Gerratana Barbara, Stapon Anthony, Townsend Craig A
Department of Chemistry, The Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA.
Biochemistry. 2003 Jul 1;42(25):7836-47. doi: 10.1021/bi034361d.
The Erwinia carotorova carA, carB, and carC gene products are essential for the biosynthesis of (5R)-carbapen-2-em-3-carboxylic acid, the simplest carbapenem beta-lactam antibiotic. CarA (hereafter named carbapenam synthetase) has been proposed to catalyze formation of (3S,5S)-carbapenam-3-carboxylic acid from (2S,5S)-5-carboxymethyl proline based on characterization of the products of fermentation experiments in Escherichia coli cells transformed with pET24a/carB and pET24a/carAB, and on sequence homology to beta-lactam synthetase, an enzyme that catalyzes formation of a monocyclic beta-lactam ring with concomitant ATP hydrolysis. In this study, we have purified recombinant carbapenam synthetase and shown in vitro that it catalyzes the ATP-dependent formation of (3S,5S)-carbapenam-3-carboxylic acid from (2S,5S)-5-carboxymethyl proline. The kinetic mechanism is Bi-Ter where ATP is the first substrate to bind followed by (2S,5S)-5-carboxymethyl proline and PPi is the last product released based on initial velocity, product and dead-end inhibition studies. The reactions catalyzed by carbapenam synthetase with different diastereomers of the natural substrate and with alternate alpha-amino diacid substrates were studied by HPLC, ESI mass spectrometry, and steady-state kinetic analysis. On the basis of these results, we have proposed a role for each moiety of (2S,5S)-5-carboxymethyl proline for binding to the active site of carbapenam synthetase. Coupled enzyme assays of AMP and pyrophosphate release in the reactions catalyzed by carbapenam synthetase with adipic and glutaric acid, which lack the alpha-amino group, in the presence and absence of hydroxylamine support the formation of an acyladenylate intermediate in the catalytic cycle.
胡萝卜软腐欧文氏菌的carA、carB和carC基因产物对于最简单的碳青霉烯β-内酰胺抗生素（5R）-碳青霉-2-烯-3-羧酸的生物合成至关重要。基于用pET24a/carB和pET24a/carAB转化的大肠杆菌细胞发酵实验产物的表征以及与β-内酰胺合成酶的序列同源性，已提出CarA（以下称为碳青霉烯胺合成酶）催化由（2S，5S）-5-羧甲基脯氨酸形成（3S，5S）-碳青霉烯胺-3-羧酸，β-内酰胺合成酶是一种催化单环β-内酰胺环形成并伴随ATP水解的酶。在本研究中，我们纯化了重组碳青霉烯胺合成酶，并在体外证明它催化由（2S，5S）-5-羧甲基脯氨酸ATP依赖性形成（3S，5S）-碳青霉烯胺-3-羧酸。动力学机制为Bi-Ter，即ATP是第一个结合的底物，随后是（2S，5S）-5-羧甲基脯氨酸，基于初速度、产物和终产物抑制研究，焦磷酸是最后释放的产物。通过高效液相色谱、电喷雾电离质谱和稳态动力学分析研究了碳青霉烯胺合成酶与天然底物的不同非对映异构体以及与替代α-氨基二酸底物催化的反应。基于这些结果，我们提出了（2S，5S）-5-羧甲基脯氨酸的每个部分在与碳青霉烯胺合成酶活性位点结合中的作用。在有和没有羟胺存在的情况下，对碳青霉烯胺合成酶与缺乏α-氨基的己二酸和戊二酸催化的反应中AMP和焦磷酸释放的偶联酶分析支持了催化循环中酰基腺苷酸中间体的形成。
