Enzymes aminomutases

Methylmalonyl CoA mutase, leucine aminomutase, and methionine synthase (Figure 45-14) are vitamin Bj2-dependent enzymes. Methylmalonyl CoA is formed as an intermediate in the catabolism of valine and by the carboxylation of propionyl CoA arising in the catabolism of isoleucine, cholesterol, and, rarely, fatty acids with an odd number of carbon atoms—or directly from propionate, a major product of microbial fer-... 

Studies on three different iron-sulfur enzyme systems, which all require S-adenosyl methionine—lysine 2,3-aminomutase, pyruvate formate lyase and anaerobic ribonucleotide reductase—have led to the identification of SAM as a major source of free radicals in living cells. As in the dehydratases, these systems have a [4Fe-4S] centre chelated by only three cysteines with one accessible coordination site. The cluster is active only in the reduced... 

Timothy J. Montavon was born in Wheelersburg, OH, in 1982. He attended Xavier s University in Cincinnati, OH, where he earned a B.S. in chemistry in 2004. While at Xavier s, he worked in the laboratory of Professor Aaron Baba developing novel fluoroiono-phores. In 2004, he began attending graduate school at Boston College working in the laboratory of Professor Steven Bruner. His current research is focused on the structure and mechanism of aminomutase enzymes used in the biosynthesis of enediyne natural products. 

For this group of aminomutases PLP is required as a second coenzyme. Third, X is attached via a carbon atom the enzymes are called mutases. Methyl-malonyl-Co mutase is required for catabolism of propionate in the human body, and is one of only two known vitamin B12-... 

Aminomutases. The enzymes i -[5-Iysine mutase (which is also D-a-Iysine mutase) and D-omithine... 

Recently, a new SAM dependent lysine 2,3-aminomutase was detected and characterized in Bacillus subtilis. Unlike the enzyme from C. subterminah SB4, the enzyme in B. subtilis apparently consists of four identical subunits each with a molecular mass of 54 kDa [30]. A PLP binding motif was identified in this amino-mutase that is also highly conserved in other lysine 2,3-aminomutases [31]. 

PLP radical was proved by application of [2-2H]lysine and [2-13C]lysine instead of lysine 24 to the reaction mixture [38]. In the first experiment the EPR signal was narrowed in the second it was broadened. The involvement of PLP in the reaction was proven by ESEEM spectroscopy. By incubation of the aminomutase with lysine, SAM, and [4 -2H]PLP a prominent doublet centered at the Lamour frequency for 2H was recognized [39], in accordance with the structure of an external aldi-mine. These findings establish a new role for PLP in enzyme reactions - PLP facilitates the radical isomerization. 

Overton and coworkers discovered a leucine 2,3-aminomutase in plant tissue cultures of Andrographis paniculata that converts (S)-leucine in (R)-f-leucine [43] (Scheme 1.6.10). The enzyme activity was investigated in cell free extracts by incubation with (S)-[U-14C]leucine and by measuring the radioactivity of the methyl ester camphanamide derivatives of the reaction mixtures by radio-GC. The stereochemistry of the /i-am i no acid was determined by radio-GC comparison of the enzyme reaction product as methyl ester camphanamide derivative with an authentic sample. The enzyme is not dependent on cobalamin, because addition of intrinsic factor does not induce its inhibition. 

Biosynthesis of (S)-/ -Tyrosine in Bacillus brevis Vm4 //-Tyrosine 43 is a constituent of the peptide antibiotics edeine A and B [60] obtained from cultures of BaciUus brevis Vm4. //-Tyrosine is derived from a-tyrosine 42 by use of a tyrosine 2,3-aminomutase [61]. The purified enzyme has properties fundamentally different from those of all other aminomutases so far mentioned. It requires ATP and Mg2+ ions, but no other cofactors. 

Besides a lysine 5,6-aminomutase, Clostridium sticklandii also has a D-ornithine 4,5-aminomutase (EC 5.4.3.5) [78, 79], D-Ornithine is generated from L-ornithine by ornithine racemase [80], The two genes encoding D-ornithine 4,5-aminomutase have been cloned, sequenced, and expressed in E. coli [81]. The enzyme is an a2/ 2-heterotetramer, consisting of 12 800 Da and 82 900 Da subunits. The protein requires Bn and pyridoxal phosphate as cofactors. Similar to the lysine 5,6-aminomutase, a conserved base-ofi)/histidine-on cobalamin binding motif is present in the 82 900 Da protein. 

So far, two types of aminomutase have been investigated in detail. Lysine 2,3-aminomutase from Clostridium subterminale SB4 is the example par excellence for the SAM-dependent type of aminomutase. Several other enzymes belonging to the same family are known. Examples are biotin synthase [82], pyruvate formate lyase [83, 84], and anaerobic ribonucleotide reductase [85]. 

In 1980 Poston (PI) proposed that vitamin B12 was required for the conversion of the branched-chain amino acid p-leucine to leucine. He found circulating P-leucine levels elevated in patients with vitamin B12 deficiency. The concentration of leucine on the other hand was found to be much lower. He suggested that 2,3-aminomutase, which catalyzes the interconversion of P-leucine and leucine, is a vitamin B12-dependent enzyme which is consequently reduced in patients with pernicious anemia. The enzyme has been found in the liver of several animals and in human leucocytes, and in vitro experiments have shown it to be adenosylcobalamin dependent (P2). 

P-Lysine aminomutase has also been shown to catalyze the transformation of D-lysine into D-2,5-diaminohexanoate, and the enzyme has been described as o/p-lysine 5,6-aminomutase (o/P-LAM). ... 

Figure 10. EPR spectral evidence for SAM- or SAH-dependent reduction of the iron-sulfur clusters of lysine 2,3-aminomutase. In this figure, AdoMet refers to S-adenosylmethionine (SAM). (A) Spectrum of enzyme in the absence of dithionite and SAM (B) spectrum of enzyme in the presence of dithionite only (C) spectrum of enzyme in the presence of dithionite and SAM (D) spectrum of enzyme in the presence of dithionite and SAH. Adapted from reference 25 with permission from the American Chemical Society.

In mammals, there are only three vitamin B12 -dependent enzymes methionine synthetase, methylmalonyl CoA mutase, and leucine aminomutase. The enzymes use different coenzymes methionine synthetase uses methylcobal-amin, and cobalt undergoes oxidation during the reaction methylmalonyl CoA mutase and leucine aminomutase use adenosylcobalamin and catalyze the formation of a 5 -deoxyadenosyl radical as the catalytic intermediate. 

The amino mutases require pyridoxal phosphate for activity, and although little is known about them mechanistically, Freyis work on lysine 2,3-aminomutase has proved very informative (Lieder et al., 1998 Frey, 1997). Lysine 2,3-aminomutase is not a 1 2 enzyme but it functions very similarly (Section 1). The enzyme uses pyridoxal phosphate to facilitate the 1,2... 

Hennig M, Griimn B, Contestabile R, John RA, Jansonius JN. Crystal structure of glutamate-1-semialdehyde aminomutase an alpha 2-dimeric vitamin B6-dependent enzyme with asymmetry in structure and active site reactivity. 1997 94 4866-4871. 

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