction endonuclease cleavage sites to facilitate gene mutagenesis by the replacement of specific gene segments with synthetic double-stranded DNA. An expression vector containing the calmodulin gene was used to transform E. coli. Purification and characterization of calmodulin (VU-1 calmodulin) expressed by these transformants showed that it lacks two posttranslational modifications: an amino-terminal blocking group and N epsilon, N epsilon, N epsilon-trimethyllysine at position 115. The cyclic nucleotide phosphodiesterase activator properties of VU-1, higher plant, and vertebrate calmodulins were not statistically different. However, VU-1 calmodulin was found to activate nicotinamide adenine dinucleotide (NAD) kinase to a maximal level that was at least 3-fold higher than that found with higher plant and vertebrate calmodulins. This higher level of activation is also characteristic of calmodulins from Dictyostelium discoideum and Chlamydomonas reinhardtii [Roberts, D. M., Burgess, W. H., & Watterson, D. M. (1984) Plant Physiol. 75, 796-798; Marshak, D. R., Clarke, M., Roberts, D. M., & Watterson, D. M. (1984) Biochemistry 23, 2891-2899]. The only common feature among Dictyostelium, Chlamydomonas, and VU-1 calmodulins not found in higher plant and vertebrate calmodulins is an unmethylated lysine at position 115. The results indicate that the lack of methylation of lysine-115 may contribute to the maximal level of NAD kinase activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the fidelity of DNA replication. Isolation of high fidelity DNA polymerase-primase complexes by immunoaffinity chromatography

Error rates for conventionally purified DNA polymerase-alpha from calf thymus, chicken, and human sources have been reported to be one in 10,000 to one in 40,000 nucleotides incorporated. Isolation of polymerase-alpha by immunoaffinity chromatography yields a multiprotein high molecular weight replication complex that contains an associated DNA primase (Wong, S. W., Paborsky, L. R., Fisher, P. A., Wang, T. S-F., and Korn, D. (1986) J. Biol. Chem. 261, 7958-7968). We have isolated DNA polymerase-primase complexes from calf thymus, from a human lymphoblast cell line (TK-6), and from Chinese hamster lung cells (V-79) using two different methods of immunoaffinity chromatography. These enzyme complexes are 12- to 20-fold more accurate than conventionally purified calf thymus DNA polymerase-alpha when assayed using the phi X174am3 fidelity assay; estimated error rates are one in 460,000 to one in 830,000 nucleotides incorporated when the enzyme complex is freshly isolated. The polymerase-primase complex from calf thymus exhibited no detectable 3'----5' exonuclease activity using a heteroduplex substrate containing a single 3'-terminal mismatched nucleotide. Upon prolonged storage at -70 degrees C, the error rate of the immunoaffinity-purified calf thymus DNA polymerase-primase complex increases to about one in 50,000 nucleotides incorporated, an error rate similar to that exhibited by conventional isolates of DNA polymerase-alpha.     

Relationship between intracellular ice formation in oocytes of the mouse and Xenopus and the physical state of the external medium--a revisit

We have previously reported that intracellular ice formation (IIF) in mouse oocytes suspended in glycerol/PBS solutions or ethylene glycol (EG)/PBS solutions and rapidly cooled to −50 °C or below occurs at temperatures where a critical fraction of the external water remains unfrozen [P. Mazur, S. Seki, I.L. Pinn, F.W. Kleinhans, K. Edashige, Extra- and intracellular ice formation in mouse oocytes, Cryobiology 51 (2005) 29-53; P. Mazur, I.L. Pinn, F.W. Kleinhans, The temperature of intracellular ice formation in mouse oocytes vs. the unfrozen fraction at that temperature, Cryobiology 54 (2007) 223-233]. For mouse oocytes in PBS or glycerol/PBS that fraction is 0.06; for oocytes in EG that fraction was calculated to be 0.13, more than double. The fractions unfrozen are computed from ternary phase diagrams. In the previous publication, we used the EG data of Woods et al. [E.J. Woods, M.A.J. Zieger, D.Y. Gao, J.K. Critser, Equations for obtaining melting points for the ternary system ethylene glycol/sodium chloride/Water and their application to cryopreservation., Cryobiology 38 (1999) 403-407]. Since then, we have determined that ternary phase diagrams for EG/NaCl/water synthesized by summing binary phase data for EG/water NaCl/water gives substantially different curves, which seem more realistic [F.W. Kleinhans, P. Mazur, Comparison of actual vs. synthesized ternary phase diagrams for solutes of cryobiological interest, Cryobiology 54 (2007) 212-222]. Unfrozen fractions at the temperatures of IIF computed from these synthesized phase diagrams are about half of those calculated from the Woods et al. data, and are in close agreement with the computations for glycerol; i.e., IIF occurs when about 92-94% of the external water is frozen. A parallel paper was published by Guenther et al. [J.F. Guenther, S. Seki, F.W. Kleinhans, K. Edashige, D.M. Roberts, P. Mazur, Extra-and intra-cellular ice formation in Stage I and II Xenopus laevis oocytes, Cryobiology 52 (2006) 401-416] on IIF in oocytes of the frog Xenopus. It too examined whether the temperatures of IIF were related to the unfrozen fractions at those temperatures. It also used the Woods et al. ternary phase data to calculate the unfrozen fractions for EG solutions. As reported here, once again the values of these unfrozen fractions are substantially different from those calculated using synthesized phase diagrams. With the latter, the unfrozen fractions at IIF become very similar for EG and glycerol.     

The pathogenic human sulfite oxidase mutants G473D and A208D are defective in intramolecular electron transfer

Mutations G473D and A208D were identified in patients with isolated sulfite oxidase (SO) deficiency, and the equivalent amino acids (G451 and A186, respectively) have been localized to the vicinity of the molybdopterin active site in the X-ray structure of chicken SO [Kisker, C., Schindelin, H., Pacheco, A., Wehbi, W., Garrett, R. M., Rajagopalan, K. V., Enemark, J. H., and Rees, D. C. (1997) Cell 91, 973-983]. To assess the effects of these mutations in human SO, steady-state kinetic studies of enzyme turnover and laser flash photolysis measurements of intramolecular electron transfer (IET) rate constants between the reduced heme [Fe(II)] and Mo(VI) centers were carried out in the recombinant G473D, G473A, G473W, G473D/R212A, and A208D human SO mutants. In the G473D and A208D mutants, the IET rate constants at pH 6.0 are decreased by 3 orders of magnitude relative to that of the wild type. Steady-state kinetic measurements indicate that the IET process is the rate-limiting step in the catalytic cycle of these two mutants. Thus, the large decreases in the IET rate constants and the kcat values, and the large increases in the Km(sulfite) values, rationalize the fatal impact of these mutations. Far-UV CD spectra of G473D indicate that the protein backbone conformation is remarkably changed, and the sedimentation equilibrium indicates that the protein is monomeric. Furthermore, EPR studies also suggest that the active site structure of the Mo(V) form of A208D is different from that of the wild type. In contrast, similar studies on G473A show that it is dimeric, that its Mo(V) active site structure is similar to that of the wild type, and that its IET rate constant is only 2.6-fold smaller than that of the wild type. IET in G473W is severely impaired, and no IET is observed for G473D/R212A. In chicken SO, the equivalent residues (G451 and A186) are both buried inside the protein. Thus, for human SO, the mutations to charged residues at the equivalent sites most likely cause crucial global or localized structural changes, and expose an alternative docking site that may compete with the Mo domain for docking of the heme, thereby retarding IET and efficient catalytic turnover of the sulfite oxidation reaction.     

Purification of an alanine racemase from Streptococcus faecalis and analysis of its inactivation by (1-aminoethyl)phosphonic acid enantiomers

An alanine racemase has been purified some 30 000-fold almost to homogeneity from Gram-positive Streptococcus faecalis NCIB 6459; the enzyme has been purified to the same extent (4000-fold) from an O-carbamyl-D-serine-resistant mutant with a 7-fold higher enzyme level in crude extract. The racemase has one pyridoxal phosphate molecule per 42-kDa subunit, has a Vmax of 3570 units/mg and a Km of 7.8 mM in the L to D direction, and has a Vmax of 1210 units/mg and a Km of 2.2 mM in the D to L direction. The Keq is 0.8 and kcat/Km values are ca. 3 X 10(5) M-1 s-1. The purified enzyme is inhibited in a time-dependent manner by both L- and D-(l-aminoethyl)phosphonates (Ala-P), confirming observations of Atherton et al. in crude extracts of this organism [Atherton, F. R., Hall, M. J., Hassal, C. H., Holmes, S. W., Lambert, R. W., Lloyd, W. J., & Ringrose, P. S. (1980) Antimicrob. Agents Chemother. 18, 897]. Studies with [1-2H]-, [1-3H]-, and [1,2-14C]Ala-P rule out enzymic activation and processing as the basis for irreversible inhibition. Thus, enzyme after exposure to [14C]Ala-P or [alpha-3H]Ala-P and gel filtration contains stoichiometric amounts of radioactive label, but denaturation quantitatively releases intact Ala-P into solution as revealed by high-performance liquid chromatography and cocrystallization with authentic material. The Ala-P isomers are slow binding inhibitors of this racemase as is the alpha,alpha'-dimethyl analogue but not the D or L isomers of the corresponding phosphinate.(ABSTRACT TRUNCATED AT 250 WORDS)